ArticlePDF AvailableLiterature Review

Advances in molecular detection of Aspergillus: An update

April 2010
Archives of Microbiology 192(6):409-25

April 2010
192(6):409-25

DOI:10.1007/s00203-010-0563-y

Source
PubMed

Authors:

M. Z. Abdin

Jamia Hamdard University

Malik Mobeen Ahmad

Integral University

Saleem Javed

Aligarh Muslim University

Filamentous cosmopolitan fungi of the genus Aspergillus can be harmful in two ways, directly they can be opportunistic pathogens causing aspergillosis and indirectly due to aflatoxin production on food products which can lead to aflatoxicosis. Therefore, a number of methods have been proposed so far for detection of the fungi with lowest possible concentration at the earliest. Molecular methods such as PCR and/or in combination with certain techniques have been found to be useful for Aspergillus detection. We discuss here various technologies that have emerged in recent years and can possibly be used for the molecular detection of Aspergillus in an efficient way. These methods like RSIC, C-probe, and inversion probe with pyrosequencing or direct ss/dsDNA detection have been used for the identification of fungal or bacterial pathogens and thus formulate a 'gold standard' for Aspergillus detection.

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MINI-REVIEW

Advances in molecular detection of Aspergillus: an update

M. Z. Abdin •Malik M. Ahmad •Saleem Javed

Received: 20 February 2009 / Revised: 1 November 2009 / Accepted: 10 March 2010 / Published online: 1 April 2010

ÓSpringer-Verlag 2010

Abstract Filamentous cosmopolitan fungi of the genus

Aspergillus can be harmful in two ways, directly they can

be opportunistic pathogens causing aspergillosis and indi-

rectly due to aﬂatoxin production on food products which

can lead to aﬂatoxicosis. Therefore, a number of methods

have been proposed so far for detection of the fungi with

lowest possible concentration at the earliest. Molecular

methods such as PCR and/or in combination with certain

techniques have been found to be useful for Aspergillus

detection. We discuss here various technologies that have

emerged in recent years and can possibly be used for the

molecular detection of Aspergillus in an efﬁcient way.

These methods like RSIC, C-probe, and inversion probe

with pyrosequencing or direct ss/dsDNA detection

have been used for the identiﬁcation of fungal or bacterial

pathogens and thus formulate a ‘gold standard’ for

Aspergillus detection.

Keywords Aspergillus Molecular detection 

PCR Polymerase chain reaction-enzyme immunoassay 

DNA ﬁngerprinting Microarray technique 

Retrotransposon insertion-site context typing STRAf 

ELISA

Introduction

Contamination with mycotoxins is a major problem of food

and feeds storage. Mycotoxins are secondary metabolites

produced by ﬁlamentous fungi. These toxic extrolites are

not crucial for sustaining the life; instead, it is believed that

they provide a competitive edge over other non-toxigenic

molds and bacteria. Contamination of food and animal

feeds with mycotoxin leads to adversely effect on human

health also and leads to economic losses. Mycotoxins

contaminate approximately 25–50% of the total crops

harvested, and since molds best thrive in tropical regions,

they damage about 80% of the crops. There are about 300

different mycotoxins, but only 20 of them are known to

have toxic effect on human when ingested along with

contaminated food (Konietzny and Greiner 2003; Table 1).

Mycotoxins like Aﬂatoxins (AF), Ochratoxins (OT),

Zeralenone (ZEN), Trichothecenes, and Fumonisins (F)

are the major mycotoxins inﬂuencing the public health

and agriculture. World Health Organization-International

Agency for Research on Cancer has categorized the

mycotoxins according to its carcinogenic nature on

humans. Aﬂatoxins have been classiﬁed as the most car-

cinogenic mycotoxin and placed under Group I type

while OT and F have been placed under Group 2B and

trichothecenes have been kept under non-carcinogenic

category of Group 3 (Hussein and Brasel 2001; WHO–

IARC 1993a,b).

Aﬂatoxins are produced by a large number of Asper-

gillus species, basically by three phylogenetically distinct

sections. The main producers are A. ﬂavus,A. parasiticus,

A. nomius, A. pseudotamarii, A. parvisclerotigenus, and

A. bombycis of section Flavi. A. ochraceoroseus and

A. rambellii from section Ochraceorosei and Emericella

astellata, E. venezuelensis from section Nidulatans also

Communicated by Axel Brakhage.

M. Z. Abdin M. M. Ahmad

Department of Biotechnology, Faculty of Science,

Jamia Hamdard, New Delhi 110062, India

S. Javed (&)

Department of Biochemistry, Faculty of Science,

Jamia Hamdard, New Delhi 110062, India

e-mail: saleemjaved70@yahoo.co.in

123

Arch Microbiol (2010) 192:409–425

DOI 10.1007/s00203-010-0563-y

Table 1 List of 20 mycotoxins responsible for food spoilage and health problems

Mycotoxin Type Producer fungal species Affected commodities References

Aﬂatoxin B (B1, B2) A. ﬂavus,A. parasiticus, A. tamarii,

A. pseudotamarii, A. bombycis,

A. parvisclerotigenus,A. nomius,

A. minisclerotigenes, A. oryzae,

A. toxicarius, A. versicolor,

A. rambellii, A. arachidicola,

A. ochraceoroseus, Emericella

astellata, E. venezuelensis

Cotton seed, peanuts, peanut butter,

pea, sorghum, rice, pistachio,

maize, oilseed rape, maize ﬂour,

sunﬂower seed, ﬁgs, spices,

meats, dairy products, fruit juices

(apple, guava)

Pildain et al. (2008), Foong-

Cunningham et al. (2006), Murphy

et al. (2006), Samson et al. (2006),

Frisvad and Samson (2004a),

Frisvad et al. (2004a), Konietzny

and Greiner (2003), Atalla et al.

(2003), Hussein and Brasel (2001),

Ito et al. (1995)

G (G1, G2) A. parasiticus, A. nomius,

A. bombycis,A. pseudotamarii,

A. terreus, A. versicolor,

A. arachidicola, A. toxicarius,

A. minisclerotigenes

Peanuts, cotton seed, sunﬂower

seed, tree nuts, pistachio, peanut

butter, maize ﬂour, pea, cereals,

corn, ﬁgs, meats, spices, dairy

products, fruit juices (apple,

guava)

Pildain et al. (2008), Foong-

Cunningham et al. (2006), Murphy

et al. (2006), Samson et al. (2006),

Frisvad et al. (2005b), Somashekar

et al. (2004), Atalla et al. (2003),

Konietzny and Greiner (2003)

Sterigmatocystin A. ﬂavus, A. versicolor,

A. ochraceoroseus, A. rambellii,

A. chevalieri, A. ruber,

A. amstelodami, E. nidulans,

E. rugulosa, E. venezuelensis

Peanuts, maize, peanut butter,

pistachio, cotton seed, oilseed

rape, tree nuts, sunﬂower seed,

soybeans, pea, cereals, ﬁgs

Foong-Cunningham et al. (2006),

Murphy et al. (2006), Frisvad and

Samson (2004a), Frisvad et al.

(2004a), Konietzny and Greiner

(2003)

Ochratoxin A (OTA) A. westerdijkiae, A. niger,

A. ostianus, A. sclerotiorum,

A. carbonarius, A. steynii,

A. sulphureus, A. sclerotionum,

A. lacticoffeatus, A. sclerotioniger,

P. verrucosum

Cereals (wheat, oats, barley), beans,

soybeans, pea, nuts, coffee, cocoa

beans, grapes, ﬁg, sunﬂower seed,

sugarcane, meats, dried fruits,

spices, vinegar, wine

Batista et al. (2009), Foong-

Cunningham et al. (2006), Murphy

et al. (2006), Samson et al. (2006),

Frisvad et al. (2005b), Frisvad et al.

(2004b)

B (OTB) A. westerdijkiae, A. alliaceus,

A. sclerotiorum, A. sulphureus,

A. petrakii, Penicillium virdicatum,

P. cyclopium, A. albertensis,

A. auricomus, A. wentii

Cereals, beans, nuts, cocoa beans,

dried fruits, spices, wine

Batista et al. (2009), Murphy et al.

(2006), Frisvad et al. (2004b),

Bennett and Klich (2003), Hussein

and Brasel (2001)

C (OTC) A. ochraceus Wheat, oats, barley, maize, nuts,

coffee, meats, dried fruits

Murphy et al. (2006), Frisvad and

Samson (2004b), Bennett and

Klich (2003)

Trichothecenes Deoxynivalenol

(DON)

Fusarium graminearum,

F. sambucinum, F. culmorum,

F. sporotrichiodies, F. solani,

Aspergillus parasiticus,

A. terreus, A. oryzae, A. versicolor,

Microdochium nivale

Barley, potatoes, wheat, silage,

maize, soybean, rye, sunﬂower,

porcessed grains, cookies,

cornmeal

Murphy et al. (2006), Lillard-Roberts

(2004), Atalla et al. (2003),

Bennett and Klich (2003), Logrieco

et al. (2003), Konietzny and

Greiner (2003), Tomczak et al.

(2002), El-Banna et al. (1984)

T2 toxin F. solani, F. sporotrichiodes,

F. acuminatum, F. lateritium,

F. chlamydosporum,

F. rigidiusculum, F. poae,

F. sambucinum, A. oryzae,

A. parasiticus

Cereal crops (wheat, barley, oats,

and rye), ﬁg, potato, processed

grains (malt, beer, bread), silage

Murphy et al. (2006), Lillard-Roberts

(2004), Bennett and Klich (2003),

Atalla et al. (2003), Logrieco et al.

(2003), Hussein and Brasel (2001)

HT2 toxin F. solani, F. chlamydosporum,

F. poae, F. sporotrichiodes,

F. acuminatum, F. sambucinum

Potato, maize, wheat, barley, oat,

rye, processed grains (malt, beer),

ﬁg, silage

Murphy et al. (2006), Lillard-Roberts

(2004), Bennett and Klich (2003),

Logrieco et al. (2003), Konietzny

and Greiner (2003), El-Banna et al.

(1984)

Diacetoxyscirpenol

(DAS)

Gibberella intricans, F. equiseti,

F. solani, F. chlamydosporum,

F. lateritium, F. sporotrichiodes,

F. poae, F. sambucinum,

F. acuminatum

Potato, maize, wheat, sunﬂower,

corn, oat, ﬁg, processed grains,

silage

Murphy et al. (2006), Lillard-Roberts

(2004), Bennett and Klich (2003),

Logrieco et al. (2003), Hussein and

Brasel (2001)

Nivalenol (NIV) F. tricinctum,F. poae,

F. crookwellense, F. equiseti,

F. graminearum, F. culmorum,

F. sambucinum, A. parasiticus,

A. versicolor

Wheat, cereals, maize, barley, corn,

oat, rye, processed grains (malt,

beer)

Murphy et al. (2006), Lillard-Roberts

(2004), Bennett and Klich (2003),

Atalla et al. (2003), Logrieco et al.

(2003), Konietzny and Greiner

(2003), Tomczak et al. (2002)

El-Banna et al. (1984)

410 Arch Microbiol (2010) 192:409–425

123

Table 1 continued

Mycotoxin Type Producer fungal species Affected commodities References

Fumonisin B1 F. nygamai, F. verticillioides,

F. culmorum, F. avenaceum,

F. roseum, F. proliferatum,

F. acutatum, F. phyllophilum

Maize, corn and products, asparagus spears,

garlic bulbs, wheat, rice

Murphy et al. (2006), Bennett and Klich

(2003), Konietzny and Greiner (2003),

Fotso et al. (2002), Hussein and Brasel

(2001)

B2 F. acutatum, F. pseudocircinatum,

F. proliferatum, F. verticillioides,

F. acutatum, A. niger

Wheat, rice, maize, corn, sorghum,

asparagus crown, onion, garlic, coffee

beans

Frisvad et al. (2007), Murphy et al. (2006),

Lillard-Roberts (2004), Bennett and

Klich (2003), Fotso et al. (2002),

Hussein and Brasel (2001)

Moniliformin

(MON)

F. chlamydosporum, F. proliferatum,

F. monoliforme, F. oxysporum,

F. equiseti, F. begoniae,

F. phyllophilum, F. subglutinans,

F. avenaceum, F. tricinctum,

F. pseudocircinatum, Microdochium

nivale

Cereals, maize, barley, oats, rice, pepper,

ﬂax, soybeans, millet, sorghum, barley,

corn, wheat

Sørensen et al. (2007), Murphy et al.

(2006), Lillard-Roberts (2004), Bennett

and Klich (2003), Fosto et al. (2002),

Tomczak et al. (2002)

Zearalenone

(ZEN)

F. graminearum, F. culmorum,

F. oxysporum, F. sporotrichiodes,

F. moniliforme, F. avenaceum,

F. crookwellense, F. equiseti,

F. heterosporum, A. oryzae

Maize, barley, oat, rye, wheat, rice,

sorghum, bread, corn, banana, sunﬂower,

walnut, cornﬂake, sweetcorn

Murphy et al. (2006), Lillard-Roberts

(2004), Bennett and Klich (2003),

Logrieco et al. (2003), Atalla et al.

(2003), Hussein and Brasel (2001)

Cyclopiazonic

acid (CPA)

Penicillium commune, P. cyclopium,

P. griseoﬂvum, P. camembertii,

P. dipodomyicola, A. ﬂavus, A.

tamarii, A. fumigatus, A. phoenicis,

A. parvisclerotigenus, A. oryzae,

A. caelatus, A. toxicarius

A. pseudotamarii,

A. minisclerotigenes

Barley, maize, cheese, peanut, rice, peeled

barley, soybean, smoke-dreid meat, coffee

beans, peanut, pistachio, millet seed

Pildain et al. (2008), Dombrink-Kurtzman

(2007), Vinokurova et al. (2007),

Murphy et al. (2006), Samson et al.

(2006), Bennett and Klich (2003),

Konietzny and Greiner (2003)

Patulin Aspergillus clavatus, A. terreus,

A. giganteus, Penicillium patulum

(urticae), P. expansum,

P. claviforme, P. griseoﬂvum,

Byssochlamys nivea,

Paecilomyces variotii

Barley, wheat, tomato, strawberry, banana,

mango, avocado, apricot, blueberry, red

raspberry, boysenberry juice, grape juice,

apple, pears

Bokhari et al. (2009), Dombrink-Kurtzman

and Engberg (2006), Murphy et al.

(2006), Frisvad et al. (2004b), Bennett

and Klich (2003), Konietzny and Greiner

(2003), Hasan (2000)

Alternariol

monomethyl

ether

(AME)

Alternaria alternata, Alternaria

brassicae, Alternaria capsici-annui,

Alternaria citri, Alternaria

cucumerina, Alternaria dauci,

Alternaria kikuchiana, Alternaria

tomato, Alternaria solani,

Alternaria longipes, Alternaria

porri, Alternaria tenuissima

Rice, olive, barley, oat, oilseed rape,

sunﬂower, maize, wheat, rye, mandarian

fruits, tomato, pepper, melon,

Logrieco et al. (2009), Ostry (2008),

Murphy et al. (2006), Lillard-Roberts

(2004), Bennett and Klich (2003),

Konietzny and Greiner (2003), Ren et al.

(1998)

Alternariol

(AOH)

Alternaria alternata, Alternaria

brassicae, Alternaria tenuissima,

Alternaria cucumerina, Alternaria

capsici-annui, Alternaria citri,

Alternaria dauci, Alternaria solani,

Alternaria longipes, Alternaria

porri

Rice, sunﬂower, mandarian fruits, olive,

oat, barley, pepper, wheat, rye, oilseed

rape, melon,

Logrieco et al. (2009), Ostry (2008),

Murphy et al. (2006), Lillard-Roberts

(2004), Bennett and Klich (2003),

Konietzny and Greiner (2003), Ren et al.

(1998)

Tenuazonic

acid

Alternaria alternata, Alternaria

tenuissima, Alternaria capsici-

annui, Alternaria citri,

A. cucumerina, Alternaria

kikuchiana, Alternaria japonica,

Alternaria longipes, Alternaria

porri, Alternaria radicina,

Alternaria tomato, Pyricularia

oryzae, Phoma sorghina

Rice, oilseed rape, olive, mandarian fruits,

rye, barley, wheat, oat, pepper, sunﬂower,

melon

Logrieco et al. (2009), Ostry (2008),

Murphy et al. (2006), Bennett and Klich

(2003), Konietzny and Greiner (2003)

Citrinin P. citrinum, P. expansum,

P. radicicola, P. verrucosum,

Monascus ruber, A. carneus,

A. terreus, A. niveus

Wheat, barely, rice, apple, medicinal plant

seeds (Hydnocarpus laurifolia, Blepharis

edulis, Piper betle, Acacia concinna,

Caesalpinea digyna, Cassia ﬁstula,

Argyreia speciosa, Embelia ribes)

Murphy et al. (2006), Frisvad et al.

(2004b), (2005b), Anderson and Frisvad

(2004), Overy and Frisvad (2003),

Bennett and Klich (2003), Konietzny and

Greiner (2003), Blanc et al. (1995)

Arch Microbiol (2010) 192:409–425 411

123

generate aﬂatoxins (Frisvad et al. 2005a). The naturally

occurring aﬂatoxins designated as aﬂatoxin B1, B2, G1,

and G2. B and G forms are referred as they emit blue or

green ﬂuorescence upon exposure to ultraviolet light

(Murphy et al. 2006). These are the most toxic and car-

cinogenic secondary metabolites among the known myco-

toxins (Ellis et al. 1991) and cause aﬂatoxicosis upon

ingestion of aﬂatoxin in contaminated food or feed. Liver is

the primary target organ for acute and chronic injury.

Modiﬁed by-product of aﬂatoxin B1 gets more toxic and

carcinogenic during detoxiﬁcation by the liver cytochrome

P-450 monoxygenase. Their epoxide form binds to guanine

residues inducing mutations (Yu et al. 2005).

Since contamination of food and feed with mycotoxi-

genic fungi is a major problem, it is crucial to develop

methods of detection for these pathogens that are relatively

rapid and highly sensitive. Besides aﬂatoxin-producing

Aspergillus species, many species like A. fumigatus,

A. ﬂavus,A. lentulus, A. niger,A. terreus, A. utus,

A. nidulans act as opportunistic pathogens (Samson, 1994).

Therefore, besides detecting them in human foods, it is also

necessary to detect them in body ﬂuids for the diagnosis of

aspergillosis and other Aspergillus-related diseases.

Aspergillus species can cause several diseases in humans

such as invasive aspergillosis, invasive pulmonary asper-

gillosis, allergic bronchopulmonary aspergillosis, and

allergic fungal sinusitis (Segal 2009; Segal and Walsh

2006; Klont et al. 2004; Stevens et al. 2000; Schubert

2009). Early detection of these fungi can serve as a

warning signal for the potential health hazard (Shapira

et al. 1997; Varga 2006). Aspergillus species can cause

several diseases in humans besides aspergillosis, such as

aspergilloma, sinusitis, and allergic bronchopulmonary

aspergillosis (Henry et al. 2000). The detection methods

that are being used, such as cultivation on media and

immunological (RIA, ELISA) methods, are time taking,

laborious, and disadvantageous in some or the other

way. However, for diagnosis of invasive aspergillosis, a

commercialized ELISA-based method (Platelia test) is now

commonly used for galactomannan detection in blood with

improved sensitivity (0.5–1 ng ml

-1

) and speciﬁcity than

the previous latex agglutination test with the same mono-

clonal antibody (Pinel et al. 2003; Giacchino et al. 2006).

Chromatographic methods use costly and highly sophisti-

cated equipments while serum-based methods work on

‘‘one substance one assay’’ concept (Konietzny and Greiner

2003; Yong and Cousin 2001). So, instead of detecting the

toxins from aspergilli after its production, an alternative

approach that can be used is to identify directly these molds

before the toxin production by molecular methods.

Due to the poor performance of immunological meth-

ods, scientists have started to diverge the attention toward

the molecular diagnostic methods. Molecular methods are

generally based on detecting the differences in sequences

of nucleic acids. Ampliﬁcation, sequencing, DNA hybrid-

ization, and gel electrophoresis are some common laboratory

methods that can be used for detection (Foong-Cunningham

et al. 2006). Molecular methods such as PCR have

facilitated the in vitro ampliﬁcation of target sequences

(Arnheim and Erlich 1992). The main advantage of PCR is

that organisms can be identiﬁed within mixtures of DNA

and without culturing the organisms. Thus, this method is

both speciﬁc and fast. Probing, in combination with

different molecular methods, has been found to be an

effective way for the identiﬁcation of speciﬁc amplicons in

a mixture with similar sizes (Sandhu et al. 1995). A large

number of probe-based methods have already been devel-

oped for the detection and enumeration of various food-

borne pathogens (Jones 1991). DNA-based diagnostic

methods have revolutionized the diagnostic technology in

the clinical, forensic science, and in the agriculture sector.

A number of DNA-based methods for the detection of

Aspergillus are described in this review (Table 2).

PCR-based detections

Several researchers have used the PCR method as probe to

identify the speciﬁc organism or contamination on food

and feeds. It is one of the easiest methods for the identi-

ﬁcation of any microorganism in samples. Manonmani

et al. (2005) using an indigenously speciﬁc primer pair for

the aﬂatoxin regulatory (aﬂR) gene assessed the presence

of aﬂatoxigenic fungi in foodstuffs. Although speciﬁcity

was assayed with both pure and mixed cultures and only

A. ﬂavus and A. parasiticus showed positive response of

ampliﬁcation, the limit of detection (LOD) of mycelium

and spores was found to be 0.5 and C100 CFU, respec-

tively. For A. ﬂavus, in groundnut and maize, the speciﬁcity

and sensitivity was as little as 100 CFU g

-1

. Shapira

et al. (1996), however, identiﬁed and sequenced three

genes, versicolorin A dehydrogenase gene (ver-1), ster-

igmatocystin-o-methyltransfersase 1 gene (omt-1), and (an

aﬂatoxin biosynthesis regulatory gene) apa-2from Asper-

gillus parasiticus. The primers only ampliﬁed A. ﬂavus and

A. parasiticus genes while the ampliﬁcation of other

Aspergillus species, Penicillum species, and Fusarium

species was not obtained. To assess the PCR’s sensitivity,

lowest level was ampliﬁed with ver-1primers of A. para-

siticus (10

spores g

-1

). Geisen (1996) and Farber et al.

(1997) also developed a PCR system targeting norsolorinic

acid reductase gene (nor-1), ver-1and omt-1genes. Both of

them had discriminated the aﬂatoxigenic Aspergillus spe-

cies from the non-producers. However, the problem still

stands with the A. ﬂavus group as some strains behave as

non-aﬂatoxigenic species. On the calmodulin gene basis,

412 Arch Microbiol (2010) 192:409–425

123

Table 2 Methods for detection of Aspergillus

Detection method Sensitivity Target species References

Polymerase chain reaction

Speciﬁc primer of aﬂR 0.5 CFU

C100 CFU

A. ﬂavus

A. parasiticus

Manonmani et al.

(2005)

Calmodulin gene 12.5 pg A. carbonicus

A. japonicus

Perrone et al.

(2004)

ver-1 gene 10

spores g

-1

A. parasiticus Shapira et al.

(1996)

18S rRNA gene 10 fg A. fumigatus Bansod et al.

(2008)

Calmodulin gene 10 pg A. niger,

A. tubingensis

Susca et al.

(2007)

18S rRNA gene 50 fg

10 fg

A. fumigatus, A. ﬂavus, A. niger, A. terreus, A. nidulans,

C. albicans

Einsele et al.

(1997)

18S rRNA gene 2 spores per reaction A. ﬂavus Zhou et al. (2000)

Hot start PCR 0.2 GE Aspergillus, Candida, Blastomyces, Histoplasma

capsulatum, Sporothrix schenckii

Sandhu et al.

(1995)

Real-time PCR of mt tRNA &

rRNA

5 copies ml

-1

A. fumigatus, A. ﬂavus, A. niger, A. terreus Bolehovska et al.

(2006)

RTi-PCR of genomic DNA SYBR Green I- 5 conidia

per reaction

TaqMan- 50 conidia per

reaction

A. carbonarius Selma et al.

(2008)

Monochrome light cycler PCR 0.1 pg

0.01 pg

A. ﬂavus

A. fumigatus

Bu et al. (2005)

Semi-nested PCR 0.1 fg A. fumigatus, Rhizopus, Absidia Bialek et al.

(2005)

Nested PCR of ITS regions 10–100 ag A. fumigatus Zhao et al. (2001)

Multiplex PCR 1–10 cells Aspergillus Luo and Mitchell

(2002)

Combined methods

LiPA–PCR ITS–50 pg

ITS1–50 fg

Aspergillus Martin et al.

(2000)

PCR-EIA 0.5 pg Aspergillus Hinrikson et al.

(2005)

PCR-EIA of 18S rRNA gene 5 pg A. fumigatus Elie et al. (1998)

Nested-speciﬁc PCR-EIA of

18S rRNA gene

1.7 ng ll

-1

A. fumigatus Golbang et al.

(1999)

PCR-EIA of mt gene 0.6 fg ml

-1

Aspergillus Jones et al. (1998)

DNA ﬁngerprinting method

RAPD – A. carbonarius Fungaro et al.

(2004)

DNA microarray

ITS region of 18S rRNA gene 10 pg Aspergillus, Candida Hsiao et al.

(2005)

Other molecular method

SPC with immuno-ﬂuorescence

labeling

2–10 hyphae per sample A. fumigatus de Vos and Nelis

(2003)

NASBA 10

–10

copies ml

-1

A. fumigatus Yoo et al. (2008)

Different methods used for detecting Aspergillus and other genus. (CFU Colony-forming unit, GE Genome equivalent, RTi-PCR quantitative

Real-time polymerase chain reaction, ITS Intergenic transcribed spacer, LiPA-PCR Line probe assay polymerase chain reaction, PCR-EIA

Polymerase chain reaction-enzyme immunoassay, RAPD Random ampliﬁed polymorphic DNA, SPC Solid phase cytometry, NASBA Nucleic

acid sequence-based ampliﬁcation)

Arch Microbiol (2010) 192:409–425 413

123

Susca et al. (2007b) identiﬁed Aspergillus niger and

Aspergillus tubingensis, while Perrone et al. (2004)

reported Aspergillus carbonicus and Aspergillus japonicus

using PCR method. Susca et al. (2007b) developed a more

sensitive detection for molds than Perrone et al. (2004)

using the same gene and the same method. Li et al. (1998)

identiﬁed the phylogenetic relationship between patho-

genic species of A. fumigatus,A. ﬂavus,A. niger,A. ter-

reus, and Emericella nidulans by PCR ampliﬁcation of the

mitochondrial cytochrome b gene. Except for Emericella

nidulans, all other strains produced the 426-bp-long frag-

ments. Species-speciﬁc nucleotides were found in each of

the ﬁve species. No difference between the strains was

found after comparing the 142-amino acid sequences

derived from the 426-bp nucleotide sequences.

Kappe et al. (1998) identiﬁed pathogenic fungi

(A. fumigatus, A. ﬂavus, Candida albicans, etc.) in human

tissue with the help of ampliﬁcation of 18S rRNA gene

fragments. Three different types of oligonucleotide primers

(TR1/CA1-TR2/AF2, UF1 and EU1) with an additional

RZY1 primer were designed. The oligonucleotide AF

hybridized with all known pathogenic aspergilli except

A. niger and A. versicolor; oligonucleotide CA paired with

three pathogenic Candida species. Similarly, targeting 18S

region, Bansod et al. (2008) made a speciﬁc detection of

Aspergillus fumigatus in patients with pulmonary tuber-

culosis by a two-step PCR. Again, Einsele et al. (1997)

developed a PCR assay to identify fungal pathogens such

as Aspergillus,Candida,Mucor,Penicillum,Trichosporon

cutaneum, and T. glabrata. The species-speciﬁc primers

and probes were designed by comparing the 18S rRNA

gene sequences of these pathogens. The Southern blot

detection system was 2–10 times more sensitive than the

simple in-gel detection with ethidium bromide. The

designed probes conﬁrmed the species-speciﬁc hybridiza-

tion and exhibited no cross-reactivity with others. How-

ever, the Aspergillus probe showed 100% identity with

H. capsulatum; hybridization with this probe was addi-

tionally tested with probe speciﬁcally hybridizing with

A. fumigatus,A. ﬂavus, and A. versicolor, with positive

results. Einsele and coworkers achieved this sensitivity,

especially for Aspergillus species, by heating-alkaline

denaturation-lysis for DNA extraction (DNA yield

increased) and by amplifying a gene found in multicopy

(C100 copy) numbers. Besides this, amplicon hybridization

with labeled internal oligonucleotide and alkaline denatur-

ation further improved the sensitivity of this assay. Sandhu

et al. (1995) developed a highly speciﬁc oligonucleotide

probe to identify the fungal infection of Aspergillus

fumigatus, A. glaucus, A. niger, A. terreus, Candida, with

Hot start PCR. Using 28S rRNA genes, 21 highly speciﬁc

oligonucleotides were synthesized for approximately 50

fungal species. The speciﬁcity was determined by adding

50 pmol of probes, radiolabelled with [

P] ATP, on

hybridization membrane under high stringency. The sensi-

tivity of PCR primers was also evaluated and was found to

be successfully ampliﬁed from as little as 0.2 genomes. In

order to measure the air fungal concentration of 17 homes

(indoor and outdoor) in Cincinnati, Meklin et al. (2007) did

mold-speciﬁc quantitative PCR (MSQPCR) and showed

that Aspergillus penicillioides followed by A. versicolor is

present more than any other Aspergillus species. In another

experiment, Zhou et al. (2000) detected ﬁve other com-

monly found fungal species along with A. ﬂavus in indoor

atmosphere and found that a minimum of two fungal spores

was needed for the successful ampliﬁcation by single

primer within a time period of 5–6 h.

Two modiﬁed semi-nested PCR assays for aspergillosis,

caused by A. fumigatus, identiﬁcation performed in parafﬁn

wax embedded tissue have been described by Bialek et al.

(2005). Primers from 18S rRNA gene sequences were

made and to raise the LOD, they introduced a third primer

in the assay. A minimum amount of 0.1 fg of plasmid DNA

(5 genome equivalent) was detected, assuming that there

are 40 copies per genome. Cruzado et al. (2004) also tested

two nested PCR methodologies on 27 strains of 16 genera

and compared the results with previously reported results,

for aspergillus detection. The detection limit obtained by

the procedure given by Yamakami et al. (1996), differs by

500-fold in comparison with the method of Cruzado et al.

who reported a limit of 65 fg instead of 1–10 fg which was

reported by Williamson et al. (2000). The fungal patho-

genic strain identiﬁcation with nested PCR technique by

amplifying the internal transcribed spacer (ITS) regions

has been developed. This method speciﬁcally detected

A. fumigatus with sensitivity of 10–100 attogram (ag) of

sample DNA (Zhao et al. 2001). For speedy detection,

multiplex PCR has been used to identify the pathogenic

fungi directly from cultures. The fungi selected for the

experiment were A. fumigatus,Candida albicans,C. glab-

rata,C. parapsilosis, and C. tropicalis. The detection was

based on the species-speciﬁc primers designed from ITS

regions of rRNA genes. The sensitivity of each set of

primers ranged from 100–1000 DNA molecules, repre-

senting the ability to amplify the appropriate amplicon from

puriﬁed genomic DNA from 1–10 cells. The procedure was

shortened by taking the 0.5 ll of fragments of hyphae,

instead of extracting DNA (Luo and Mitchell 2002). Araujo

et al. (2009) characterized A. fumigatus strains with

microsatellite-based multiplex PCR and found that it is a

simple technique to perform and shows high discriminatory

power with excellent reproducibility (de Valk et al. 2008).

Scherm et al. (2005) differentiated the aﬂatoxin pro-

ducers from non-aﬂatoxin producers of A. ﬂavus and

A. parasiticus strains, with the reverse transcription-poly-

merase chain reaction (RT–PCR) technique. Total RNAs of

414 Arch Microbiol (2010) 192:409–425

123

13 strains were analyzed using speciﬁc primers based on

the conserved regions of 9 structural genes (aﬂD,aﬂG,

aﬂH,aﬂI,aﬂJ,aﬂK,aﬂL,aﬂM,aﬂN,aﬂO,aﬂP, and aﬂQ)

and 2 regulatory genes aﬂS and aﬂR of the aﬂatoxin B1

biosynthetic pathway. All these genes expression varied in

response of aﬂatoxin production and growth. Likewise,

Degola et al. (2009) discriminated the aﬂatoxin-producing

A. ﬂavus (Alfa

) from the non-aﬂatoxigenic (Aﬂa

)

through the same process; however, they classiﬁed some of

the strain as ‘‘slow aﬂatoxin accumulators’’ as these strains

produced the mycotoxin after 10 days of growth.

A few assays are successfully using the real-time PCR

method like LightCycler

technique in combination with

rapid in vitro ampliﬁcation of DNA and detection (Buch-

heidt and Hummel 2005). Contamination problem and false

results of PCR had made the real-time PCR assay, a suit-

able choice against the PCR methods. A well-designed

assay, good primer choice, and probe sequence provide

both sensitivity and speciﬁcity (Goebes et al. 2007). A

large number of Aspergillus species as well as other genus

have been detected from air and clinical samples using

these methods. The threshold values have been reported

between 500 pg and 50 fg. Real-time PCR (RTi-PCR) has

also been used for the quantiﬁcation of Aspergillus car-

bonarius in wine grapes. Genomic DNA from 52 fungal

strains was extracted from the reference sample and food

isolates to evaluate the speciﬁcity of the two primers and a

probe. Comparatively, the LOD for SYBR Green I RTi-

PCR was found to be 10 times higher than the TaqMan

Bangara et al. (2000) devised a SYBR Green quantitative

PCR system for detecting aﬂatoxin producer species of

A. ﬂavus in black pepper based on the nor-1gene sequence.

The sensitivity showed by SYBR Green

was 4.5 910

cells g

-1

of pepper. In a similar way, Mayer et al. (2003)

artiﬁcially infected maize, pepper, and paprika to deter-

mine the minimal cell number for its infection by Taq-

Man

quantitative PCR. The detection limit for the PCR

was little more sensitive (10

cells) than reported previ-

ously. In both cases, the infected commodities had shown a

higher nor-1gene copy number than the spore counts while

the non-infected one had always negative result. Therefore,

the RTi-PCR assay can be used for the prediction of

probable toxigenic risk, even when there is very low levels

of infection present (Selma et al. 2008). However, it had

been suggested that high amount of plant genomic DNA

interferes with the fungal DNA concentration and hinders

the reaction. Therefore, careful DNA extraction is a crucial

step that determines the sensitivity of the method. Light-

Cycler real-time ﬂuorescence PCR, used by Imhof et al.

(2003) and Bu et al. (2005), rapidly detected fungal

infection in clinical tissue samples. Both species-speciﬁc

hybridization assay and direct sequencing of ampliﬁcation

products identiﬁed A. fumigatus. Melting curve analysis of

Aspergillus species gave single products at 90°C. Suanthie

et al. (2009) while performing multiplexed real-time PCR

for Aspergillus, Fusarium, and Penicillium species

obtained a sensitivity of 1 pg to 10 ng in distiller’s grain

(DG). They had designed pairs of broad-spectrum primers

and probes from the ITS region of rDNA. In another

experiment, Bolehovska et al. (2006) detected 103 clinical

invasive aspergillosis samples out of 354 tested positive

with this method. To identify the sensitivity of up to 5

copies mL

-1

, they had used two hybridization probes along

with the primers. Schabereiter-Gurtner et al. (2007) pro-

posed a novel real-time PCR assay for detecting 11

aspergilli and candida species in clinical samples. The

analytical sensitivity varied according to the samples iso-

lated. Sensitivity of pure culture was 1 CFU per PCR while

that of blood- or CSF-isolated samples were 5–10

CFU mL

-1

and 0.05 CFU lL

-1

, respectively. They had

aligned the ITS2 regions of several pathogenic aspergilli

species to obtain a consensus sequence for designing a

single set of primer and increased the sensitivity using

biotinylated probes. Species-speciﬁc detection of A. car-

bonarius and A. niger was done with real-time PCR by

targeting the ITS, calmodulin, and polyketide synthase

(pks) gene regions (Haugland et al. 2004; Mule

´et al. 2006;

Auoti et al. 2007).

Combined methods

For fast and sensitive determination of pathogenic fungal

infections, several workers have combined two different

techniques. The one is mostly PCR. Recently, single-

stranded conformational polymorphisms (SSCP) together

with PCR were used to screen the 11 Aspergillus species by

the detection of calmodulin nucleotide variations (Samson

et al. 2007a). SSCP indirectly detects a single base dif-

ference thereby affecting the strand’s mobility through a

gel by altering the intra-strand base pairing and its resulting

three-dimensional conformations (Susca et al. 2007c;

Fujita and Silver 1994). For determining the medically

important fungi from cultural or clinical samples, fungal-

speciﬁc probes were designed by taking the ITS1 and ITS2

regions of 5.8S gene. All the tested strains Aspergillus,

Candida, and Cryptococcus neoformans were unambigu-

ously differentiated with the exception of cross-contami-

nation from two Candida species. However, of all 21

specimens, the sensitivity was 100% where the species-

speciﬁc probes were added in the PCR-reverse line blot

(RLB) hybridization assay (Zeng et al. 2007). Martin et al.

(2000) designed a method combining reverse hybridization

line probe assay (LiPA) and PCR for the ampliﬁcation of

ITS regions of Aspergillus, Candida, and Cryptococcus.

The assay is based on reverse hybridization principal.

Arch Microbiol (2010) 192:409–425 415

123

Speciﬁc probes are immobilized at identiﬁed sites on a

nitro-cellulose membrane strip (Bosma et al. 2004) and are

hybridized with the biotinylated PCR products of organ-

ism’s DNA. The amalgams formed are consequently

detected through colorimetric method (Rossau et al. 1997).

This mingled method had 10-fold lower detection limit

than the agarose gel electrophoresis for DNA. Cross-con-

tamination results of speciﬁc probes with other pathogens

were found in the experiment such as CD3 probe designed

for C. albicans, paired with C. dubliniensis. However, with

this assay, A. fumigatus can be detected in as low quantity

as 50 fg ml

-1

(1GE) and for C. albicans, threshold value

was 2–10 cells ml

-1

of blood.

Polymerase chain reaction-enzyme immunoassay (PCR-

EIA), a method that uses enzyme-bound antibody to detect

the amplicons, was found to be speciﬁc and sensitive for A.

ﬂavus,A. fumigatus,A. nidulans,A. niger,A. terreus, and

A. versicolor identiﬁcation (Hinrikson et al. 2005). PCR

products were hybridized with digioxigenin-labeled probes

on microtiter plate coated with streptavidin. Amplicons,

designed from ITS1 and ITS2 regions, were detected on a

single colorimetric enzyme immunoassay method. Thus,

the aforementioned method proved to be ten times more

sensitive than the conventional PCR detection. de Aguirre

et al. (2004) had also found the same threshold value in

their experiment with pathogenic fungal species. The

beneﬁt of using PCR-EIA was that only small amount of

DNA (in picogram) is needed for identiﬁcation, DNA

probes can reproducibly be synthesized and are very spe-

ciﬁc, probes can be stored ready for use, and the evaluation

of results are instant (colorimetric and spectrophotometric

measurements). Elie et al. (1998), however, had found the

detection limit 10 times less than Hinrikson et al. (2005)

for A. fumigatus 18S rDNA gene in a microtiter plate EIA.

In another experiment, a nested-speciﬁc PCR-EIA, for

A. fumigatus using 18S rDNA gene sequence, Jones with

co-workers (1998) have got a much more sensitive method

than that of Golbang et al. (1999). This may be because of

the presence of high copy number of mitochondrial gene.

Restriction fragments of different ampliﬁed regions

(PCR–RFLP) have shown a distinction between A. niger,

A. tubingensis,A. carbonarius, and A. aculeatus isolates

(Medina et al. 2005; Bau et al. 2006; Zanzotto et al. 2006;

Martinez-Culebras and Ramson 2007). Similar results have

also been obtained using species-speciﬁc primers in PCR

for species level distinction. Targeting the calmodulin

region, Perrone et al. (2004) and Susca et al. (2007a) dif-

ferentiated A. carbonarius from A. niger while aiming the

ITS regions, Haugland and Vesper (2002) detected A.

carbonarius and A. niger and Gonzalez-Salgado et al.

(2005) did species-speciﬁc identiﬁcation for a number of

black aspergilli. In diagnosing the invasive aspergillosis

disease, Mirhendi et al. (2007) tested a single set of primer

and designed a restricted PCR proﬁle for ﬁve most path-

ogenic Aspergillus species. They also reviewed that both

ITS regions are easy target areas of nucleotide sequence for

discriminating the standard Aspergillus species.

DNA ﬁngerprinting-based methods

For typing of Aspergillus species, a number of molecular

techniques have been developed recently. RFLP ﬁnger-

printing, MLPs, and MLST are being thought to have

discriminatory power and reliability (Varga 2006; de Valk

et al. 2008). Microsatellite length polymorphism (MLP), as

the name suggests, microsatellites are ampliﬁed by PCR.

These simple sequence repeats are present all over the

DNA (Krackow and Ko

¨nig 2008; de Valk et al. 2008). The

simplest example of a microsatellite is a (CA)

repeats,

where n is variable between alleles. These markers often

present high levels of inter- and intra-speciﬁc polymor-

phism, particularly when tandem repeats number are ten or

greater (Queller et al. 1993). Multilocus sequence typing

(MLST) is a technique in molecular biology for the typing

of multiple loci. MLST involves sequencing of internal

fragments of multiple (usually seven) housekeeping genes.

MLST directly measures the DNA sequence variations in a

set of housekeeping genes and characterizes strains by their

unique allelic proﬁles. The principle of MLST is simple:

the technique involves PCR ampliﬁcation followed by

DNA sequencing. Nucleotide differences between strains

can be checked at a variable number of genes (generally

seven) depending on the degree of discrimination desired

(Maiden et al. 1998). Several workers had performed RFLP

of mtDNA, rDNA, pectin lyase (pelA), and pyruvate kinase

(pki) genes to distinguish between different black aspergilli

species (Varga et al. 1993; Varga et al. 1999; Parenicova

et al. 2001; de Vries et al. 2005). Likewise, Perrone et al.

(2006) have analyzed the polymorphism of ampliﬁed

fragments so as to distinguish all the black aspergilli spe-

cies at molecular level. However, to estimate the level of

aﬂatoxins in the Aspergillus ﬂavus, Baird et al. (2006) used

DNA ampliﬁcation ﬁngerprinting (DAF) method to deter-

mine regions of ITS1 and ITS2. Aspergillus carbonarius

was identiﬁed using an RAPD-based method (Fungaro

et al. 2004). A random gene fragment was selected whose

speciﬁc oligonucleotides primers were synthesized and

used for ampliﬁcations. An 809-bp fragment was detected

only from the A. carbonarius DNA. Once this marker was

sequenced, it was transformed into a unique and robust

PCR-based marker. With the experiment, it was found that

there exists no relationship between RAPD genotyping and

mycotoxigenic properties of strains. When compared to

conventional method, results were obtained within 24 h of

time. Comparing the three DNA ﬁngerprinting methods

416 Arch Microbiol (2010) 192:409–425

123

(RFLP, MLP, and RAPD), Bart-Delabesse et al. (2001)

found that out of total 67 isolates of A. fumigatus tested, 49

genotypes with 37 unique types were differentiated by

RFLP, while MLP analysis yielded 28 unique patterns in

43 distinct genotypes whereas RAPD showed only 31 types

with 17 unique types. Therefore, the polymorphisms were

in order of RFLP [MLP [RAPD. The MLP and RFLP

methods are based on speciﬁc DNA hybridization between

either a probe or primer and target DNA. Thereby, they

have higher reproducibility than that of the RAPD. RFLP

of mitochondrial DNA of Aspergillus section Flavi has

been used for their taxonomic differentiation. Mitochon-

drial DNA restricted with HaeIII, AreI, or DraI gave

unique band with each aspergillus species; however, with

some restriction enzyme(s), similarity patterns may arise in

bands (Quirk and Kupinski 2002).

In another study, ampliﬁed fragment length polymor-

phism (AFLP) and genomic DNA sequencing were used to

identify 77 black aspergilli strains from grapes. It was

observed that they shared \25% of AFLP similarity and

belonged to four main distinct divisions were identiﬁed.

Perrone et al. (2006) suggested that AFLP could be utilized

as a tool for studying genetic diversity of Aspergillus

species. Thus, with the help of the AFLP method, genetic

relatedness and/or resolving relationships within or in

between two closely related groups or species could be

evaluated.

McAlpin and Mannarelli (1995) designed a DNA

probe for differentiating A. ﬂavus strains. The repetitive

DNA sequences have proven to be a useful and reliable

character in evaluating genetic relatedness of strains at

different levels of taxonomic classiﬁcation, and the probe

was created by DNA ﬁngerprinting technique. The 6.5-kb

pAF28 DNA probe hybridized with DNA of closely

related varieties including A. ﬂavus var. oryzae,A. ﬂavus

var. parasiticus,A. ﬂavus var. sojae,andA. nomius.

However, no hybridization was observed when the probe

was hybridized with the other fungal species DNA such

as A. ochraceus NRRL402, A. auricomus NRRL391,

A. alliaceus NRRL315, F. moniliforme NRRLA-28160,

and P. thomii NRRL6218.

DNA microarray-based methods

Microarray has rapidly become one of the standard labo-

ratory tools for identiﬁcation as well as quantiﬁcation of

many speciﬁc DNA sequences in complex nucleic acid

samples (Chen et al. 1997; Jayapal and Melendez 2006).

Microarray-based proﬁling is a powerful approach for the

diagnosis of disease targets. The array detection for gene

expression can serve both as markers in addition of

detection of expression level (Jayapal and Melendez 2006).

For the ﬁrst time, Leinberger et al. (2005) applied DNA

microarray technology for the recognition and identiﬁca-

tion of fungal mycoses. Twelve Aspergillus and Candida

species, which most frequently cause this disease, were

targeted. For detection, 16–24 bases of oligonucleotide

probes were designed by exploiting the conserved nature of

ITS1 and 2, thereby, making these probes as genus speciﬁc.

Although the cross-hybridization had been observed in

Candida species which can be resolved by designing

additional species-speciﬁc probe, yet this method have

been found to be better than any other technique. The

whole procedure takes only 4 h for determination of 12

species, after DNA extraction. In a similar way, Hsiao et al.

(2005) also identiﬁed 228 pathogenic fungi. ITS regions of

18S rRNA genes were PCR-ampliﬁed, and products were

used for designing the probes. The ﬂuorescently labeled

probes were then immobilized on the nylon membrane of a

microtiter plate. With this DNA chip technology, 64 clin-

ically important fungal species of 32 genera were deter-

mined. From isolating the colonies to the testing of array,

the whole procedure was ﬁnished in 24 h, with a detection

limit of fungal genomic DNA as low as 10 pg.

To identify fungal pathogens in neutropenic patients, an

assay of multiplex PCR in combination with DNA micro-

array hybridization was used by Spiess et al. (2007).

Conserved regions of 18S, 5.8S, and ITS1 of rRNA genes

were used for PCR primer designing and ‘capture’ probes.

Hybridizing the fungal genomic DNA with capture probes

resulted in the species-speciﬁc identiﬁcation of A. fumig-

atus,A. ﬂavus,A. terreus, and other pathogenic fungal

species. Schmidt-Heydt and Geisen (2007) used the same

microarray technology in order to monitor the mycotoxin

production in food. The basis of microarray was to detect

the activation of all gene clusters under favorable condi-

tions and to add newly identiﬁed pathway genes, at any

time. The resulting signals were speciﬁc under hybridiza-

tion conditions. Schultz et al. (2008) showed a novel

ﬂuorescent biosensor which detects 16 molecules lm

which ﬁnally can be reduced to 1 mol lm

if the system is

conﬁned to photon noise. However, Laitala et al. (2007)

used decay time, provided by decay probe (europium as

donor and organic ﬂuorophore as acceptor), to discriminate

hybridized probe population from unhybridized population.

In a similar way, for fungal identiﬁcation, Wang et al.

(2008) also obtained ﬂuorescent signals from as low as

0.5 nM of oligonucleotide signals and detected 3 ng of

ampliﬁed product.

For detection of black aspergilli (A. carbonarius, A.

ibericus,and A. japonicus) on grapes, Bufﬂier et al. (2007)

and Susca et al. (2007a) manufactured a new type of low-

complexity oligonucleotide microarray: OLISA

OLIgo

Sorbent Arrays. This biochip consists of a series of 16

probes at each well of a microtiter plate. The specimen is

Arch Microbiol (2010) 192:409–425 417

123

PCR-ampliﬁed with biotin-labeled PCR primers, dena-

tured, and then speciﬁcally hybridized to any single

nucleotide polymorphisms of an ampliﬁed DNA target.

The spot pattern allows the automated identiﬁcation

of the species present in specimen. Identiﬁcation of

multiple species of Aspergillus simultaneously can be

achieved by combining multiple ampliﬁcation and

detection method.

Chip assays have proved their superiority by requiring

small amount of ampliﬁed products and generating huge

amount of data, and its analysis is also rapid and can be

automated. Array systems have been designed for the use

in biomedical analysis, environment monitoring, or bio-

terror agent’s detection. The basic purposes of these

microarray biosensors are portability, automatic recogni-

tion, robustness, and cost-effectiveness. The cost of these

microarray chips can be reduced to minimum level if glass

slides are used for its manufacturing.

Methods based on retrotransposon insertion-site

context (RSIC) typing

de Ruiter et al. (2007) used a novel type of PCR alternative

which is less time-consuming and easier than the RFLP or

Southern blot analysis. It has been shown earlier that ret-

rotransposons offer very stable ﬁngerprints. Therefore, they

can be employed for typing-like assays. It is a hemi-nested

ligation-dependent sort of PCR assay. RSIC utilizes the

occurrence of a multicopy element dispersed in the whole

genome and indents to amplify the ﬂanking sequences of

retrotransposon elements. Retrotransposons were charac-

terized by the presence of two LTR’s encircling the three

coding regions. After the genomic DNA isolation, a com-

bined restriction-ligation procedure was used to join the

adapters. The restriction-ligation was PCR-ampliﬁed, and

then the amplicons were analyzed. The data obtained were

further studied with bioinformatic softwares. On analyzing

the 55 A. fumigatus samples from 15 patients, 20 bands

were obtained with a range of 60–300 bp. This means that

they belonged to same genotype. Comparatively, Asper-

gillus, isolated from respiratory samples, had shown dif-

ferent genotyping within the individual patients. The major

difference between the AFLP and RSIC and STRAf (Short

tandem repeat with reference to Aspergillus fumigatus is

known as STRAf) is that the later ones are much more

sensitive than the AFLP. RSIC and STRAf can determine

two different genotypes from two patients while AFLP

cannot distinguish the difference between them (de Ruiter

et al. 2007; Klaassen and Osherov 2007). Retrotransposons

are also present in other aspergilli species such as Asper-

gillus ﬂavus and Aspergillus nidulans.

Other potential molecular methods

With solid phase cytometry (SPC) in combination with

immunoﬂuorescence labeling, distinction between Asper-

gillus species and other pathogenic fungi can be made

within an hour (de Vos and Nelis 2003). The same tech-

nique was used by de Vos et al. (2006) with addition of

laser scanning for the identiﬁcation of Aspergillus fumig-

atus hyphae in respiratory secretions. Depending upon the

enzymatic ‘viability’ staining, with carboxyﬂuorescein

diacetate, speciﬁc and non-speciﬁc detections of fungal

hyphae were performed. Speciﬁc detection needs pre-

incubation at 45°C with viability staining whereas for non-

speciﬁc identiﬁcation, only staining procedure is required.

As low as 2–10 A. fumigatus hyphae per sample have been

diagnosed in spiked sputum using non-speciﬁc and speciﬁc

staining in 2.5 and 8.5 h, respectively. Yoo et al. (2008)

used quantitative real-time (RTi) nucleic acid sequence-

based ampliﬁcation (NASBA) for identifying Aspergillus

fumigatus. They constructed an internal control (IC) RNA

from Calvibacter michiganensis subsp. sepedonicus

strains. Internal control RNA contained a 6-carboxy-X-

rhodamine (6-ROX) ﬂuorescent dye whereas A. fumigatus

beacon was labeled with 6-carboxyﬂuorescien (6-FAM)

at its 50-end. Internal control RNA was detectable in the

ranges of 10

–10

copies ml

-1

, which helped in pre-

dicting the unknown concentrations of target RNA using

titration curve. This method showed the sensitivity of

96% of identiﬁcation of A. fumigatus. NASBA-LF was

10 times more sensitive than competitive-PCR and 1000

times more than RT–PCR. Its result had led the authors

to consider the test as ‘gold standard’ with 100% spec-

iﬁcity and sensitivity (Olmos et al. 2007). To detect

intraspeciﬁc variations of aspergillus group, Kumeda and

Asao (2001) developed a novel heteroduplex panel

analysis (HPA) using PCR-ampliﬁed regions of ITS. This

method involves a panel heteroduplex formation which

was compared with a set of standard species-speciﬁc

panel. This typing discloses its discriminatory power

by detecting even a single base pair difference within

species.

Because of the high discriminatory power and high

throughput, short tandem repeats (STRs) has been

increasingly used as genetic markers. Their overall pres-

ence in most higher organisms and polymorphisms have

made them important for strain identiﬁcation and dis-

crimination in a wide variety of microorganisms. Ciardo

et al. (2007) proposed to develop an algorithm for the

identiﬁcation of various molds on the basis of molecular

and phenotypic methods. They have created an IMM

database covering almost all the medically signiﬁcant

molds based on ITS regions.

418 Arch Microbiol (2010) 192:409–425

123

Future aspects

In recent years, numerous additional novel ways have

emerged to improve the protocols for diagnosis of the

pathogens and to overcome the drawbacks. Sophisticated

molecular techniques are being developed that may be used

to screen the particular fungus within a range of popula-

tion. But before performing the molecular detections, it is

imperative to extract the nucleic acid from the samples. For

this, the samples may be divided into two categories: (1)

medical sample material such as blood or bronchoalveolar

lavage (BAL) samples (2) contaminated agricultural food

and feed samples as peanut, wheat, cereals. For medical

samples, no universal method has yet been obtained for the

source of sample collection and nucleic acid extraction

protocol. However, Loefﬂer et al. (2000) suggested the

whole blood in EDTA (Klingspor and Loefﬂer 2009)-

containing tubes could be a better sample for fungal DNA

isolation purpose. The advantage of this sample is that both

free- and the cell-associated DNA can be easily assessed,

EDTA in the tubes inhibit DNases activity present freely in

the blood without interfering the PCR assay (Garcia et al.

2002). DNA isolation is, currently, seen as the target of

choice due to its relative stability and ease of extraction

against RNA; still the extraction methods provide varia-

tions in DNA concentration. In the similar way, for con-

taminated plant materials, no universal method is present

for DNA isolation. However, the type of sample collection

is not a problem here as one can take only the contaminated

part of the plant. The major problem is the PCR inhibitors

present in the plant material itself. Besides this, the quan-

tity of fungal concentration on the contaminated material is

also a problem. To eradicate this problem or to increase the

quantity, enrichment procedures are used. But that needs

3–5 days of time causing further delay in detection.

Therefore, a single and consensus method should be gained

for extraction of DNA for both medical as well agricultural

materials which does not require enrichment procedure

(Table 3).

In a recent research, Balajee et al. (2007) and Samson

et al. (2007b) recommended the use of ITS regions as a

convenient universal marker for fungal species identiﬁca-

tion. Microsatellites or STRs under all altered conditions

produced a detectable signal. Thus, the STR assay had

proved to be an extremely robust method (de Valk et al.

2007; Klaassen and Osherov 2007) to determine the pres-

ence of Aspergillus species.

Identiﬁcation and typing of Aspergillus from cultures or

environmental samples have resulted in development of

more and more PCR-related technologies or other molec-

ular approaches. However, a molecular ‘gold standard’

assay is yet to be produced (Buchheidt and Hummel 2005).

The assurance of PCR and related methods to identify

Aspergillus has subsided the potential disadvantages. As

suggested by Niessen (2007), the new assays should be ﬁrst

checked through in silico method for the cross-reactivity

and the feasibility of the reaction to obtain good results.

Contamination in the PCR mixtures, interference of related

fungi with the target fungus (Klingspor and Loefﬂer 2009),

shared genes for different mycotoxins (sterigmatocystin

and aﬂatoxin), disrupted gene presence without the pro-

duction of toxin are few problems that may provide false

results. In addition, dead cells or pieces of DNA may also

provide results. Thus, methods are to be devised that

may provide on-site detection and are cost effective and

easier to use. DNA microarray and probe technology can

be quite useful to make the aﬂatoxigenic fungus estimation

ready-to-use, cost effective, and less time-consuming

procedure.

Few recently developed methods can be taken into

consideration for this purpose and can be modiﬁed

accordingly (Table 3). Varallyay et al. (2007) utilized LNA

(Locked nucleic acid) probes for estimating microRNAs

(miRNAs). This method allows sensitive and highly spe-

ciﬁc detection of mature miRNAs. Circularized oligonu-

cleotide probes (C-probe) have proved to be a considerable

advancement in the area of nucleic acid detection (Zhang

et al. 2006). Several authors’ have reported the real-time

technology of RCA and RAM (Faruqi et al. 2004; Nilsson

et al. 2002; Tyagi and Kramer 1996).

Inversion probes along with pyrosequencing for

detecting Mycobacterium tuberculosis can also be used

for fungal identiﬁcation, as it is a very sensitive tech-

nique. The detection limit for M. tuberculosis DNA was

found to be 500 fg (Novais et al. 2008). Besides direct

detection of ssDNA or dsDNA, the technique is also more

accurate and much faster than culture-based method.

Sequence-enabled reassembly with green ﬂuorescent

protein (GFP), b-lactamase (LAC), or mCpG can also be

utilized directly to quantify the dsDNA (Ghosh et al.

2006).

In an attempt to estimate the fungal pathogenic nucleic

acids, Wang et al. (2008) devised a microﬂuidic microarray

kit. Compared to the PCR, which detects 3 ng of DNA, this

device provides ﬂuorescent signals from as small as

0.5 nM (1 lM) of DNA. Dore et al. (2006) reported an

ultra sensitive and sequence-speciﬁc DNA detection sys-

tem. They had shown that a suitable ﬂuorophore with

exc

=530 nm and k

=575 nm increases the sensitivity

dramatically, allowing the detection limit in zeptomolar

(10

-21

) concentrations in just only 5 min without any prior

target ampliﬁcation.

These newer techniques can be used for the Aspergillus

detection, which will further enhance the sensitivity of

identiﬁcation. Though recent techniques are quite good and

efﬁcient yet they need to be modiﬁed for the sake of

Arch Microbiol (2010) 192:409–425 419

123

identiﬁcation of fungal genomes and need to be further

improved before they are used for diagnosis purposes.

Further, a thorough study of all human pathogenic

Aspergillus species is needed to improve the understanding

about the virulence and variations present in the natural

isolates.

Table 3 Future applications

Technique name Working Detection limit References

MIP ?Pyrosequencing Pyrosequencing is a real-time DNA sequencing.

Instead of simple primers, MIPs (circularized

ss-oligonucleotides) are used. With

ampliﬁcation, PPi is released turning AMP in

ATP by ATP sulfurylase enzyme. The ATP

provides energy to luciferase for oxidizing

luciferin to generate light. This light is

quantiﬁed by pyrosequencer

500 fg Novais et al. (2008)

Signal detection of ss/ds DNA It is a type of DNA sensor based on electrostatic

interactions between positively charged optical

transducer and ﬂuorescently labeled negative

DNA probe. Transducer gets quenched after

joining with ssDNA probe which then

illuminates when a complementary

oligonucleotide sequence is added to it

3 zM Dore et al. (2006)

Ampliﬁcation by C- probe Ampliﬁcation starts by DNA polymerase extends

single forward primer bound to C-probe

through RCA. RAM utilizes two primers.

Forward primer is same as RCA to generate

ssDNA which the reverse primer, having

identical sequence to C-probe, uses for second-

round extension.

– Zhang et al. (2006)

Detection through LNA probe In Northern blot assay, LNA probes are used to

increase the sensitivity by enhancing the

hybridization capacity. Modiﬁed LNA probe

detection is sensitive to miRNA by about 10-

folds than regular DNA

– Varallyay et al. (2007)

Detection by DNA probes The ampliﬁed PCR product is used for

generating the DNA probes that are to be used

with ISH. Bearham et al. (2008) tested the

DNA probes for detection of Minchinia sp.

from rock oysters

10 fg of template DNA in

250 ng of host DNA

Bearham et al. (2008)

RIDA It is a type of probe ampliﬁcation where speciﬁc

synthetic ssDNA (RP) joins the template. The

restriction nicking endonuclease cuts the

recognized site resulting in the reduction in Tm

and subsequently releasing two smaller ssDNA

probes. The same RP is used to cleave again

and again generating large amount of ssDNA

copies. This method can be used for both DNA

and RNA. It can speciﬁcally detect samples

within 5–15 min

– Gao et al. (2008)

NASBA-LF It is an isothermal ampliﬁcation method for RNA

basically. First primer attaches to the

complementary end of 3’ template to

synthesize cDNA by reverse transcriptase.

Second primer attaches to 5’ end of DNA and

T7 RNA polymerase again synthezises cRNA.

With lateral ﬂow-through, enhanced binding

rate reduces hybridization time

2 copies of DNA Olmos et al. (2007)

(MIP molecular inversion probe, zM zeptoMolar (10

-21

M), C-probe circularized probe, RCA rolling circle ampliﬁcation, RAM ramiﬁcation

mechanism, LNA probe locked nucleic acid probe, miRNA microRNA, ISH in situ hybridization, RIDA rapid isothermal detection assay, RP

reporting probe, NASBA-LF nucleic acid sequence-based ampliﬁcation-lateral ﬂow-through)

420 Arch Microbiol (2010) 192:409–425

123

Acknowledgments SJ is thankful to Department of Science and

Technology, Government of India for ﬁnancial assistance.

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Natural inhibitors: A sustainable way to combat aflatoxins

Article

Full-text available

Dec 2022

Among a few hundred mycotoxins, aflatoxins had always posed a major threat to the world. Apart from A. flavus, A. parasiticus, and A. nomius of Aspergillus genus, which are most toxin-producing strains, several fungal bodies including Fusarium, Penicillium, and Alternaria that can biosynthesis aflatoxins. Basically, there are four different types of aflatoxins (Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin G1 (AFG1), Aflatoxin G2 (AFG2)) are produced as secondary metabolites. There are certainly other types of aflatoxins found but they are the by-products of these toxins. The fungal agents generally infect the food crops during harvesting, storing, and/or transporting; making a heavy post-harvest as well as economic loss in both developed and developing countries. And while ingesting the crop products, these toxins get into the dietary system causing aflatoxicosis, liver cirrhosis, etc. Therefore, it is imperative to search for certain ways to control the spread of infections and/or production of these toxins which may also not harm the crop harvest. In this review, we are going to discuss some sustainable methods that can effectively control the spread of infection and inhibit the biosynthesis of aflatoxins.

In vitro Evaluation of Rhizobacterial Strains from Groundnut on Growth of Aflatoxigenic Aspergillus Flavus

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Jun 2023

Comparative Analysis of Microbial and Mycotoxin Contamination in Korean Traditional Soybean Paste and Soy Sauce Production with and without Starter

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Jun 2023

Doenjang and ganjang are traditional, Korean fermented foods. They are manufactured by fermenting jangs, either through the traditional natural fermentation, using straw, or the commercial inoculating starter cultures. However, both Bacillus cereus and aflatoxins have been detected in jangs, compromising their safety. Aflatoxins have been detected on numerous occasions. However, studies are yet to be conducted on whether these safety issues differ depending on the manufacturing method. In this study, we evaluated whether the manufacturing methods of doenjang and ganjang alter their safety. Samples of traditional and commercial doenjang and ganjang were analyzed for aflatoxin and B. cereus contamination. Microbiome taxonomic profiling was performed to assess microbial composition. The experimental methodology involved sample collection from various stages of production, including the use of starter cultures and natural fermentation processes. Aflatoxin levels were determined using regulatory limits, and B. cereus content was evaluated against specific thresholds. Aflatoxins were detected in both traditional and commercial doenjang and ganjang, with either the total aflatoxin (15 μg/kg) or aflatoxin B1 (10 μg/kg) exceeding the regulatory limits. However, ochratoxin A was not detected in any of the samples. B. cereus was detected in some samples, within the regulatory limit (4 log CFU/g), and was not influenced by the manufacturing method. Analysis at the production stage showed that aflatoxin increased alongside the fermentation time in traditional doenjang. However, in ganjang, no significant differences were associated with the fermentation period. When using starter cultures, the fermentation period did not affect the toxin level in both foods. Both methods showed lower aflatoxin content in the initial doenjang and ganjang samples than in meju. B. cereus was not detected in either method, as its content decreased over the fermentation period. Microbiome taxonomic profiling confirmed that even when using starter cultures, B. cereus was not a dominant species and was considerably affected by the environment. Staphylococcus aureus and Pseudomonas, pathogenic in nature, were detected in products manufactured using the traditional method; hence, the potential risk of this method was higher than that of the commercial method. The experimental methodology employed in this study contributes to understanding the microbial composition and toxin contamination levels in doenjang and ganjang, contributing to the overall knowledge of their safety and quality control.

Isolation and Molecular Characterization of Collar Rot Causing Pathogen Aspergillus niger on Groundnut in Rajasthan, India

Article

Jun 2023

Groundnut, a major pulse crop also grown as an oil seed over 90 countries worldwide, is important to the Indian economy. The states of Andhra Pradesh, Gujarat, Rajasthan and Tamil Nadu are the main producers of groundnut. In 2021, disease incidence with symptoms similar to collar rot was reported from Jaipur district of Rajasthan. The fungal strain was isolated from the collar region of susceptible groundnut variety TAG-24. White colonies with mycelia were observed in 3-4 days which eventually turned dark brown in color. Pathogenicity tests were performed on healthy groundnut and collar rot symptoms developed within 7-8 days of inoculation. Sequencing of PCR products amplified with the18S rRNA (NS1F-NS8R) and ITS (NS1F-ITS4R) primers indicated 99.4% and 100% identity respectively with a reference strain of Aspergillus niger CBS 554.65 (ATCC 16888) in the NCBI-Genebank database. Based on the morphological and molecular characteristics, the fungal pathogen was identified as Aspergillus niger and submitted to Gene Bank with the accession nos. ON954789 and OQ653131. The the culture is submitted to NCMR-NCCS (accession number MCC 9892).

Aflatoxins

Chapter

Full-text available

Feb 2023

The aflatoxin producing fungi Aspergillus flavus, A. parasiticus, and A. nomius, although they are also produced by other species of Aspergillus as well as by Emericella spp.(Telemorph). There are many types of aflatoxins, but the four main ones are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2, while aflatoxin M1 (AFM1) and M2 (AFM2) are the hydroxyl-ated metabolites of AFB1 and AFB2. Aflatoxin B1, which is a genotoxic hepatocarcinogen, which presumptively causes cancer by inducing DNA, adducts leading to genetic changes in target liver cells. Cytochrome-P450 enzymes to the reactive intermediate AFB1–8, 9 epoxide (AFBO) which binds to liver cell DNA, resulting in DNA adducts, metabolize AFB1 Ingestion of contaminated food is the main source of exposure to aflatoxins, which adversely affect the health of both humans and animals. The compounds can cause acute or chronic toxic effects of a teratogenic, mutagenic, carcinogenic, immunotoxic or hepatotoxic character. You can reduce your aflatoxin exposure by buying only major commercial brands of food and by discarding that look moldy, discolored, or shriveled.

Rice Weevil (Sitophilus oryzae L.) Gut Bacteria Inhibit Growth of Aspergillus flavus and Degrade Aflatoxin B1

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May 2024
J. Fungi

In this study, bacteria residing in the gut of the rice weevils (Sitophilus oryzae L.) (Coleoptera: Curculionidae) feeding on aflatoxin-contaminated corn kernels were isolated and evaluated for their ability to suppress Aspergillus flavus and to remove/degrade aflatoxin B1 (AFB1). Four morphologically distinct S. oryzae gut-associated bacterial isolates were isolated and identified as Bacillus subtilis (RWGB1), Bacillus oceanisediminis (RWGB2), Bacillus firmus (RWGB3), and Pseudomonas aeruginosa (RWGB4) based on 16S rRNA gene sequence analysis. These bacterial isolates inhibited A. flavus growth in the dual culture assay and induced morphological deformities in the fungal hyphae, as confirmed by scanning electron microscopy. All four bacterial isolates were capable of removing AFB1 from the nutrient broth medium. In addition, culture supernatants of these bacterial isolates degraded AFB1, and the degradation of toxin molecules was confirmed by liquid chromatography-mass spectrometry. The bacterial isolates, B. subtilis RWGB1, B. oceanisediminis RWGB2, and P. aeruginosa RWGB4, were capable of producing antifungal volatile organic compounds that inhibited A. flavus growth. These results suggest that the bacterial isolates from S. oryzae gut have the potential to bind and/or degrade AFB1. Further research on their application in the food and feed industries could enhance the safety of food and feed production.

Reflectance Multispectral Imaging for Fungal Growth Level Estimation

Conference Paper

Jan 2024

The Menace of Aflatoxin: Understanding the Effects of Contamination by Aspergillus Species on Crops and Human Health and Advancements in Managing These Toxic Metabolites

Chapter

Full-text available

Apr 2023

The Menace of Aflatoxin: Understanding the Effects of Contamination by Aspergillus Species on Crops and Human Health and Advancements in Managing These Toxic Metabolites

Chapter

Full-text available

Apr 2023

Food security and safety are essential global issues that require collaboration from governments, private industry, and individuals to ensure there is enough safe and nutritious food to meet the needs of a growing population. The three main elements impacting food security and safety are the availability of food, access to safe food, and the utilization of food for a healthy life. Aflatoxins, harmful mycotoxins produced by certain fungi, damage a significant proportion of the world’s food supply, which is a factor in food insecurity. Effective strategies to prevent and manage aflatoxin contamination in crops include promoting sustainable and diversified agricultural practices, improving crop management, post-harvest handling and storage, and strict regulation and monitoring of food quality and safety. To date, there have been 20 different types of aflatoxins identified, with B1, B2, G1, and G2 being the most prevalent and dangerous. To mitigate the impact of aflatoxins, it is important to understand the mechanisms of contamination, the impact of aflatoxins, and the management strategies that can be employed to reduce contamination. An updated review on aflatoxin contamination, its impact and management strategies can provide valuable information for researchers, policymakers, and food safety professionals.

Aflatoxin Contamination, Its Impact and Management Strategies: An Updated Review

Article

Full-text available

Apr 2022

Aflatoxin, a type of mycotoxin, is mostly produced by Aspergillus flavus and Aspergillus parasiticus. It is responsible for the loss of billions of dollars to the world economy, by contaminating different crops such as cotton, groundnut, maize, and chilies, and causing immense effects on the health of humans and animals. More than eighteen different types of aflatoxins have been reported to date, and among them, aflatoxins B1, B2, G1, and G2 are the most prevalent and lethal. Early detection of fungal infection plays a key role in the control of aflatoxin contamination. Therefore, different methods, including culture, chromatographic techniques, and molecular assays, are used to determine aflatoxin contamination in crops and food products. Many countries have set a maximum limit of aflatoxin contamination (2–20 ppb) in their food and agriculture commodities for human or animal consumption, and the use of different methods to combat this menace is essential. Fungal infection mostly takes place during the pre- and post-harvest stage of crops, and most of the methods to control aflatoxin are employed for the latter phase. Studies have shown that if correct measures are adopted during the crop development phase, aflatoxin contamination can be reduced by a significant level. Currently, the use of bio-pesticides is the intervention employed in many countries, whereby atoxigenic strains competitively reduce the burden of toxigenic strains in the field, thereby helping to mitigate this problem. This updated review on aflatoxins sheds light on the sources of contamination, and the on occurrence, impact, detection techniques, and management strategies, with a special emphasis on bio-pesticides to control aflatoxins

GOOD FOOD PROJECT: DETECTION OF ASPERGILLUS CARBONARIUS AND OTHER BLACK ASPERGILLI ON GRAPES BY DIRECT MULTI-DETECTION DNA MICROARRAY

Article

Full-text available

Interspecific mitochondrial DNA restriction fragment length polymorphisms in Aspergillus section Flavi

Article

Nov 2002

Mitochondrial DNA restriction fragment length polymorphisms (RFLPs) are described for 64 isolates, representing 11 species of Aspergillus section Flavi. Mitochondrial DNA haplotypes were identified following digestion of total cellular DNA with the restriction enzymes HaeIII, AseI, or DraI. In general, isolates of the same species possessed identical mitochondrial DNA haplotypes. Three haplotypes were found in multiple, closely related species: one in A. flavus, A. oryzae, and A. subolivaceus; a second in A. parasiticus and A. sojae; and a third in A. tamarii and A. flavofurcatis. Four distinct haplotypes were each associated with a single species: A. nomius, A. avenaceus, A. leporis, and A. zonatus. Mitochondrial DNA haplotypes complement traditional morphological and growth criteria in making taxonomic decisions within Aspergillus section Flavi.

Current Systematics of the Genus Aspergillus

Chapter

Jan 1994

Robert A Samson

In their monograph of the genus Aspergillus, Raper and Fennell (1965) accepted 132 species subdivided into 18 groups. The generic and species concepts were well circumscribed and this monograph is still valuable and used. Several sections of the genus which remained problematic for identification have been reinvestigated, including the subgenus Aspergillus section Aspergillus (= the Aspergillus glaucus group = Eurotium) (Blaser, 1975; Pitt, 1985; Kozakiewicz, 1989), section Nigri (Al-Mussallam, 1980; Kozakiewicz, 1989 Kusters-van Someren et al., 1990, 1991), section Circumdati (Christensen, 1982), section Nidulantes = Emericella (Horie, 1980; Christensen and States, 1982), section Versicolores (Klich, 1993), section Flavi (Murakami, 1971; Murakami et al., 1982; Christensen 1981; Klich and Pitt, 1985, 1988a), section Fumigati (Frisvad and Samson, 1990; Samson et al, 1990, Nielsen and Samson, 1992) and section Restricti (Pitt and Samson, 1990b). Samson (1979, 1992, 1993) provided a compilation of the species and varieties described since 1965 with a critical review of the validity of the published taxa. The genus now contains more than 180 species, with about 70 named teleomorphs.

Naturally occurring aflatoxins (Group 1), aflatoxins M1 (Group 2B)

Article

Jan 1993

I.A.R.C. Aflatoxins

Genotypic and phenotypic variability among black Aspergilli

Article

Jan 2000

Real-time monitoring of rolling-circle amplification using a modified molecular beacon design

Article

Jul 2002
NUCLEIC ACIDS RES

Mats Nilsson

We describe a method to monitor rolling‐circle replication of circular oligonucleotides in dual‐color and in real‐time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling‐circle amplification (RCA) reaction. Modified molecular beacons were made of 2′‐O‐Me‐RNA to prevent 3′ exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter‐molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.

Ratio-based decisions and the quantitative analysis of cDNA microarray images

Article

Jan 1997

Yidong Chen

Mycotoxins and other extrolites produced by species in Penicillium subgenus Penicillium

Article

Jan 2004
STUD MYCOL

Cited By (since 1996):137, Export Date: 18 October 2014

Rapid Detection of Mycotoxigenic Molds and Mycotoxins in Fruit Juice

Article

Jan 2006

Many commodities and products can become contaminated with mycotoxigenic molds and mycotoxins during harvest, storage and handling, and processing. Health hazard posed by mycotoxins to animals and humans is well recognized and over 100 countries have established regulations for major mycotoxins in food. Rapid detection and quantification of molds and mycotoxins in food commodities and processed food products is necessary for assuring safety and quality of food and implementation of HACCP. This paper provides a brief review of novel approaches and technologies for mycotoxin analysis including enzyme linked immunosorbent assays (ELISA), flow through membrane based immunoasays, chromatographic techniques, nucleic acid amplification assays, biosensors, and microarrays for detection of molds and mycotoxins. Some methods have already been commercialized while others innovative tests have been reported in the literature and have the potential for providing real-time test for mold and mycotoxin testing. The review focuses on methods for molds and mycotoxins in fruit juices.

DNA probes: Applications in the food industry

Article

Dec 1991
TRENDS FOOD SCI TECH

J. Leighton Jones

DNA probes have revolutionized diagnostic technology in the clinical and forensic fields. Their power in detecting and characterizing specific organisms and material derived from the organisms is now being applied to problems in the food industry. So far, uses include the detection and assessment of foodborne pathogens; the diagnosis of crop and livestock diseases; the improvement of crop and livestock breeding practices; and authenticity testing of meat products, plant material, and even documents and labels. Improvements in the user-friendliness of probe-based assays and wider appreciation of their applicability will further their adoption by the food science community.

Advances in molecular detection of Aspergillus: An update

Abstract and Figures

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