Rapid Detection of Shiga toxin-Producing E. Coli in Animal Origin Foods Using Loop-Mediated Isothermal Amplification (LAMP) Assay

Abstract

The aim of this study was comparative evaluation of loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assay for rapid and inexpensive detection of shiga toxin-producing E. coli in animal origin foods by targeting stx1 and stx2 genes. The LAMP assay was performed using a water bath. The standardized LAMP assay was evaluated on 122 E. coli field isolates obtained from various animal origin food samples to ensure its reliability and usefulness. The result showed that conventional PCR could detect 68 (55.73%) and 75 (61.47%) positive E. coli isolates for stx1 and stx2 genes. Whereas, LAMP showed higher sensitivity by detecting 79 (64.75%) and 87 (71.31%) positive isolates of E. coli for stx1 and stx2 genes, respectively. LAMP assay was found to be highly specific and 10 times more sensitive as it could detect 1.11 × 10² cfu/ml for both stx1 and stx2 genes of E. coli isolates, whereas conventional PCR could detect 1.85 x 10³ cfu/ml for both stx1 and stx2 genes of E. coli isolates. The rapidness, sensitivity, specificity, easiness and cost-effectiveness of LAMP assays will be very useful for the detection of foodborne pathogens for improving food sanitation and maintaining food safety.

Full text

1 23
Agricultural Research
ISSN 2249-720X
Volume 8
Number 4
Agric Res (2019) 8:490-496
DOI 10.1007/s40003-018-0391-x
Rapid Detection of Shiga toxin-Producing
E. Coli in Animal Origin Foods Using
Loop-Mediated Isothermal Amplification
(LAMP) Assay
P.Baraily, R.J.Zende, D.P.Kshirsagar,
V.M.Vaidya, R.N.Waghamare,
A.M.Paturkar, R.P.Todankar &
A.H.Shirke

1 23
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FULL-LENGTH RESEARCH ARTICLE
Rapid Detection of Shiga toxin-Producing E. Coli in Animal
Origin Foods Using Loop-Mediated Isothermal Amplification
(LAMP) Assay
P. Baraily
1,2
•R. J. Zende
1,2
•D. P. Kshirsagar
1,2
•V. M. Vaidya
1,2
•
R. N. Waghamare
1,2
•A. M. Paturkar
1,2
•R. P. Todankar
1,2
•A. H. Shirke
1,2
Received: 23 February 2018 / Accepted: 11 September 2018 / Published online: 15 October 2018
ÓNAAS (National Academy of Agricultural Sciences) 2018
Abstract The aim of this study was comparative evaluation of loop-mediated isothermal amplification (LAMP) and
polymerase chain reaction (PCR) assay for rapid and inexpensive detection of shiga toxin-producing E. coli in animal
origin foods by targeting stx1 and stx2 genes. The LAMP assay was performed using a water bath. The standardized LAMP
assay was evaluated on 122 E. coli field isolates obtained from various animal origin food samples to ensure its reliability
and usefulness. The result showed that conventional PCR could detect 68 (55.73%) and 75 (61.47%) positive E. coli
isolates for stx1 and stx2 genes. Whereas, LAMP showed higher sensitivity by detecting 79 (64.75%) and 87 (71.31%)
positive isolates of E. coli for stx1 and stx2 genes, respectively. LAMP assay was found to be highly specific and 10 times
more sensitive as it could detect 1.11 910
2
cfu/ml for both stx1 and stx2 genes of E. coli isolates, whereas conventional
PCR could detect 1.85 x 10
3
cfu/ml for both stx1 and stx2 genes of E. coli isolates. The rapidness, sensitivity, specificity,
easiness and cost-effectiveness of LAMP assays will be very useful for the detection of foodborne pathogens for improving
food sanitation and maintaining food safety.
Keywords Loop-mediated isothermal amplification (LAMP) Shiga toxin-producing E. coli Food safety 
Polymerase chain reaction (PCR) Sensitivity Specificity
Introduction
Escherichia coli is among the first bacterial species to
colonise in intestine during infancy [14]. On the basis of
their virulence and disease manifestation, there are five
distinct groups of E. coli, which include toxin-producing
strains like enterotoxigenic (ETEC), enterohaemorrhagic
(EHEC) or verocytotoxigenic E. coli (VTEC), enteroag-
gregative (EAggEC), non-toxic strains like enteropatho-
genic (EPEC) and enteroinvasive (EIEC) E. coli [3]. These
groups are associated with diarrhoea, haemorrhagic colitis
(HC), dysentery, bladder and kidney infections, surgical
wound infection, septicaemia, haemolytic uraemic syn-
drome (HUS), pneumonia and meningitis, and some of
these conditions result in death. Pathogenic types of E. coli
also occur in animal origin foods, and in particular, vero-
cytotoxigenic E. coli (VTEC) are zoonotic agents that
cause severe diseases and are responsible for many food-
borne outbreaks worldwide [17].
According to the World Health Organization (WHO)
report, approximately 11 million children under the age of
5 years died because of E. coli-mediated gastroenteritis
[23]. Shiga toxin-producing E. coli (STEC), also known as
Vero toxin-producing E. coli (VTEC), comprises a sero-
logically diverse group of pathogens that cause disease in
humans and animals characterized by the production of
cytotoxins that disrupt protein synthesis within host cells.
These toxins are synonymously either called verocytotox-
ins (VT), because of their activity on Vero cells, or Shiga
&D. P. Kshirsagar
drdpk04v@gmail.com
1
Department of Veterinary Public Health & Epidemiology,
Bombay Veterinary College, Parel, Mumbai 12, India
2
Maharashtra Animal and Fishery Science University, Nagpur,
India
123
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Author's personal copy

toxins (Stx) because of their similarity with the toxin pro-
duced by S.dysenteriae. The two main groups consist of
Stx1, which is nearly identical to the toxin of S. dysenteriae
type 1 and Stx2, which shares less than 60 percentage
amino acid sequence with Stx1 [1]. There are at least 100
serotypes of E. coli that produce Shiga toxins [9] The cattle
are considered the primary reservoir of both O157 and non-
O157 STEC bacteria [2].
Most of the infections are caused due to the ingestion of
contaminated foods, particularly undercooked ground beef.
Other foods of bovine origin including roast beef, raw
unpasteurized milk and other dairy products like yogurt,
curd, cheese and foods are derived from other species,
including pork, chevon, mutton, fish, shellfish meat of wild
or exotic mammals. In India, there is little information
available on the prevalence of Shiga toxin-producing E.coli
across the country. The STEC from non-diarrhoeic animal
sources in India was first isolated in 1999 [18].
In the past few decades, several molecular methods have
been developed to overcome the shortcomings of the
classical diagnostics methods, especially the in vitro
amplification of a pathogen-specific nucleic acid sequence.
Loop-mediated isothermal amplification technology
developed by Notomi et al. [10] is a novel DNA amplifi-
cation method which can amplify target gene under
isothermal conditions with high efficiency and sensitivity
[24]. Developing countries like India require low-cost
detection techniques for detection of these pathogens at
district, block as well as at field level. Loop-mediated
isothermal amplification (LAMP) has attracted a lot of
attention as a potentially rapid, accurate and cost-effective
novel nucleic acid amplification method.
Materials and Methods
Isolation of E. coli from Animal Origin Foods
A total of 298 animal origin food samples comprising 139
chicken, 52 buffalo meat, 32 mutton, 39 pork, 16 milk and
10 each of fish and eggs were collected from retail shops
located in and around Mumbai city over a period of
6 months during 2015–16. These samples were further
processed for isolation of E. coli spp. following standard
technique as per IS 5887(Part 1): 1976.
All of these positive isolates were further characterized
by biochemical tests and the results were interpreted and
validated as per bacteriological analytical manual for
E. coli (2007). Further, 122 positive E. coli isolates were
subjected for detection by standardized conventional
polymerase chain reaction (PCR) and loop-mediated
isothermal amplification (LAMP) methods.
Bacterial Strains and DNA Extraction
The reference strain E. coli (MTCC 443) was procured
from Institute of Microbial Technology (MTCC), Chandi-
garh, India. Additionally, 122 field isolates of E. coli iso-
lated were also included in the study.
Genomic DNA of E. coli was extracted as per the pro-
tocol [12] with slight modifications. A colony of E. coli
isolate on nutrient agar was picked and mixed with 1000 ll
of NSS in centrifuge tube. It was then centrifuged at
10,000 rpm for 10 min. After centrifugation, the pellet
formed was dissolved in 100 ll of nuclease-free water
(NFW), vortexed and further boiled at 100 °C for 10 min.
The centrifuge tube was subjected to rapid cooling in ice
which was followed by centrifugation at 10,000 rpm for
10 min. Then, the upper aqueous phase which contained
DNA was transferred to sterile micro-centrifuge tube.
These extracted DNAs were further used for amplification.
Until use, these were stored at freezing temperature
(-20 °Cto-80 °C) in sterile micro-centrifuge tube.
Primer Used for LAMP and PCR Reactions
Each LAMP primer set used in this study consisted of two
outer (F3, B3), two inner (FIP, BIP) and two loop primers
(Loop F, Loop B), which recognized eight different regions
of the gene target and were commercially synthesized by
Integrated DNA Technologies (IDT) obtained from Sigma
Aldrich, Bangalore, India. The LAMP primer sets for each
of the VTEC gene targets (stx1 and stx2) were selected
from previous study [4]. The primers used in PCR for the
specific detection of E. coli were previously described [18]
for stx1 gene and [5]stx2 genes. The sequences of the
primers are summarized in Table 1.
Optimization of LAMP Assay
The optimization of LAMP assay was carried out by con-
ducting the trials at different temperatures 58 °C, 60 °C,
62 °C, 63 °C, 65 °C and 66 °C and 58 °C, 60 °C, 62 °C,
63 °C, 65 °C, 65.2 °C and 66 °C for both stx1 and stx2
genes, respectively, and also at different time periods
50 min, 60 min and 70 min for both the genes. The LAMP
reaction mixture was optimized using different concentra-
tions of inner primers, outer primers, MgSO
4
and dNTPs.
However, 65 °C was chosen as the optimal temperature
since there was presence of significant visual turbidity due
to formation of large amount of by-product, pyrophosphate
ion, being produced, yielding an insoluble white precipitate
of magnesium pyrophosphate in reaction mixture and flu-
orescence on addition of SYBR green dye under ultraviolet
illumination. After completion of LAMP, amplified DNA
was analysed by electrophoresis on 1.5% agarose gel at
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98 V for 45 min. A 100 bp DNA ladder was run along with
LAMP products.
Optimization of PCR
The PCR assay for the detection of E. coli was standard-
ized as per the method of [5,19] with slight modifications.
PCR was performed using a Thermocycler PCR machine
(Eppendorf Mastercycler gradient, Germany). The cycle
times are standardized for both stx1 and stx2 genes of
E. coli positive isolate (Table 2). Following the last cycle,
there was 7-min incubation at 72 °C for the final elonga-
tion. The tubes were then held at 4 °C for both the genes.
Amplified PCR products were analysed by agarose gel
electrophoresis on 1.5% agarose gel.
Sensitivity of the LAMP Assay
Sensitivity (detection limit) of LAMP assay was evaluated
using 18-h-old E. coli culture on trypton soya agar, incu-
bated for overnight at 37 °C. Tenfold serial dilution was
carried out in PBS up to 10
-7
dilutions. 10
-4
–10
-7
dilu-
tions were used for both LAMP and PCR assays. To
determine the total viable count (TVC) of each dilution, the
culture was plated onto nutrient agar. After incubation at
37 °C for 18 h, the numbers of colonies were counted.
Specificity of LAMP Assay
The specificity of LAMP assay was tested using standard
E. coli DNA template and four other templates from non-
E. coli strain. The DNA templates were prepared as
described previously. The specificity of E. coli-specific
Table 1 Oligonucleotide sequences of LAMP primers used in this study
S. No. Target gene Primer Sequence (50-30)
1. Stx1 LAMP Primers
F3: Forward outer primer
B3: Backward outer primer
FIP: Forward inner primer
(F1c-F2)
BIP: Backward inner primer (B1c-B2)
PCR Primers
F3: GCT ATA CCA CGT TAC AGC GTG
B3: ACT ACT CAA CCT TCC CCA GTT C
FIP: GCT CTT GCC ACA GAC TGC ACA
TTC GTT GAC TAC TTC TTA TCT GG
BIP: CTG TGA CAG CTG AAG CTT TAC
GCG AAA TCC CCT CTG AAT TTG CC
F:: CAG TTA ATG TGG TGG CGA AGG
R: CAC CAG ACA ATG TAA CCG CTG
2. Stx2 LAMP Primers
F3: Forward outer primer
B3: Backward outer primer
FIP: Forward inner primer
(F1c-F2)
BIP: Backward inner primer (B1c-B2)
PCR Primers
F3: CAG TTA TAC CAC TCT GCA ACG TG
B3: CTG ATT CGC CGC CAG TTC
FIP: GCT CTT GAT GCA TCT CTG GTA
CAC TCA CTG GTT TCAT CAT ATC TG
BIP: CTG TCA CAG CAG AAG CCT TAC G
GAC GAA ATT CTC CCT GTA TCT GCC
F: CTT CGG TAT CCT ATT CCC GG
R: GGA TGC ATC TCT GGT CAT TG
Table 2 Details of steps and conditions of thermal cycling for different primer pairs in PCR assay
Steps Stx1 gene Stx2 gene
Temperature (°C) Time (min) Temperature (°C) Time (min)
Initial denaturation 94.0 4.0 94.0 5.0
Denaturation 94.0 1.0 94.0 1.0
Annealing 63.7 1.0 55.0 1.0
Extension 72.0 1.0 72.0 1.5
Final extension 72.0 7.0 72.0 7.0
Cycles 35 32
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LAMP was performed by testing it with four other bacterial
species viz. Pseudomonas aeruginosa,Salmonella spp.,
Proteus vulgaris and Klebsiella pneumoniae. The reaction
was performed at 65 °C for 60 min, and the results of this
assay were compared with conventional PCR assay.
Results and Discussion
Standardization of LAMP
LAMP was standardized for the detection of stx1 and stx2
genes of E. coli from foods of animal origin. The LAMP
conditions optimized for the amplification after standard-
ization were 65 °C for 60 min followed by 80 °C for 2 min
for termination of the reaction for both stx1 and stx2 genes.
The presence of significant visual turbidity and fluores-
cence on addition of SYBR green dye was observed at
65 °C (Fig. 1). LAMP products observed under UV tran-
silluminator of gel documentation system exhibited speci-
fic ladder-like pattern in case of DNA amplification
(Fig. 2a, b). The PCR was standardized for stx1 and stx2
gene (348 and 478 bp, respectively) using reference strain
(Fig. 3a, b).
Analysis of Animal Origin Food Samples
In the present study, 122 (40.93%) positive isolates of
E. coli were recovered from 298 animal origin food sam-
ples analysed (Table 3). Out of 122 E. coli analysed for
virulence gene characterization using conventional PCR
and LAMP. It was observed that conventional PCR could
detect 68 and 75 (55.73% and 61.47%) positive stx1 and
stx2 genes of E. coli isolates, whereas LAMP showed
higher sensitivity by detecting 79 (64.75%) and 87
(71.31%) positive isolates of E. coli for stx1 and stx2 genes,
respectively.
After successful standardization of LAMP, all the pos-
itive E. coli isolates (122) were subjected to LAMP tech-
nique. After subjecting all the 122 positive E. coli isolates
to LAMP, it was observed that all of the isolates were
found 79/122 (64.75%) and 87/122 (71.31%) positive for
stx1 and stx2 genes of E. coli, respectively, using LAMP
technique. The results of the present study are in agreement
with the previous findings who could detect all the 24
strains (100%) of stx-producing E. coli. However, six
strains of non-stx-producing E. coli were not detected by
LAMP technique [4]. Similarly, LAMP technique
Fig. 1 Visualization of LAMP products under UV light for fluores-
cence. A tube: DNA amplification indicated by fluorescence due to
SYBR green dye. B tube: No DNA amplification
Fig. 2 a Ladder-like pattern of LAMP products on 1.5% agarose gel
(stx1 gene). Lane 1–4: Ladder-like pattern of LAMP products of stx1
gene of E. coli, Lane 5: Negative control showing no ladder-like
pattern, Lane M: TrackIt
TM
100bp DNA ladder (Invitrogen, Cat. No.
10488-058). bLadder-like pattern of LAMP products on 1.5%
agarose gel (stx2 gene). Lane 1–6: Ladder-like pattern of LAMP
products of stx2 gene of E. coli, Lane 7: Negative control showing no
ladder-like pattern, Lane M: TrackIt
TM
100bp DNA ladder (Invitro-
gen, Cat. No. 10488-058)
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developed for iapH gene of Shigella and enteroinvasive
E. coli detected 38 out of 38 enteric pathogens [13]. This
may be attributed to difference in the target gene of E. coli
and primers used changing the sensitivity of detection.
The PCR technique could detect 68/122 (55.73%) and
75/122 (61.47%) of stx1 and stx2 genes of E. coli isolates,
respectively. LAMP technique could detect 79/122
(64.75%) and 87/122 (71.31%) isolates positive for stx1
and stx2 genes of E. coli. This may be attributed to the
presence of four specific primers targeting six distinct sites
on the stx1 and stx2 genes of E. coli.
However, 71% positive isolates by PCR for stx gene was
compared to 100% by LAMP method [8]. Moreover, in
case of Salmonella 90% and 72.72% detection of positive
Salmonella isolates by PCR, LAMP technique successfully
identified all the Salmonella spp. analysed (100%) [11,15].
Determination of Detection Limits (Sensitivity)
and Specificity of LAMP
Sensitivity of LAMP
The sensitivity (detection limit) of LAMP was evaluated by
using tenfold serial dilution method. The total viable count
(TVC) of undiluted culture was 1.11 x 10
9
by calculation
using plate-counting method.
Similar protocol of DNA dilution was adopted for
evaluating sensitivity (detection limit) of conventional
PCR assay. The sensitivity (detection limit) of the LAMP
assay was noted to be tenfold greater than that of con-
ventional PCR as LAMP could detect 1.11 x 10
2
cfu/ml
for both stx1 and stx2 genes of E. coli isolates, whereas
conventional PCR could able to detect 1.85 x 10
3
cfu/ml
for both stx1 and stx2 genes of E. coli isolates. The sen-
sitivity (detection limit) of the LAMP assay was noted to
be tenfold greater than that of conventional PCR as LAMP
could detect 1.11 x 10
2
cfu/ml for both stx1 and stx2 genes
of E. coli isolates, whereas conventional PCR could able to
detect 1.85 x 10
3
cfu/ml for both stx1 and stx2 genes of
E. coli isolates. The results are in accordance with a study
conducted using LAMP assay for detection of E. coli from
diarrhoeal stool, who reported that the LAMP assay could
detect 10
2
cfu/ml, whereas the PCR could detect 10
3
cfu/
ml of E. coli indicating that LAMP was 10 times more
sensitive than PCR [13].
Sensitivity of LAMP assay is ten times higher than the
PCR-based method, and these findings are also in agree-
ment with previous study reports [16], [20], [21] and [22].
They further stated that LAMP was more sensitive tech-
nique than PCR. However, some of the authors reported
that the LAMP test developed for E. coli was 100 times
more sensitive than conventional PCR [6]. This variation
Fig. 3 a Standardization of PCR for stx1 gene of E. coli. Lane 1–7:
348 bp PCR products of stx1 gene of E. coli isolates. Lane M:
TrackIt
TM
100bp DNA ladder (Invitrogen, Cat. No. 10488-058).
bStandardization of PCR for stx2 gene of E. coli. Lane 1 and 5: 478
bp PCR products of stx2 gene of E. coli, Lane N: Negative control for
E. coli, Lane M: TrackIt
TM
100bp DNA ladder (Invitrogen, Cat.
No.10488-058)
Table 3 Details of samples positive for E. coli
S. No. Type of food sample Number of samples examined Number of E. coli isolates recovered Prevalence (%)
1. Chicken 139 68.00 48.92
2. Buffalo meat 52 21.00 40.38
3. Mutton 32 8.00 25.00
4. Pork 39 18.00 46.15
5. Fish 10 7.00 70.00
6. Egg 10 Nil Nil
7. Milk 16 Nil Nil
Total 298 122 40.93
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may be attributed to the difference in target gene and
LAMP primers used in their study.
Specificity of LAMP
In the present study, the specificity of LAMP assay was
tested using standard E. coli DNA template and four other
templates from non-E. coli strains viz. P. aeruginosa,
Salmonella spp., P.vulgaris and K.pneumoniae. The
LAMP was carried out as per the standard protocol at
65 °C for 60 min in water bath. It was found that the
LAMP assay successfully amplified E. coli DNA only,
while it did not amplify any non-E. coli organisms. Simi-
larly, the PCR detected E. coli successfully and did not
give any positive result with non-E. coli strains. Thus, the
specificity of both LAMP and conventional PCR was found
to be 100%.
The present study indicated that LAMP could differen-
tiate and specifically detect the E. coli from other non-
E. coli strains. However, both LAMP and PCR assays were
successfully able to identify only E. coli without giving any
false-positive results for non-E. coli strains showing 100%
specificity for both the assays. The specificity results
(100%) observed in present study are also in accordance
with [7] who reported that LAMP technique could amplify
all the 35 enteric bacteria successfully but none of non-
E. coli standard strains used under study viz. P.aerugi-
nosa,Salmonella spp., P.vulgaris and K.pneumoniae.
amplified using LAMP technique.
Conclusions
Shiga toxin-producing E. coli (STEC) strains are zoonotic
foodborne pathogen of significant public health concern
due to its frequent involvement in outbreak of haemor-
rhagic colitis (HC) and ability to cause life-threatening
complications such as haemorrhagic uraemic syndrome
(HUS) and thrombotic thrombocytopenic purpura. The
LAMP method gives results similar to that of gold standard
microbiological culture method. To ease the odds faced by
PCR, LAMP stands out to be good and effective diagnostic
test for empowering in developing countries as it does not
require sophisticated equipment like thermocycler for
DNA amplifications and well-trained personnel. Thus, this
LAMP assay can help in improving food sanitation,
maintaining food safety as well as developing international
trade.
Acknowledgements This work was supported by grants from Indian
Council of Agricultural Research, New Delhi, under the Project ‘‘All
India Co-Ordinated Research Project on Post Harvest Engineering
and Technology’’ implemented at Bombay Veterinary College, Parel,
Mumbai.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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