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Science Archives (2020) Vol. 1 (3), 98-101
98
http://dx.doi.org/10.47587/SA.2020.1304
Molecular techniques of viral diagnosis
Mohammed Ahmed Mustafa1,2* , Marwan Q AL-Samarraie3 & Marwa T. Ahmed4
1,3Department of Pathological Analysis, College of Applied Sciences, University of Samarra
2Department of Medical Laboratory Techniques, College of Technology, University of Imam Jafar Al-Sadiq Dujail
4Department of Microbiology, College of Medicine, University of Tikrit, Iraq
*Corresponding author: mohammed.alsad3@gmail.com
Received: Nov 19, 2020 / Revised: Dec 2, 2020/ Accepted: Dec 11, 2020
Abstract
Viral infections are the cause of very serious problems in humans all over the globe. The recent outbreak of coronavirus disease
2019 caused by SARS-CoV-2 is a perfect example of how viral infection could pose a great threat to global public health and
economics. Therefore, to defeat the viral pathogens, we need to get a reliable diagnosis. Detection of the viral presence fast and
accurate in the patient is important for appropriate treatment, control, and prevention of epidemics. Diagnosis of infectious
microbes became easier by the development of molecular techniques, especially with the emerging of the polymerase chain
reaction (PCR). The easiness and high sensitivity of the technique made this method reliable enough to be used to detect any
known genetic sequence has led to its wide application in the life sciences. Currently, real-time PCR has made remarkable
contributions, with an additional fluorescent probe detection system that has increased the sensitivity of this assay over
conventional PCR, with the ability to verify the amount of amplification product and to quantitate the target concentration.
Further, nucleotide sequence analysis of the amplification products has encouraged epidemiological studies of contagious disease
in outbreaks, and the follow up of treatment for infections, particularly the viruses with a high tendency to mutations.
Keywords Virus, Molecular diagnosis, Molecular microbiology, Nucleic acid, PCR techniques, Viral laboratory diagnosis
Introduction
Viruses are tiny microscopic parasites with the genetic material
of either deoxyribonucleic acid (DNA) or ribonucleic acid
(RNA) surrounded by protein coal called (envelope). Viruses
only replicate in a living cell because they are known as
obligate intracellular parasites. Once viruses enter the host
cells, they take over and emerge in the cells’ biosynthetic types
of machinery for the replication of their genomes and other
elements (Coboet et al., 2006; Owen et al., 2013). Viruses are
relatively the most common causative agent of human
diseases. Millions are still dying from AIDS (HIV) virus and
hepatitis viruses worldwide. Initiating viruses are also causing
serious issues in the human population. Human viral infections
have significantly high morbidity and mortality rates (Souf,
2016). Therefore, efficient diagnostic strategies are required to
detect these viral infections as early and accurately as possible.
Early and accurate detection of viral presence in patients plays
an important role in choosing appropriate therapy on time,
minimizing therapy costs, minimizing unnecessary loss of
human lives, and controlling the disease. It also helps to
develop appropriate disease prevention and treatment
strategies, like the development of antiviral vaccines and new
therapeutic agents (Shen et al, 2020; Cella et al., 2013; Landry
et al., 2016).
Traditionally, laboratory diagnoses of medical viruses are
carried out by isolating viruses in embryonated chicken eggs,
in tissue culture, or laboratory animals and visual examination
of viral particles in a sample using electron microscopy among
others (Cella et al., 2013). Many conventional diagnostic tools
tend to be indolent, time-consuming, expensive, and poorly
performed (Acharya et al., 2013; Kumar, 2013). In contrast,
molecular techniques have facilitated diagnostic virology by
detecting the existence of viral nucleic acids in the viral sample
(Acharya et al., 2013). Here, we describe some of the
molecular diagnostic techniques for the detection of viruses.
Review Article
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ISSN: 2582-6697
Science Archives (2020) Vol. 1 (3), 98-101
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Molecular techniques for identification and characterization
Reliable detection of causative agents like viruses in viral
diseases, distinctive genetic sequences in viral diseases, and
protein entity are very objectively required for the management
of these diseases with taking into accounts the specificity and
sensitivity as important tools in diagnosis. Regular molecular
techniques like classical PCR and blotting technique albeit
assumed good in diagnosis. Anyway, nowadays molecular
techniques like gene and peptide sequencer, real-time PCR
may identify in even more accurate and explicit ways without
consuming much time.
Molecular diagnostic techniques of viruses
Nucleic acid-based molecular detection techniques have
developed diagnostic virology with their faster, highly
sensitive, and a highly specific diagnosis (Stone et al., 2014;
Canberk et al., 2016). Since these techniques detect specific
nucleic acid sequences, nucleic acid-based diagnostic tests can
be applied for the detection of any infection that affects
humans (Cobo et al., 2006).
Polymerase Chain Reaction (PCR)
PCR is a model example of genetic amplification assay. It has
developed the level of molecular diagnosis since developed by
Mullis and Faloona (1987). The PCR main principle depends
on the extraction and purification of DNA molecule and
evidential amplification of the targeted sequence, using a
thermostable DNA polymerase and two specific
oligonucleotide primers. After the PCR reaction, the amplified
product can be detected by several techniques, including gel
electrophoresis, colorimetric methods, and sequencing (Cobo,
2012; Levine et al., 2011). Since its introduction, PCR started
to be used for the detection of human viral infections with total
clinical sensitivity ranging from 77.8% to 100% and clinical
specificity ranging from 89% to 100% (Hassan et al., 2006;
Demmler et al. 1988; Myerson et al., 1993; Sundaramurthy et
al., 2018). These reports recommend that PCR can also be used
for the detection of viruses in many types of specimens. PCR
is an exceptionally multilateral technique. Several variants of
the conventional PCR have been developed, however; the main
variants are reverse transcription-PCR and real-time PCR
(Coboet et al., 2006; Souf, 2016). The first method was
invented to amplify ribonucleic acid (RNA) targets (Coboet et
al., 2006); the second technique was introduced to quantify
deoxyribonucleic acid (DNA) in real-time throughout the PCR
reactions (Gruber et al., 2001).
Reverse Transcription-PCR (RT-PCR)
RT-PCR was devised to amplify RNA targets. In this
technique, reverse transcriptase (RT) is used to convert viral
RNA targets into complementary DNA (cDNA), and then
amplify the resulting cDNA by conventional PCR. Since its
development, RT-PCR has been employed for the diagnosis of
human infection by RNA viruses. Conventional RT-PCR
demonstrated overall sensitivity ranging from 73% to 100%
and specificity ranging from 99% to 100% in the detection of
viral infection (Maignan et al., 2019; Falsey et al., 2002;
Formentry et al., 2006). These data indicate that RT-PCR is an
exceptional technique for the diagnosis of human infection by
RNA viruses. Currently, however, the method is not used
commonly in clinical specimens because of its high cost and
time-consuming process (Shen et al., 2020).
Multiplex PCR
In a similar response combination, at least two or more primer
sets prepared for amplification of a variety of targets are
utilized (Chamberlain et al., 1988). In a clinical sample, more
than one target sequence can be co-amplified in a single tube.
However, the primers used must be carefully selected for them
to have similar annealing temperatures and less
complementarily. This sort of PCR is less sensitive than PCR
with a single primer set. Multiplex PCR assays have been
developed and commercialized for viral respiratory pathogens
and detection of viral infections of the central nervous system
(Boriskin et al., 2004; Templeton et al., 2004).
Real-Time PCR
Real-time PCR is a simple, quantitative assay for amplification
of DNA sequence. It was clarified firstly by, Higuchi et al.
(1993). It depends on using fluorescent-labeled probes to
verify and quantify the PCR products as they are being
generated in real-time. The real-time PCR, which has three
novel features as temperature cycling occurs considerably
faster in cooperation to ordinary PCR assays, hybridization of
specific DNA probes are added continuously during the
amplification process and a fluorescent dye is cogitated to the
probe and fluoresces only when hybridization takes place.
Lack of post PCR processing of amplified products would
make this technique convenient. The production of amplified
products is automatically observed by real-time monitoring of
fluorescence. A small-signal can be produced within 30–45
min depending on the amount of target gene. Since the tubes
do not have to be opened at the time of reaction, the risk of
contamination decreased considerably. In recent years, some
automated real-time PCR systems have been available
commercially. (Light Cycler &TaqMan). Using these systems,
such as the Light Cycler TM and the Smart Cycler, they
perform the real-time fluorescence monitoring by using
fluorescent dyes such as SYBR-Green I, which binds non-
specifically to double-stranded DNA that was previously
generated during the PCR amplification (Fakruddin et al.,
2012; Holland et al., 1991).
Nested-PCR
This type of PCR possesses relatively high sensitivity and
specificity (Haqqi et al., 1988). This technique uses two pairs
of amplification primers and two rounds of PCR. In the first
round, it uses one primer pair for 15 to 30 cycles. The
product's first-round amplification is submitted to the second
round of amplification coupled with the second pair of
primers. The major setback of nested PCR is the high rates of
contamination.
Science Archives (2020) Vol. 1 (3), 98-101
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Transcription-based amplification methods
Transcription-based amplification method based on two
procedures, nucleic acid sequence-based amplification
(NASBA) and transcription-mediated amplification (TMA).
NASBA and TMA are not different in terms of principle. They
are isothermal amplification methods. The entire amplification
process is performed at a temperature of 41°C. In both
methods, the viral RNA target is first converted into cDNA
with RT and then multiple copies of viral RNA products are
synthesized by RNA polymerase. The sole difference between
TMA and NASBA in the amplification process is two enzymes
(RT and RNA polymerase). They are used in the case of TMA
while NASBA utilizes three enzymes (avian myeloblastosis
virus reverse transcriptase (AMV-RT), RNase H, and T7 RNA
polymerase) (Fakruddin et al., 2012; Cobo, 2012).
Transcription-based amplification methods have several
advantages, for example, they do not require a thermal cycler,
so developing countries and budget-restricted laboratories can
afford to perform the assays, they are rapid (requires fewer
cycles), and they produce a single-stranded RNA product that
is suitable for detection by various techniques (Mohammadi-
Yeganeh et al., 2012; Yu et al., 2012). Transcription-based
amplification assays are convenient for the diagnosis of human
infections caused by RNA viruses because they are capable of
amplifying viral genomic RNA, messenger RNA, or ribosomal
RNA (Cobo, 2012; Mohammadi-Yeganeh et al., 2012).
Ayeleet et al. (2004) developed a NASBA assay that uses gag-
based molecular beacons to differentiate between HIV-1
subtype C (C and C′) occurring in Ethiopia. The assay has high
levels of sensitivity and specificity (90.5% sensitivity, 100%
specificity for the C beacon, and 100% sensitivity, 95.2%
specificity for the C′ beacon) by considering sequencing as the
gold standard for genotyping.
Conclusion
The introduction of nucleic acid-based diagnostic tests into
diagnostic virology has made a remarkable improvement in the
detection of human viral infections. Since nucleic acid-based
diagnostic tests are highly sensitive and specific, they play a
distinctive role in the diagnosis and control of viral infection.
Molecular diagnostic methods diagnose viral infections by
detecting viral RNA or DNA. Therefore, these techniques can
detect infected individuals before the antibody response is
raised against the particular virus. This is especially important
in young, elderly, and immune-suppressed patients. However,
they are unreachable for resource-limited nations due to their
high cost, instrumentation complexity, and the requirement for
technical expertise.
Authors’ contributions
All authors have contributed significantly to the conception
and design of the study, the interpretation of data, and the
drafting and revision of the manuscript. All authors read and
approved the final manuscript .
Conflict of Interest
The authors hereby declare no conflict of interest.
Consent for publication
The authors declare that the work has consent for publication
Funding support
The authors declare that they have no funding support for this
study
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