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ES Food Agrofor., 2021, 3, 4-16
4 | ES Food Agrofor., 2021, 3, 4-16 © Engineered Science Publisher LLC 2021
ES Food and Agroforestry DOI: https://dx.doi.org/10.30919/esfaf437
Identification of Human Coronavirus: An Overview on Conventional, Newly Developed and Alternative Methods
Zhe Wang1,* Amit Nautiyal,2 Xiaozhou Huang,3 Rui He3 and Pei Dong3,*
Abstract
The recent respiratory disease (COVID-19 caused by novel coronavirus, SARS-CoV-2) outbreak, responsible for current pandemic, is causing a great risk to a public health. The rapid and accurate identifying methods is the key to prevent this pandemic that helps with selecting suitable treatment and saving people’s life. Polymerase chain reaction (PCR) is regarded as a gold standard test diagnosis of these infections with high sensitivity and specificity. It is the first nucleic acid amplification method. With advancement in research there are number of other nuclide acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence-based amplification, strand displacement amplification and rolling cycle amplification etc. These isothermal nucleic acid amplification methods are considered as promising methods due to its quick procedure time at constant temperature thus preventing the use of thermal cyclers. The various existing, improved, newly developed and alternative methods/approaches that can be used for accurate detection are being summarized in this review to assist researchers and clinicians in developing better methods for timely and effective detection of coronavirus.
Keywords: Coronavirus; Polymerase chain reaction; Nucleic acid amplification. Received: 10 February 2021; Accepted date: 16 March 2021.
Article type: Review article.
1. Introduction
Coronaviruses are large, enveloped virus (80-200nm) contains
single-stranded Ribonucleic acid (RNA) with positive polarity.
The characteristic feature of all coronaviruses is the presence
of few hundreds of club shaped projections having 10-20nm
in length that form the ‘corona’, thus their name. Human
coronavirus (HCoV) has been noted to have only one type of
projection, they are dimer of two dissimilar proteins that form
tetramer to produce surface spike. Several coronaviral species
are known to infect mammals and birds, based on their
genome sequence analysis, they were divided into three
groups. It is group I and II viruses that can infect mammals
whereas group III viruses were found exclusively in birds.[1,2]
They may cause variety of diseases in animal such as
gastroenteritis and respiratory tract, however, in humans, it
causes respiratory and neurological diseases. [3] The first
coronaviral infection in humans was reported in mid 1960s.[4–
6] The infectious sample was found to be negative for human
pathogens known at the time. Laboratory experiments showed
it can cross bacteria-tight filter and was antibiotic resistant but
ether sensitive. These tests along with electron microscopy
showed it was an enveloped virus, first indication of its
structure. The detection methods used at the time was tissue
culture and inoculation of healthy adult volunteers.
Serologically, they were categorized as HCoV-229E and
HCoV-OC43 based on their genome sequences they were
separated into two groups I and II, respectively.[7,8] It was until
2003 when another coronavirus was identified as Severe Acute
Respiratory Syndrome coronavirus (SARS-CoV) that causes
infectious disease, SARS. Initially, it was inferred that the
disease was caused by other known pathogens, however
research findings confirmed the new coronavirus is
responsible for SARS disease.[9–11] The cell culture analysis
was initially performed and found that infected cells were not
reacting to routine panel of immunological reagents used to
identify virus isolates. They were not responding in reverse
transcription polymerase chain reaction (RT-PCR) assays as
well. The electron microscopy revealed the structure of virus
1 Department of Chemistry, Oakland University, Rochester Hills, MI
48309 USA. 2 Department of Chemistry, Xavier University of Louisiana, New
Orleans, LA 70125 USA. 3 Department of Mechanical Engineering, George Mason University,
Fairfax, VA 22030 USA.
*Email: [email protected] (Z. Wang); [email protected] (P. Dong)
ES Food & Agroforestry Review article
© Engineered Science Publisher LLC 2021 ES Food Agrofor., 2021, 3, 4-16 | 5
Fig. 1 Basic structure of coronavirus with detection methods showing either positive or negative report.
particles with surface characteristics like coronavirus. Based
on this, universal coronavirus primers were designed by
research groups based on RNA-dependent RNA polymerase
gene of other coronaviruses.[10] Using these primers, different
strategies were adopted in detection such as amplification of
virus from cell culture with random RT-PCR, applied
differential display primers and cloned PCR fragments or
utilizing degenerated primers under less stringent conditions.
All these adopted strategies and genome sequence indicated
that SARS-CoV is a distinct member of coronavirus. It is the
first highly pathogenic human coronavirus to emerge with
mortality rate of 9.6% worldwide.[12] Again in 2003, another
group I coronavirus (HCoV-NL63) was detected in the
Netherlands where 7-months old child was suffering with
conjunctivitis, bronchiolitis and fever. The initial diagnostics
performed on nasopharyngeal aspirate sample was negative
for other known pathogens including HCoV-229E and HCoV-
OV43. The virus was enveloped confirmed by chloroform
sensitivity and acid liability tests. The full genome sequence,
analyzed by the sequence-independent Virus Discovery
cDNA-amplified fragment length polymorphism (VIDISCA)
method, indicates HCoV-NL63 was previously unknown
group I coronavirus.[13]
In 2004, novel coronavirus was identified as HCoV-HKU1
in China, where 71 years old man with pneumonia was
admitted to hospital. The tests conducted were negative for
several respiratory viruses, influenza A virus, human
metapneumovirus and SARS-CoV. No Cytopathic effect (CPE)
was observed in cell culture assay. The RT-PCR amplification
using universal primer revealed the sequence clusters with
coronaviridae in phylogenetic analysis. The full genome
sequence confirmed that HCoV-HKU1 was previously
unknown group II coronavirus.[14] The Middle East
Respiratory Syndrome (MERS) disease was reported during
summer 2012 in Jeddah, Saudi Arabia. A 60year old man was
admitted to hospital with fever, cough and shortness of breath.
The initial diagnostics performed for influenza A and B viruses,
parainfluenza virus types 1 to 3, respiratory syncytial virus,
and adenovirus were all negative. The sequence dependent RT-
PCR identified the virus that has been previously unknown to
group II coronavirus.[15] The isolated virus was previously
named as HCoV-EMC (Erasmus Medical Center, however
after broad consultation and careful consideration Coronavirus
Study Group of International Committee of Taxonomy of
Viruses named HCoV-MERS.[16]
The coronavirus is a common cold virus that causes in
infection in our nose or sinuses or may lead to a disease known
as COVID. It affects our upper respiratory tract (sinuses, nose,
and throat) or lower respiratory tract (windpipe and lungs).
Most coronaviruses are not dangerous however, it spreads
through person-to-person via respiratory droplets and
infections ranges from mild to severe that may lead to death.
The main symptoms of the disease include fever, cough, sore
throat, headache or shortness of breath. It can lead to
pneumonia, respiratory failure, liver and heart problems and
septic shock. These symptoms are often mistaken as common
cold and can lead to person’s death.
There were six coronavirus that have been identified until
now, namely, HCoV-229E, HCoV-OC43, HCoV-NL63,
HCoV-HKU1, severe acute respiratory syndrome coronavirus
(SARS-CoV), Middle East respiratory syndrome coronavirus
(MERS-CoV), only SARS-CoV and MERS-CoV have once
caused pandemic (more than 20 countries were affected). The
major event of coronavirus detected was listed in Table 1. A
novel coronavirus (SARS-CoV-2) was detected in Wuhan,
China (December 2019) have caused another outbreak of
coronavirus disease (COVID-19) worldwide that led to the
current pandemic we are in. There are more than 100million
confirmed COVID-19 cases worldwide including two million
deaths (more than 500,000 in US).[17] Therefore, early
detection of the SARS-CoV-2 should be fast and accurate that
should be beneficial in controlling the source of infection and
help preventing in widespread of the disease. In this study, we
reviewed currently available approaches, including PCR and
new alternative methods in detecting or identifying
coronavirus that may assist researchers to develop new, rapid
and accurate techniques.
2. Techniques
Currently, there are several techniques available for
identification of coronaviruses. This section provides a brief
overview on methods that can be used for successful detection
and identification of coronavirus such as cell culture, electron
microscopy, serology, PCR based methods, nucleic acid
amplification method, microarray based. These methods are
discussed with newly developed methods for detection of
Review article ES Food & Agroforestry
6 | ES Food Agrofor., 2021, 3, 4-16 © Engineered Science Publisher LLC 2021
human coronavirus. The alternative methods that have been
developed already but haven’t been used for CoV detection
were also discussed (Table 2).
2.1 Cell culture
This method is used to detect the appearance of cytopathic
effect by inoculating the samples from patients into other cells
and can demonstrate the species of cells that could be infected.
For example, it was found that HCoV-NL63 will not infect
human lung fibroblasts Medical Research Council cell strain
5 (MRC-5s) while the cytopathic effect showed in two
epithelial cell lines, Rhesus Monkey Kidney Epithelial Cells
(LLC-MK2) and Vero-B4 respectively.[18] Cell culture usually
have a relative high sensitivity by observing the cytopathic
effects. Reports suggested that only 3 of 10 samples showed
negative cytopathic effect after 2 weeks response time for
HCoV-229E.[19] However, cell culture cannot be used as
detection method alone as infection cannot be recognized with
naked eyes. Therefore, it is used with other detections,
including electron microscopy, enzyme-linked
immunosorbent assay (ELISA), PCR, etc. for successful
detection of coronavirus to avoid misdiagnosis.
2.2 Electron microscopy
It is one of the conventional methods that provides a direct
image of cells with or without the appearance of the virus. It
is the first method to be used to provide the structure of a virus
to differentiate coronavirus with other pathogens. It has been
used to identify different coronaviruses.[5,6,9,10,20] In addition,
immune electron microscopy (sometimes, immunoelectron
microscopy) has been used to identify these viruses with
sensitivity up to 50 times higher than conventional electron
microscope.[21–23] Moreover, transmission electron microscope
(TEM) is another microscopic method that has been used for
rapid viral diagnosis.[24] It is considered as one of the most
convenient method for coronavirus detection however,
preparation of the samples by cell culture takes several days to
finish that limit its the application for rapid detection.
2.3 Serology
This method uses viral antigens prepared from the infected
cells that have specific reaction with viral antibodies from
serum samples and signal difference indicates the positive or
negative results. The serology detection technology contains
several types, including rapid diagnostic test, ELISA,
neutralization assay, chemiluminescent immunoassay,
immunofluorescence assay, etc. A rapid diagnostic test have
the fast response time (10-30 mins), ELISA is one of the most
common methods for lab detection.[25,26] Most of coronaviruses
can be detected by this methods.[10,27–29] However, its sensitivity
is highly dependent on the period after the symptom’s onset.
For example, during MERS pandemic, less than 50% of
samples showed a positive result for MERS patients with 11-
15 days symptom while nearly 100% positive results come
with over 21 days from onset.[30] In addition to the days from
onset of symptoms, the nasopharyngeal aspirate samples gives
accurate results compared with urine or fecal samples.[31]
While it is still being used as a common method for COVID-
19 detection due to its low cost and convenience, there are
other methods that are provide more information than these
methods do.
3. PCR based method
PCR is widely used method used to produce billions of copies
of gene by separating the two strands of DNA. It allows
researchers and scientist to amplify small quantity of samples
helping them to study in detail. This method requires an
enzyme that polymerizes the DNA strands and is regarded as
a ‘gold standard’ for virus detection due to its high sensitivity
and sequence specificity.[32,33] Typically, cDNA is transferred
from RNA template using an enzyme known as reverse
transcriptase and the process is termed as reverse transcription
(RT). The reverse transcription is followed by PCR and
product formed is detected using detection
methods/instruments.
3.1 RT-PCR based on universal coronavirus primers
It is the most specific method where a sequence of short
fragment of sDNA, known as primers, is used to amplify any
member of coronavirus family (Fig. 2). The main advantage
of this method is to identify previously unknown coronavirus
by quick screening of pathogens in single assay. Adachi et
al.[34] showed the sequencing analysis on several clinical
samples obtained from patients with probable or suspected
SARS disease. The sequence analysis not only used for SARS-
CoV detection also for HCoV-229E and HCoV-OC43 as well.
Fig. 2 Schematics for steps involved in the amplification of viral RNA by RT-PCR.
ES Food & Agroforestry Review article
© Engineered Science Publisher LLC 2021 ES Food Agrofor., 2021, 3, 4-16 | 7
The PCR product was detected by electrophoresis and have
shown high sensitivity towards three coronaviruses used in the
study. Previously, it was described that RT-PCR was unable to
detect HCoV-NL63 due to mismatches with sequences.
Moreover, HCoV-NL63 and HCoV-229E are of same virus
serotype, their antibodies might cross react with each other
and results in misdiagnosis.[13] Therefore, Moës et al.[35]
modified those primer sequence based on HCoV-NL63 and
other coronavirus prototypes sequence alignment. The study
was conducted on young patients ranged in age from 1 month
to 16 years. Although, sensitivity found out to be lower than
expected they were able to detect all HCoV-N63 positive
samples with modified primer sequence. The commonly used
methods for detecting PCR product are gel visualization and
sequencing under the UV light. Sometimes the primers target
sequences show unexpected variation that can lead to missed
detection. Therefore, an alternative approach to rapidly detect
infectious disease with high sensitivity was presented. The
study introduced the Triangulation Identification for the
Genetic Evaluation of Risks (TIGER), using mass
spectroscopy the base composition of PCR product can be
analyzed.
The main principal of this method is ‘intelligent PCR
primers’ that target diverse microbial genomes. This method
can help in detecting all types of microbes: bacteria, virus,
fungi and protozoa that helps in preventing the biological
weapons attack. This unique method is capable of distinguish
each coronavirus from the mixture of SARS-CoV, HCoV-
229E and HCoV-OC43.[36,37] Despite all this, these methods are
not used in clinical samples due to their time-consuming
sample preparations steps and high cost. Therefore, new
methods were developed to address these issues.
3.2 Real time RT-PCR
The real time RT-PCR (rRT-PCR) is one of the most widely
used method to detect coronavirus as a facile quantitative
assay. This method is more sensitive than conventional RT-
PCR that helps in early detection for the virus due to specific
primer design. Lu et al.[38] developed the rRT-PCR assay for
detecting MERS-CoV by targeting their nucleocapsid (N)
gene and compared with previously developed assay
(targeting upstream enveloped gene, upE of the virus) for
identification of MERS-CoV infection in the samples. This
method showed enhanced sensitive for detecting the virus due
to the abundance of N-gene, a sub-genomic mRNA produced
during virus replication. Another study was reported by Noh
et al.[39] to detect HCoV and bat coronaviruses, simultaneously
where a developed duplex (rRT)-PCR method target the
conserved spike S2 region of SARS-CoV and MERS-CoV.
This method requires laborious sample handling and post PCR
analysis that makes it prone to contamination.
3.3 Nucleic Acid amplification
PCR was the first nucleic acid amplification method invented
by Mullis and is preferred method for its ease in methodology,
availability of reagents and equipment.[40] However, due to its
high cost of equipment, possibility of contamination and
sensitivity towards contaminants leads to false positive that
necessitates to develop new alternative methods such as loop
meditated isothermal amplification (LAMP), nucleic acid
sequence-based amplification (NASBA), strand displacement,
rolling circle amplification (RCA) etc. Many of these methods
are isothermal and offers the major advantage over PCR that
requires thermal cycler.
3.4 Regular loop meditated isothermal amplification
It is a facile, faster and cost-effective isothermal methods that
depend on auto cycling strand displacement DNA synthesis.
The typical mechanism of loop amplification (LAMP) method
includes production of starting material, cycling amplification
and recycling. This synthesis is carried out at a constant
temperature ranges from 60-65 °C in the presence of high
strand displacement activity DNA polymerase, specific
primers (two inner and two outer) that detect six dissimilar
sequence of target DNA.[41] The LAMP assay was
demonstrated to detect SARS-CoV.[42] The six different
primers were used to accelerate the amplification reaction of
ORF1b region of SARS-CoV and amplified products were
analyzed by gel electrophoresis. It can detect SARS-CoV in
64% of samples infected with SARS disease and detection rate
increases as disease progresses. The sensitivity of this method
is comparable to those of conventional PCR based methods.
The same research group demonstrated the real time
monitoring of LAMP.[43] The amplification product can be
detected by monitoring the production of white precipitate of
pyrophosphate ion formed as a byproduct in amplification of
DNA. This increases the turbidity of the solution and
indication of large amount of DNA synthesized thus removing
a step of endpoint detection. In clinical samples, Reverse
Transcription LAMP (RT-LAMP) has been used for early
diagnosis and rapid detection of SARS-CoV, MERS-CoV with
higher sensitivity than conventional RT-PCR assay.[44,45] The
HCoV-NL63 was detected by LAMP too where it was
analyzed by agarose gel electrophoresis with high sensitivity
and specificity in clinical samples.[46] However, solution
turbidity (pyrophosphate during polymerization), fluorescent
dye intercalated in dsDNA amplified product or some
unexpected from primer dimer reactions they all can interfere
in the signal as a noise that may result in false positive. All
these non-specific signals can be separated by having
sequence specific method. Recently, in clinical settings, RT-
LAMP was performed on suspected patients infected from
SARS-CoV-2 that have high sensitivity and specificity. This
method does not require any expensive equipment that reduces
time and cost of detection and can contribute to disease control
where laboratories capacities are limited.[47,48]
3.5 Nucleic acid sequence-based amplification
NASBA is a single step isothermal transcription-based
amplification method specifically designed for RNA detection,
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8 | ES Food Agrofor., 2021, 3, 4-16 © Engineered Science Publisher LLC 2021
however in some cases, DNA can be amplified too. A
predefined constant temperature is maintained to allow
reaction to proceed soon after amplification intermediate
formed and makes it easier to use. Its amplification kinetics is
faster than DNA-amplification (binary increase per cycle) due
to multiple replication of RNA copies from DNA product.[49]
The amplification process uses group of three enzyme that led
to single strand RNA amplified product. The amplified
product can be easily detected with high efficiency using
existing methods such as enzymatic based detection,
electroluminescent (ECL), molecular beacon and fluorescent
spectroscopy.
Huang et al.[50] developed a method that combine RT-
LAMP and vertical flow visualization strip to detect N gene of
MERS-CoV. The developed assay results were visible by the
naked eye in 5min and completes the detection in mere 35min
(Fig. 3). The method has high specificity due to no cross
reactions with multiple coronaviruses (SARS, HKU, 229E,
OC43). The quenching probe 3G (QProbe) showed the
improvement in RT-LAMP assay by blocking primer sequence
by fluorescent dye. This avoids non-specific amplification
signals and can detect primer driven signals only. This method
can detect MERS-CoV with no cross reactions with other
respiratory viruses. In clinical samples, the efficiency of the
assay was similar to that of real time RT-LAMP. The
developed assay is rapid, convenient to handle as dry reagents
and portable fluorometer can be used to detect signals.[51]
3.6 Strand displacement amplification
The specificity of the LAMP detection can be further
improved by replacing fluorescent dye with one-step strand
displacement (OSD), which includes strand exchange reaction.
This strand displacement amplification (SDA) enables a one-
pot assay that can rapidly (30-50min) detect MERS-CoV and
related coronaviruses. The OSD-RT-LAMP method offers
several advantages such as no postprocessing of LAMP
reactions, reduces the possibility of cross-reactions due to
aerosolization of templates, reduce cost, save time and can be
used in real time detection (Fig. 4).[52–54]
The OSD-RT-LAMP can be used to directly convert
MERS-CoV template into glucose signals that can be easily
read by commercial glucometer. It can detect as few as 20
nucleic acid templates equivalent to atto-molar levels.[55]
Another way to reduce cost and enhance sensitivity is to use
human chorionic gonadotropin (hCG) as a signal for
identification using off-the-shelf pregnancy test strip. The
hCG reporter protein was engineered to yield a single
attachment point for oligonucleotide probes to hCG. The
presence of virus can be identified in a simple positive or
negative response in LAMP based assay. This method can
easily detect smaller number of virus templates (as low as 20
copies) in human serum and saliva.[56] Although its
performance is optimal at 60-65 °C, sensitivity can be
comparable at lower temperature (40 °C) using
phosphorothioated primers.[57] The strand displacement
method is useful in generating probes for microarrays to
produce highly pure DNA. Its sensitivity is high and can be
carried out directly on biological samples.[58]
3.7 Rolling circle amplification
RCA is an isotherm amplification method that can readily
detect few target specific circularized probes in the sample. It
has the capability of amplifying DNA probe sequence more
than 109-fold both in solution and solid phase.[59] The RCA
reaction involves the enzyme (DNA polymerase) that start the
replicating the sequence of circular DNA repeatedly until
process is terminated. Unlike PCR and other isothermal
methods, it is resistant to contamination and requires little to
no assay optimization.[60,61] Xu et al.[62] combined RCA with
qPCR in one step to enhance the sensitivity and specificity of
the assay. detection limit reached as low as 500aM and can be
used for quantification of other small RNAs. The SARS-CoV
RNA was detected by RCA in both solution and solid phase.
The sensitivity was high in solid state phase due to reduction
in background signals.[63] This confirms the ultrasensitive
nature of the assay and can be used in detecting other
coronaviruses. The ease in quantification with high accuracy
makes it suitable for portable automation with high
throughput.[64]
Fig. 3 Schematics illustration of RT-LAMP-VF. A) amplification of RT-LAMP products, B) visual detection of RT-LAMP products
(Reproduced with the Permission from [50], Copyrights 2018 the authors).
ES Food & Agroforestry Review article
© Engineered Science Publisher LLC 2021 ES Food Agrofor., 2021, 3, 4-16 | 9
Fig. 4 Schematics representation of RT-LAMP amplification with detection via OSD. (Reprinted with the permission from reference
[53] Copyright 2015, American Chemical Society).
Table 1. Major event of coronavirus detections. Coronavirus Methods References
HCoV-229E and
HCoV-OC43
Tissue culture and
inoculation, Electron
microscopy
[7,8,34]
Universal coronavirus
primers, PT-PCR
[9,10,11,34]
[36,37]
rRT-PCR [39]
SARS-CoV LAMP assay, RT-LAMP [42,44]
RCA [59,62]
Microarray system, PCR [67,69]
Virus discovery based on
cDNA-AFLP* [13]
HCoV-NL63 Universal coronavirus
primers, PT-PCR [35]
LAMP [46]
HCoV-HKU1 Universal coronavirus
primers, PT-PCR [14]
Sequence dependent PT-PCR [15]
HCoV-MERS RT-LAMP [45]
RT-LAMP & Vertical flow
visualization strip [50]
OSD-RT-LAMP [55]
Serology [31]
SARS-CoV-2 RT-LAMP [47,48]
Microarray [75]
NASBA & RT-PCR [87]
CRISPR [88]
SARS-CoV-2 Molecular POC [89]
Next Generation Sequencing [90]
POC Biosensor [91]
* (amplified fragment length polymorphism) (VIDISCA)
3.8 Microarray based method
The DNA microarray is a novel technology that are capable of
accurately and rapidly detecting a variety of viruses.[65–67] It can
identified from thousands of sample at one time.[68] This
method has the potential to mitigate the pandemic disease
situation in virus detection aspect. For human coronavirus, the
RNA is used to produce complementary DNA (cDNA) by
reverse transcription. During reverse transcription, a specific
sequence of cDNA was labeled for characterization. This
labeled cDNA is loaded onto each microarray spot for
hybridization with the ssDNA fixed on the microarray
followed by rinsing to remove free DNAs. The microarray can
be used to detect and quantify coronavirus RNA by detection
spots. Based on this technology, a variety of works have been
reported, however mutation of the coronavirus remains one of
the biggest challenges in its detection. Long et al.[67] developed
a universal microarray system that can identify virus with high
mutation rates. The Zip Codes (3’ end) were covalently of
fluorophore, silver, and chemiluminescence label in the
attached to a slide and their cZip Codes (5’ end) remain
constant. This would help in tagging target sequence to make
universal microarray. They performed PCR on 16 primers
specific to SARS-CoV and ligase detection reaction (LDR) to
eliminate interference of mutant virus resulting a universal
detection of SARS coronavirus. Meanwhile, Guo et al.[69]
developed a DNA microarray to detect the single nucleotide
polymorphism (SNP) and PCR was used to amplify the
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10 | ES Food Agrofor., 2021, 3, 4-16 © Engineered Science Publisher LLC 2021
cDNAs produced by SARS-CoV strains. More than 20 SNPs
can be detected and determine the strain type (100% accuracy)
using this microarray. Moreover, a PCR-free gold nanoparticle
(Au-NP) based microarray was developed to identify avian
influenza virus H5N1, H1N1 and H3N2.[65] Compared with the
fluorescent dye detection, this method involves coupling of
Au-NP with silver and probe the light-scattering signal from
silver shell resulting in higher sensitivity. A flow-based
chemiluminescence microarray could detect viruses and
bacteria.[70] The detection limit of such method is comparable
to the qPCR analysis and can detect multiple samples
simultaneously.
4. Newly developed methods
4.1 Reverse-Transcription Recombinase-Aided
Amplification (RT-RAA)
It is a single tube, isothermal method that can replace PCR
because of their similarities. It can use reverse transcription
and fluorescence system for real time detection. It utilizes
mixture of enzymes (recombinase UvsX and DNA polymerase)
and protein (ssDNA binding protein) and has the ability to
detect less concentrated samples that are often missed by PCR
or LAMP methods.[71] The amplified product can be analyzed
at lower temperature (~40 °C) within 35min without opening
the tube, reducing the possibility of contamination. It was
developed as probe-directed recombinase amplification
(PDRA) for rapid detection of A1298C polymorphism related
to heart disease.[72] The same group used this method for
detection of respiratory syncytial viruses (RSV). The use of
recombinase reduces the false positive results due to their
inherent proofreading capabilities.[73,74] Recently, this method
was used for the identification of SARS-CoV-2 in patients
suffering for COVID-19. They created two assays for S and
orf1ab gene of SARS-CoV-2 using clinical samples for
validation. The assays performed on those sample have results
that were in agreement with real time RT-PCR, proving it a
beneficial and specific tool for SARS-CoV-2 detection.[75]
4.2 RNA targeting CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR) is a newly developed gene editing technology
where organism’s genetic material can be added, removed or
changed at a location in the genome. It is an effective tool for
rapid, cheap and accurate detection of nucleic acid and can be
used for disease diagnosis.[76,77] It is an adaptive immune
system where enzyme that cleave the phosphodiester bond
within a polynucleotide chain and can be programed for
CRISPR-based diagnostics (CRISPR-Dx). These enzymes are
known as CRISPR-associated (Cas) enzymes. Gootenberg et
al.[78] reprogrammed Cas13a with CRISPR RNAs (crRNA)
and its ‘collateral’ activity was detected using their developed
platform, termed as SHERLOCK (Specific High Sensitivity
Enzymatic Reporter Unlocking). It combines isothermal pre-
amplification with Cas-13a to detect single molecule of DNA
or RNA. It can detect Dengue or Zika virus ssRNA in liquid
biopsy samples.[79] Recently, it was showed to detect SARS-
CoV-2 with more than 1 × 104 copies/mL and no cross
reactivity to other coronaviruses. It was demonstrated that by
running reaction within one single tube without opening the
lid, the aerosol contamination and reduce the false positive
rate can be avoided (Fig. 5).[80] The main advantage of this
technology is its portability, multiplexable and rapid
quantitative detection of nucleic acid.[81,82]
Fig. 5 Schematics illustration of steps involved in RT-RAA and CRISPR detection of coronavirus. [1) Extraction of virus genome,
2) Isothermal amplification (RT-RAA) and programmed for CRISPR detection, 3) CRISPR assisted (CAS) detection and 4) Detection
signal obtained from fluorescence reader or direct observation under blue light.] (reproduced with the permission from [80,
Copyright 2020, The Author(s)).
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© Engineered Science Publisher LLC 2021 ES Food Agrofor., 2021, 3, 4-16 | 11
4.3 Electrochemical detection
Compared with those PCR-based methods, the
electrochemical-based detection has advantage in its facile use,
lower cost, higher efficiency, and relatively simple data
analysis. A variety of virus detections using electrochemical-
based method have been reported. Layqah et al.[83] used gold
nanoparticles (Au NP) to modify an array of carbon electrodes.
The square wave voltammetry (SWV) measurements were
performed by using ferrocyanide/ferricyanide as a probe. The
linear range were 0.001 to 100 ng mL-1 and 0.01 to 10000 ng
mL-1 with limit of detection (LOD) of 0.4 and 1.0 pg mL-1 for
HCoV and MERS-CoV, respectively. Meanwhile, Liu et al.[84]
developed an electrochemical immunosensor to detect Alpha
fetoprotein (AFP) by using platinum NP anchored cobalt
oxide/graphene nanosheets as the label of antibodies, which is
beneficial to amplify the signal and obtain better
electrochemical performance. The Au NP was also used to
accelerate electron transfer and capture primary antibodies in
the system. As a result, the sensor showed a wide linear range
from 0.1 pg mL-1 to 60 ng mL-1 with low LOD of 0.029 pg
mL-1 for AFP. The method could potentially be used for
SARS-Cov-2 detection.
Moreover, Wei et al.[86] developed an electrochemical
system to detect carcinoembryonic antigen (CEA) by using Au
NP modified Cu2S-CuS/graphene composite as matrix and
carboxyl-Au NP supported toluidine blue (TB) as label. The
system was duel-signaling respond that the increase of CEA
concentration leads the decrease of the oxidation peak current
of Cu2S-CuS and increase of oxidation peak current of TB.
The linear range is 0.001-100 ng/mL with LOD of 0.78 pg/mL.
Similar approach was reported the use graphene and gold
nanoparticles (Au NPs) in rapid, accurate and ultrasensitive
detection of SARS-CoV-2 coronavirus in less than 5min. They
used ssDNA probes coupled with Au NPs to selectively target
viral gene, nucleocapsid phosphoprotein. These capped
sensing probes were then immobilized on paper based
electrochemical sensor chip. The signal response was
increased when Au NPs (configuration 2) were used instead of
gold electrode (configuration 1) due to increased reactivity of
ssDNA coupled with Au NPs (Fig. 6). This favors the electron
transfer because of high surface area of nanoparticles that
improves the interaction of ssDNA with viral RNA which
amplifies the signal.[85]
There are methods have been developed already but have
not been used in detection of coronaviruses. These alternative
methods can be useful in detecting coronavirus that helps in
mitigating the effect of respiratory disease like COVID-19 on
the world.
Fig. 6 Schematics representation of fabrication of paper based electrochemical sensor for SARS-CoV-2 detection. The response
signal in both configurations. {Configuration 1: sensing probes were directly coupled with gold electrode, configuration 2: sensing
probes were capped on gold nanoparticles and were deposited on the electrode.} (Reprinted with the permission from [85] Copyright
2020 American Chemical Society).
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Table 2. Human coronavirus detection methods.
Techniques Sensitivity Response time Limit of detection Coronavirus
detected References
Cell culture High
Depends on
cytopathic effect
of the virus,
usually several
days
Long response time
Need to combine
with other
detection
methods
[18,19]
Electron
Microscope High Low
Not convenient, need
long preparation time All [21–24]
Serology Medium ~ 1 day Sensitivity is not very
high All [10,26–31]
RT-PCR 5 × 103 per µL
sample 1-3 day
HCoV-NL63,
HCoV-229E [35]
RT-PCR with
mass spectroscopy ≈1 PFU/mL ~ 1 day
SARS-CoV,
HCoV-229E,
HCoV-OC43
[36]
Regular LAMP 100x greater than
PCR <1hr 0.01 PFU
HCoV-NL63,
SARS-CoV,
MERS-CoV
[45,46]
NASBA 2 × 101 per µL
sample 5-30min 1 × 101 per µL sample
SARS-CoV,
MERS-CoV [50]
SDA Order of attomolar 25min 0.06PFU MERS-CoV [55,56]
RCA High <90min ~500aM SARS-CoV [63,96]
RNA targeting
CRISPR
1 × 104 - 1 × 105
copies/mL 30-60min >1 × 104 copies/mL SARS-CoV-2 [80]
RT-RAA High 10-30min
10 copies (S gene) and 1
copy (orf1ab gene) per
reaction
SARS-CoV-2 [75]
Electrochemical 0.001-100 ng/mL Within 20min 0.4-1.0 pg/mL MERS-CoV [83]
4.4 Ligase Chain Reaction.
While RCA, as discussed above, uses DNA polymerase
enzyme to replicate the sequence, ligase chain reaction (LCR)
uses both DNA polymerase and DNA ligase enzyme to start
the reaction. It is similar to PCR with only difference is that it
amplifies the probe molecules instead of producing amplicons
via polymerization. The process involves the hybridization of
two pairs of complementary oligonucleotides to the target
molecule. These two pairs of oligonucleotides are then ligated
by DNA ligase, which then serve as template for future
ligation. The thermal circler starts the reaction and target
molecule got doubled at each cycle. The amplified product is
then detected by techniques like electrophoresis, ELISA etc. [92,93] This method can have higher specificity than PCR based
methods and can be used for multiplex reaction suitable for
detection by microarrays.[94] However, specificity of ligase
reaction is restricted to region of ligase junction. Further, it can
detect DNA from dead pathogen that may leads to false
positive results.
4.5 Helicase Dependent Amplification
It is an isothermal nucleic acid in vitro amplification method
that uses fork mechanism for replication. In this method, DNA
helicase was used to separates the dsDNA and DNA
polymerase to create ssDNA. The sequence specific primers
were then hybridized at each end of target DNA. The DNA
polymerase then extend the hybridized primer to the template
to produce dsDNA. These two new dsDNA are then used as a
substrate by helicase to start the next cycle. This method
results in exponential amplification of the target and
reproduction of multiple cycles at single incubation
temperature, eliminating thermocycling equipment.[95] The
amplified products can be detected by gel electrophoresis and
real time format. HDA have several advantages over
isothermal DNA amplification methods; reaction scheme is
simple, high yield in shorter time (microgram scale of DNA
can be obtained from nanogram of input DNA within an hour),
primers are not required. All these characteristics shows great
potential in developing a diagnostic tool to detect pathogens at
the point-of-care.
5. Summary
It is of great importance select appropriate diagnostic tool to
detect pathogens like novel coronavirus to prevent the future
pandemics. Each method describe here has its own unique
advantage over the other and disadvantage too. PCR is widely
used identification methods that has high sensitivity and
specificity. However, it requires a complex equipment that can
be operated by educated personnel with expertise in this type
of analysis. Nucleic acid amplification methods are
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ultrasensitive that can detect small amount of DNA or RNA in
shorter period of time. Even though the amplification process
requires isothermal condition, the high temperatures limits is
application. The microarray-based methods often do not
require high temperature, but their high cost limits its use
widely. The electrochemical approach shows potential with
high sensitivity and stability as it a promising detection
method however, it still lacks wide application. The methods
such as LCR and HAD are great alternative to existing
detection methods that can also be used for rapid identifying
coronavirus. By incorporating real time detection methods
such as electro-chemiluminescent can make them useful for
future and can be applicable as PCR.
Acknowledgments
This publication was made possible by the supports from the
Army Research Office under Grant Number W911NF-18-1-
0458, National Science Foundation (CHE-1832167 and HRD-
1700429); P. D. acknowledges the financial support of the US
Department of the Interior Bureau of Reclamation
(R19AC00116) and 4VA (a collaborative partnership for
advancing the Commonwealth of Virginia).
Conflict of interest
There are no conflicts to declare.
Supporting information
Not applicable
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Author information
Zhe Wang is an assistant professor at
Oakland University. He earned a B.S. in chemistry in 2001 and received his Ph.D.
in chemistry and material science in
2007 at the Lanzhou University. From 2007-2009, Dr. Wang served as a
postdoctoral fellow at the University of
California Los Angeles and Oakland University, where he worked on the multifunctional materials
for advanced engineering and electrochemistry sensor projects. Before he joined Oakland, Dr. Wang was an
Assistant Professor at Xavier University of Louisiana.
Currently, he is serving as Associate Editor of "Engineered
Science Materials & Manufacturing" and Editor Board of
“Advanced Composites and Hybrid Material” His research
focuses on the interfacial material and phenomena study, particularly on the molecular reactions at the
electrode/liquid/gas interface for energy conversion, green synthesis, and biomedical diagnosis area.
Amit Nautiyal is a research associate in the NIH research center at the Xavier
University of Louisiana. He received his bachelor’s in Polymer Science at Delhi
College of Engineering, India and
master’s in Materials Science at Indian Institute of Technology, Bombay. He
obtained his Ph.D. in Polymer & Fiber Engineering at Auburn
University in 2018. Dr. Nautiyal’s research interests include multifunctional coatings, polymer nanocomposite,
microwave-assisted synthesis, and conducting polymers.
Xiaozhou Huang is a Ph.D. student at
George Mason University. He received his master’s degree in Nanotechnology
from Rice University, and his B.S. degree in Material Science and Engineering
from Hefei University of Technology. He
is interested in exploring applications for novel materials and using technology
emerging from the lab to better human
life.
Rui He is a Ph.D. student in the
Department of Mechanical Engineering, George Mason University. He received
his bachelor's degree in the Department of Polymer material and Engineering,
Nanjing Tech University, and master's
degree in Material Science and Nano Engineering, Rice University. His current research interest is
on advanced materials for water treatment and batteries.
Pei Dong is an assistant professor in the
Department of Mechanical Engineering at George Mason University. She
obtained her B.S. in Microelectronics
from Nankai University and her Ph.D. in
Mechanical Engineering from Rice
University. She then did her postdoctoral research in the Department of Materials
Science and NanoEngineering at Rice
University. She received the Franz and Frances Brotzen Fellowship Award. Her current research
interests include the design, synthesis, and applications of advanced materials in energy, water, and biomedical areas.
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