Identification of Human Coronavirus: An Overview on ...Fig. 1 Basic structure of coronavirus with detection methods showing either positive or negative report. particles with surface

ES Food Agrofor., 2021, 3, 4-16

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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


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.



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,



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).



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



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)).



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).



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



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

References

[1] M. Lai, Annu. Rev. Microbiol., 1990, 44, 303–333, doi:

10.1146/annurev.micro.44.1.303.

[2] J. M. González, P. Gomez-Puertas, D. Cavanagh, A. E.

Gorbalenya and L. Enjuanes, Arch. Virol., 2003, 148, 2207–

2235, doi: 10.1007/s00705-003-0162-1.

[3] V. M. Corman, I. Eckerle, T. Bleicker, A. Zaki, O. Landt,

M. Eschbach-Bludau, S. van Boheemen, R. Gopal, M.

Ballhause, T. M. Bestebroer, D. Muth, M. A. Müller, J. F.

Drexler, M. Zambon, A. D. Osterhaus, R. M. Fouchier and C.

Drosten, Eurosurveillance, 2012, 17, 1–6, doi: 10.2807/ese.17.39.20285-en

[4] D. A. J. Tyrrell and M. L. Bynoe, Br. Med. J., 1965, 1,

1467–1470, doi: 10.1136/bmj.1.5448.1467.

[5] D. Hamre and J. J. Procknow, Proc. Soc. Exp. Biol. Med., 1966, 121, 190–193, doi: 10.3181/00379727-121-30734.

[6] K. McIntosh, W. B. Becker and R. M. Chanock, Proc. Natl.

Acad. Sci., 1967, 58, 2268–2273, doi: 10.1073/pnas.58.6.2268.

[7] V. Thiel, J. Herold, B. Schelle and S. G. Siddell, J. Gen.

Virol., 2001, 82, 1273–1281, doi: 10.1099/0022-1317-82-6-

1273.

[8] J. R. St-jean, M. Desforges, A. Vabret and P. J. Talbot, J.

Virol., 2004, 78, 8824–8834, doi: 10.1128/JVI.78.16.8824.

[9] C. Drosten, S. Gunther, W. Preiser, S. van der Werf, H.-R.

Brodt, S. Becker, H. Rabenau, M. Panning, L. Kolesnikova, R.

A. M. Fouchier, A. Berger, A. Burguière, J. Cinatl, M.

Eickmann, N. Escriou, K. Grywna, S. Kramme, J. Manuguerra,

S. Müller, V. Rickerts, M. Stürmer, S. Vieth, H. Schmitz and

H. W. Doerr, N. Engl. J. Med., 2003, 248, 1967–1976, doi:

10.1056/NEJMoa030747.

[10] T. G. Ksiazek, D. Erdman, C. S. Goldsmith, S. R. Zaki, T.

Peret, S. Emery, S. Tong, C. Urbani, J. A. Comer, W. Lim, P.

E. Rollin, K. H. Nghiem, S. Dowell, A.-E. Ling, C. Humphrey,

W.-J. Shieh, J. Guarner, C. D. Paddock, P. Rota, B. Fields, J.

DeRisi, J.-Y. Yang, N. Cox, J. Hughes, J. LeDuc, W. J. Bellini

and L. J. Anderson, N. Engl. J. Med., doi:

10.1056/NEJMoa030781.

[11] J. S. M. Peiris, S. T. Lai, L. L. M. Poon, Y. Guan, L. Y. C.

Yam, W. Lim, J. Nicholls, W. K. S. Yee, W. W. Yan, M. T.

Cheung, V. C. C. Cheng, K. H. Chan, D. N. C. Tsang, R. W. H.

Yung, T. K. Ng and K. Y. Yuen, Lancet, 2003, 361, 1319–1325,

doi: 10.1016/s0140-6736(03)13077-2.

[12] World Health Organization,

https://www.who.int/csr/sars/country/2003_07_11/en/ and

https://covid19.who.int/.

[13] L. Van Der Hoek, K. Pyrc, M. F. Jebbink, W. Vermeulen-

Oost, R. J. M. Berkhout, K. C. Wolthers, P. M. E. Wertheim-

Van Dillen, J. Kaandorp, J. Spaargaren and B. Berkhout, Nat.

Med., 2004, 10, 368–373, doi: 10.1038/nm1024.

[14] P. C. Y. Woo, S. K. P. Lau, C. Chu, K. Chan, H. Tsoi, Y.

Huang, B. H. L. Wong, R. W. S. Poon, J. J. Cai, W. Luk, L. L.

M. Poon, S. S. Y. Wong, Y. Guan, J. S. M. Peiris and K. Yuen,

J. Virol., 2005, 79, 884–895, doi: 10.1128/jvi.01244-13.

[15] A. M. Zaki, S. Van Boheemen, T. M. Bestebroer, A. D. M.

E. Osterhaus and R. A. M. Fouchier, N. Engl. J. Med., 2012,

367, 1814–1820, doi: 10.1056/NEJMoa1211721.

[16] R. J. de Groot, S. C. Baker, R. S. Baric, C. S. Brown, C.

Drosten, L. Enjuanes, R. A. M. Fouchier, M. Galiano, A. E.

Gorbalenya, Z. A. Memish, S. Perlman, L. L. M. Poon, E. J.

Snijder, G. M. Stephens, P. C. Y. Woo, A. M. Zaki, M. Zambon

and J. Ziebuhr, J. Virol., 2013, 87, 7790–7792, doi:

10.1128/jvi.01244-13.

[17] World Health Organization,

https://www.who.int/dg/speeches/detail/who-director-

general-s-opening-remarks-at-the-media-briefing-on-covid-

19---11-march-2020.

[18] O. Schildgen, M. F. Jebbink, M. de Vries, K. Pyrc, R.

Dijkman, A. Simon, A. Müller, B. Kupfer and L. van der Hoek,

J. Virol. Methods, 2006, 138, 207–210, doi: 10.1016/j.jviromet.2006.07.023.

[19] S. Myint, S. Johnston, G. Simpson and H. Sanderson,

Mol. Cell. Probes, 1994, 8, 357–364, doi: 10.1006/mcpr.1994.1052.

[20] R. A. M. Fouchier, N. G. Hartwig, T. M. Bestebroer, B.

Niemeyer, J. C. De Jong, J. H. Simon and A. D. M. E.

Osterhaus, Proc. Natl. Acad. Sci. USA., 2004, 101, 6212–6216,

doi: 10.1073/pnas.0400762101.

[21] J. M. Kim, Y. S. Chung, H. J. Jo, N. J. Lee, M. S. Kim, S.

H. Woo, S. Park, J. W. Kim, H. M. Kim and M. G. Han, Osong Public Heal. Res. Perspect., 2020, 11, 3–7, doi: 10.24171/j.phrp.2020.11.1.02.

[22] A. Z. Kapikian, H. D. James, S. J. Kelly and A. L. Vaughn,

Infect. Immun., 1973, 7, 111–116, doi: 10.1128/iai.7.1.111-

116.1973.

[23] S. Dea and S. Garzon, J. Vet. diagnostic Investig., 1991,

3, 297–305, doi: 10.1177/104063879100300405.



[24] M. H. B. Catroxo, L. B. Miranda, A. Lavorenti, S.

Petrella, N. A. Melo and A. M. C. P. R. F. Martins, Int. J.

Morphol., 2010, 28, 549–555, doi: 10.4067/S0717-

95022010000200035.

[25] A. Kobokovich, R. West and G. Gronvall,

https://www.centerforhealthsecurity.org/resources/COVID-

19/serology/Serology-based-tests-for-COVID-19.html.

[26] J. A. Al-Tawfiq and Z. A. Memish, Travel Med. Infect.

Dis., doi: 10.1016/j.tmaid.2020.101785.

[27] M. J. Mäkelä, T. Puhakka, O. Ruuskanen, M. Leinonen,

P. Saikku, M. Kimpimäki, S. Blomqvist, T. Hyypiä and P.

Arstila, J. Clin. Microbiol., 1998, 36, 539–542, doi: 10.1128/jcm.36.2.539-542.1998.

[28] S. K. P. Lau, X. Che, P. C. Y. Woo, B. H. L. Wong, K.

Chan and J. S. M. Peiris, Emerg. Infect. Dis., 2005, 11, 7–10.

doi: 10.3201/eid1107.041045.

[29] S. Khan, R. Nakajima, R. Ramiro de Assis, A. Jasinskas,

J. Obiero, O. Adenaiye, S. Tai, F. Hong, D. Milton, H. Davies

and P. Felgner, doi: 10.1101/2020.03.24.006544.

[30] S. W. Park, R. A. P. M. Perera, P. G. Choe, E. H. Y. Lau,

S. J. Choi, J. Y. Chun, H. S. Oh, K. Song, J. H. Bang, E. S.

Kim, H. B. Kim, W. B. Park, N. J. Kim, L. L. M. Poon, M.

Peiris and M. D. Oh, Eurosurveillance, 2015, 20, 1–5, doi:

10.2807/1560-7917.ES.2015.20.41.30042.

[31] S. K. P. Lau, P. C. Y. Woo, B. H. L. Wong, H. W. Tsoi, G.

K. S. Woo, R. W. S. Poon, K. H. Chan, W. I. Wei, J. S. Malik

Peiris and K. Y. Yuen, J. Clin. Microbiol., 2004, 42, 2884–

2889, doi: 10.1128/JCM.42.7.2884-2889.2004.

[32] A. Balboni, L. Gallina, A. Palladini, S. Prosperi and M.

Battilani, Sci. World J., doi: 10.1100/2012/989514.

[33] C. Uhlenhaut, J. I. Cohen, S. Pavletic, G. Illei, J. C. Gea-

Banacloche, M. Abu-Asab, T. Krogmann, L. Gubareva, S.

Mcclenahan and P. R. Krause, Transpl. Infect. Dis., 2012, 14,

79–85, doi: 10.1111/j.1399-3062.2011.00657.x.

[34] D. Adachi, G. Johnson, R. Draker, M. Ayers, T. Mazzulli,

P. J. Talbot and R. Tellier, J. Virol. Methods, 2004, 122, 29–36.

doi: 10.1016/j.jviromet.2004.07.008.

[35] E. Moës, L. Vijgen, E. Keyaerts, K. Zlateva, S. Li, P.

Maes, K. Pyrc, B. Berkhout, L. van der Hoek and M. Van

Ranst, BMC Infect. Dis., 2005, 5, 1–10, doi: 10.1186/1471-

2334-5-6.

[36] R. Sampath, S. A. Hofstadler, L. B. Blyn, M. W. Eshoo,

T. A. Hall, C. Massire, H. M. Levene, J. C. Hannis, P. M.

Harrell, B. Neuman, M. J. Buchmeier, Y. Jiang, R. Ranken, J.

J. Drader, V. Samant, R. H. Griffey, J. A. McNeil, S. T. Crooke

and D. J. Ecker, Emerg. Infect. Dis., 2005, 11, 373–379, doi:

10.3201/eid1103.040629.

[37] S. A. Hofstadler, R. Sampath, L. B. Blyn, M. W. Eshoo,

T. A. Hall, Y. Jiang, J. J. Drader, J. C. Hannis, K. A. Sannes-

Lowery, L. L. Cummins, B. Libby, D. J. Walcott, A. Schink, C.

Massire, R. Ranken, J. Gutierrez, S. Manalili, C. Ivy, R.

Melton, H. Levene, G. Barrett-Wilt, F. Li, V. Zapp, N. White,

V. Samant, J. A. McNeil, D. Knize, D. Robbins, K. Rudnick,

A. Desai, E. Moradi and D. J. Ecker, Int. J. Mass Spectrom.,

2005, 242, 23–41, doi: 10.1016/j.ijms.2004.09.014.

[38] X. Lu, B. Whitaker, S. K. K. Sakthivel, S. Kamili, L. E.

Rose, L. Lowe, E. Mohareb, E. M. Elassal, T. Al-sanouri, A.

Haddadin and D. D. Erdman, J. Clin. Microbiol., 2014, 52, 67–

75, doi: 10.1128/JCM.02533-13.

[39] J. Y. Noh, S. W. Yoon, D. J. Kim, M. S. Lee, J. H. Kim,

W. Na, D. Song, D. G. Jeong and H. K. Kim, Arch. Virol., 2017,

162, 1617–1623, doi: 10.1007/s00705-017-3281-9.

[40] K. B. Mullis, Sci. Am., 1990, 262, 56–65, doi:

10.1038/scientificamerican0490-56.

[41] T. Notom, H. Okayama, H. Masubuchi, T. Yonekawa, K.

Watanabe, N. Amino and T. Hase, Nucleic Acid Res., 2000, 28,

E63, doi: 10.1093/nar/28.12.e63.

[42] L. L. M. Poon, C. S. W. Leung, M. Tashiro, K. H. Chan,

B. W. Y. Wong, K. Y. Yuen, Y. Guan and J. S. M. Peiris, Clin. Chem., 2004, 50, 1050–1052, doi:

10.1373/clinchem.2004.032011.

[43] Y. Mori, K. Nagamine, N. Tomita and T. Notomi,

Biochem. Biophys. Res. Commun., 2001, 289, 150–154, doi:

10.1006/bbrc.2001.5921.

[44] K. Shirato, T. Yano, S. Senba, S. Akachi, T. Kobayashi, T.

Nishinaka, T. Notomi and S. Matsuyama, Virol. J., 2014, 11,

1–11, doi: 10.1186/1743-422X-11-139.

[45] H. Thi, C. Thai, M. Q. Le, C. D. Vuong, M. Parida, H.

Minekawa, T. Notomi and F. Hasebe, J. Clin. Microbiol., 2004,

42, 1956–1961, doi: 10.1128/JCM.42.5.1956-1961.2004.

[46] K. Pyrc, A. Milewska and J. Potempa, J. Virol. Methods,

2011, 175, 133–136, doi: 10.1016/j.jviromet.2011.04.024.

[47] Y. H. Baek, J. Um, K. J. C. Antigua, J. H. Park, Y. Kim,

S. Oh, Y. il Kim, W. S. Choi, S. G. Kim, J. H. Jeong, B. S. Chin,

H. D. G. Nicolas, J. Y. Ahn, K. S. Shin, Y. K. Choi, J. S. Park

and M. S. Song, Emerg. Microbes Infect., 2020, 9, 998–1007,

doi: 10.1080/22221751.2020.1756698.

[48] C. Yan, J. Cui, L. Huang, B. Du, L. Chen, G. Xue, S. Li,

W. Zhang, L. Zhao, Y. Sun, H. Yao, N. Li, H. Zhao, Y. Feng,

S. Liu, Q. Zhang, D. Liu and J. Yuan, Clin. Microbiol. Infect.,

2020, 26, 773–779, doi: 10.1016/j.cmi.2020.04.001.

[49] R. Sooknanan and L. T. Malek, Bio/Technology, 1995, 13,

563–564, doi: 10.1038/nbt0695-563.

[50] P. Huang, H. Wang, Z. Cao, H. Jin, H. Chi, J. Zhao, B. Yu,

F. Yan, X. Hu, F. Wu, C. Jiao, P. Hou, S. Xu, Y. Zhao, N. Feng,

J. Wang, W. Sun, T. Wang, Y. Gao, S. Yang and X. Xia, Front. Microbiol., 2018, 9, 1–9, doi: 10.3389/fmicb.2018.01101.

[51] K. Shirato, S. Semba, S. A. El-Kafrawy, A. M. Hassan, A.

M. Tolah, I. Takayama, T. Kageyama, T. Notomi, W. Kamitani,

S. Matsuyama and E. I. Azhar, J. Virol. Methods, 2018, 258,

41–48, doi: 10.1016/j.jviromet.2018.05.006.

[52] B. Li, X. Chen and A. D. Ellington, Anal. Chem., 2012,

84, 8371–8377, doi: 10.1021/ac301944v.

[53] Y. S. Jiang, S. Bhadra, B. Li, Y. R. Wu, J. N. Milligan and

A. D. Ellington, Anal. Chem., 2015, 87, 3314–3320, doi:

10.1021/ac504387c.

[54] S. Bhadra, Y. S. Jiang, M. R. Kumar, R. F. Johnson, L. E.

Hensley and A. D. Ellington, PLoS One, 2015, 10, 1–21, doi:

https://doi.org/10.1371/journal.pone.0123126.

[55] Y. Du, R. A. Hughes, S. Bhadra, Y. S. Jiang, A. D.

Ellington and B. Li, Sci. Rep., 2015, 5, 1–14, doi:

10.1038/srep11039.

[56] Y. Du, A. Pothukuchy, J. D. Gollihar, A. Nourani, B. Li

and A. D. Ellington, Angew. Chemie - Int. Ed., 2017, 56, 992–

996, doi: 10.1002/anie.201609108.

[57] S. Cai, C. Jung, S. Bhadra and A. D. Ellington, Anal.



Chem., 2018, 90, 8290–8294, doi:

10.1021/acs.analchem.8b02062.

[58] R. S. Lasken and M. Egholm, Trends Biotechnol., 2003,

21, 531–535, doi: 10.1016/j.tibtech.2003.09.010.

[59] V. V Demidov, Expert Rev. Mol. Diagn, 2002, 2, 542–548,

doi: 10.1586/14737159.2.6.542.

[60] E. J. Cho, L. Yanq, M. Lew and A. D. Ellinqton, J. Am.

Chem. Soc., 2005, 127, 2022–2023, doi: 10.1093/nar/gkx480.

[61] S. C. Chapin and P. S. Doyle, Anal. Chem., 2011, 83,

7179–7185, doi: 10.1021/ac201618k.

[62] M. Xu, J. Ye, D. Yang, A. A. Abdullah AL-maskri, H. Hu,

C. Jung, S. Cai and S. Zeng, Anal. Chim. Acta, 2019, 1077,

208–215, doi: 10.1016/j.aca.2019.05.028.

[63] B. Wang, S. J. Potter, Y. Lin, A. L. Cunningham, D. E.

Dwyer, Y. Su, X. Ma, Y. Hou, N. K. Saksena, W. E. T. Al and

J. C. L. I. N. M. Icrobiol, J. Clin. Microbiol., 2005, 43, 2339–

2344, http://hdl.handle.net/10453/3749.

[64] Y. Gusev, J. Sparkowski, A. Raghunathan, H. Ferguson,

J. Montano, N. Bogdan, B. Schweitzer, S. Wiltshire, S. F.

Kingsmore, W. Maltzman and V. Wheeler, Am. J. Pathol.,

2001, 159, 63–69, doi: 10.1016/S0002-9440(10)61674-4.

[65] J. Zhao, S. Tang, J. Storhoff, S. Marla, Y. P. Bao, X. Wang,

E. Y. Wong, V. Ragupathy, Z. Ye and I. K. Hewlett, BMC

Biotechnol., 2010, 10, 16–19, doi: 10.1186/1472-6750-10-74.

[66] N. Thanthrige-Don, O. Lung, T. Furukawa-Stoffer, C.

Buchanan, T. Joseph, D. L. Godson, J. Gilleard, T. Alexander

and A. Ambagala, J. Virol. Methods, 2018, 261, 51–62, doi:

10.1016/j.jviromet.2018.08.010.

[67] W. H. Long, H. S. Xiao, X. M. Gu, Q. H. Zhang, H. J.

Yang, G. P. Zhao and J. H. Liu, J. Virol. Methods, 2004, 121,

57–63, doi: 10.1016/j.jviromet.2004.06.016.

[68] Q. Chen, J. Li, Z. Deng, W. Xiong, Q. Wang and Y. Q.

Hu, Intervirology, 2010, 53, 95–104, doi: 10.1159/000264199.

[69] X. Guo, P. Geng, Q. Wang, B. Cao and B. Liu, J.

Microbiol. Biotechnol., 2014, 24, 1145–1454, doi:

https://doi.org/10.4014/jmb.1404.04024.

[70] A. Kunze, M. Dilcher, A. Abd El Wahed, F. Hufert, R.

Niessner and M. Seidel, Anal. Chem., 2016, 88, 898–905, doi:

10.1021/acs.analchem.5b03540.

[71] C. Escadafal, J. T. Paweska, A. Grobbelaar, C. le Roux,

M. Bouloy, P. Patel, A. Teichmann, O. Donoso-Mantke and M.

Niedrig, PLoS Negl. Trop. Dis., 2013, 7, 1–7, doi:

10.1371/journal.pntd.0002244.

[72] S. Duan, G. Li, X. Li, C. Chen, T. Yan, F. Qiu, L. Zhao,

M. Zhao, L. Wang, Z. Feng and X. Ma, Biotechniques, 2018,

64, 211–217, doi: 10.2144/btn-2018-2010.

[73] C. Chen, X. na Li, G. xia Li, L. Zhao, S. xia Duan, T. fei

Yan, Z. shan Feng and X. jun Ma, Diagn. Microbiol. Infect. Dis., 2018, 90, 90–95, doi:

10.1016/j.diagmicrobio.2017.10.005.

[74] J. Qi, X. Li, Y. Zhang, X. Shen, G. Song, J. Pan, T. Fan,

R. Wang, L. Li and X. Ma, Arch. Virol., 2019, 164, 1843–1850,

doi: 10.1007/s00705-019-04230-z.

[75] G. Xue, S. Li, W. Zhang, B. Du, J. Cui, C. Yan, L. Huang,

L. Chen, L. Zhao, Y. Sun, N. Li, H. Zhao, Y. Feng, S. Liu, Q.

Zhang, X. Xie, D. Liu, H. Yao and J. Yuan, Anal. Chem., 2020,

92, 9699–9705, doi: 10.1021/acs.analchem.0c01032.

[76] E. S. Lander, Cell, 2016, 164, 18–28, doi:

10.1016/j.cell.2015.12.041.

[77] P. Hsu, E. S. Lander and F. Zhang, Cell, 2014, 157, 1262–

1278, doi: 10.1016/j.cell.2014.05.010.

[78] J. S. Gootenberg, O. O. Abudayyeh, J. W. Lee, P.

Esslezbichler, A. J. Dy, J. Joung, V. Verdine, N. Donghia, N.

M. Daringer, C. A. Freije, C. Myhrvold, R. P. Bhattacharya, J.

Livny, A. Regev, E. V. Koonin, B. T. Hung, P. C. Sabeti, J. J.

Collins and F. Zhang, Science, 2017, 356, 438–442, doi:

10.1126/science.aam9321.

[79] J. S. Gootenberg, O. O. Abudayyeh, M. J. Kellner, J. J.

Collins and F. Zhang, Science, 2018, 360, 439–444, doi:

10.1126/science.aaq0179..

[80] L. Guo, X. Sun, X. Wang, C. Liang, H. Jiang, Q. Gao, M.

Dai, B. Qu, S. Fang, Y. Mao, Y. Chen, G. Feng, Q. Gu, R. R.

Wang, Q. Zhou and W. Li, Cell Discov., 2020, 6, 4–7, doi:

10.1038/s41421-020-0174-y.

[81] L. Curti, F. Pereyra-Bonnet and C. A. Gimenez, bioRxiv, ,

doi: 10.1101/2020.02.29.971127.

[82] H. C. Metsky, C. A. Freije, T.-S. F. Kosoko-Thoroddsen,

P. C. Sabeti and C. Myhrvold, bioRxiv, 2020,

2020.02.26.967026, doi: 10.1101/2020.02.26.967026.

[83] L. A. Layqah and S. Eissa, Michrochimica Acta, 2019,

186, 1–10, doi: 10.1007/s00604-019-3345-5.

[84] L. Liu, L. Tian, G. Zhao, Y. Huang, Q. Wei and W. Cao,

Anal. Chim. Acta, 2017, 986, 138–144, doi:

10.1016/j.aca.2017.07.025.

[85] M. Alafeef, K. Dighe, P. Moitra and D. Pan, ACS Nano,

2020, 14, 17028–17045, doi: 10.1021/acsnano.0c06392.

[86] Y. Wei, H. Ma, X. Ren, C. Ding, H. Wang, X. Sun, B. Du,

Y. Zhang and Q. Wei, Sensors Actuators, B Chem., 2018, 256,

504–511, doi: 10.1016/j.snb.2017.10.136.

[87] A. Tahamtan and A. Ardebili, Expert Rev. Mol. Diagn.,

2020, 20, 453–454, doi: 10.1080/14737159.2020.1757437.

[88] T. Hou, W. Zeng, M. Yang, W. Chen, L. Ren, J. Ai, J. Wu,

Y. Liao, X. Gou, Y. Li, X. Wang, H. Su, B. Gu, J. Wang and T.

Xu, PLoS Pathog., 2020, 16, 1–12, doi:

10.1371/journal.ppat.1008705.

[89] K. Green, S. Graziadio, P. Turner, T. Fanshawe and J.

Allen, Oxford COVID-19 Evid. Serv., 2020, 1–7.

[90] R. Yelagandula, A. Bykov, A. Vogt, R. Heinen, E. Özkan,

M. M. Strobl, J. C. Baar, K. Uzunova, B. Hajdusits, E. Suljic,

A. Kurtovic-kozaric, S. Izetbegovic, J. Schaefer, A. Zoufaly, T.

Seitz, M. Födinger, F. Allerberger, A. Stark, L. Cochella and

U. Elling, medRxiv., Nov. 30, 2020, doi:

10.1101/2020.10.28.20217778.

[91] J. R. Choi, Front. Chem., 2020, 8, article 517 doi:

10.3389/fchem.2020.00517.

[92] G. Lisby, Mol. Biol., 1999, 12, 75–99, doi:

10.1385/MB:12:1:75.

[93] G. Csako, Clin. Chim. Acta 363, 2006, 363, 6–31, doi:

10.1016/j.cccn.2005.07.009.

[94] N. P. Gerry, N. E. Witowski, J. Day, R. P. Hammer, G.

Barany and F. Barany, J. Mol. Biol., 1999, 292, 251–262, doi:

10.1006/jmbi.1999.3063.

[95] M. Vincent, Y. Xu and H. Kong, EMBO Rep., 2004, 5,

795–800, doi: 10.1038/sj.embor.7400200.

[96] M. Xu, J. Ye, D. Yang, A. A. Abdullah AL-maskri, H. Hu,

C. Jung, S. Cai and S. Zeng, Anal. Chim. Acta, 2019, 1077,



208–215, doi: 10.1016/j.aca.2019.05.028.

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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and institutional affiliations.

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Identification of Human Coronavirus: An Overview on ...Fig. 1 Basic structure of coronavirus with detection methods showing either positive or negative report. particles with surface