1
Background
The COVID-19 pandemic, caused
by the SARS-CoV-2 virus, has led to more than three million confirmed
cases, with over 211,000 deaths globally, as of April 27, 2020. The
living and working conditions of billions of people worldwide have
been significantly disrupted due to different forms of social distancing
and lockdowns in many cities. The world economy has been remarkably
weakened as a result of business shutdowns and major restrictions
on travel. Widespread availability of accurate and rapid testing procedures
is extremely valuable in unraveling the complex dynamics involved
in SARS-CoV-2 infection and immunity. To this end, laboratories, universities,
and companies around the world have been racing to develop and produce
critically needed test kits.
One of the many challenges for
containing the spread of COVID-19 is the ability to identify asymptomatic
cases that result in spreading of the virus to close contacts. A study
of the passengers on a Diamond Princess Cruise ship forced into temporary
quarantine from an early outbreak of COVID-19, estimated the asymptomatic
proportion (among all infected cases) at 17.9% (95%CrI: 15.5–20.2%).
1
Therefore, the actual number of SARS-CoV-2-infected
individuals may be much higher than currently accounted for based
on positive test results.
2
Having accurate,
convenient, and rapid testing for widespread deployment can aid in
eliminating the silent spread of COVID-19 by asymptomatic viral carriers.
Because COVID-19 exhibits a range of clinical manifestations, from
mild flu-like symptoms to life-threatening conditions, it is important
to have efficient testing during the early stages of infection to
identify COVID-19 patients from those with other illnesses. This avoids
unnecessary quarantines of negative individuals and the spread of
infection by positive individuals. Early diagnosis permits physicians
to provide prompt intervention for patients who are at higher risk
for developing more serious complications from COVID-19 illness. More
complicated diagnostic testing based on viral genomic sequencing is
an essential tool for determining the rate and degree of mutational
variability associated with SARS-CoV-2 and for identifying newly emerging
strains of the virus for more effective vaccine development. Until
a commercial vaccine becomes available, it is important to identify
individuals who have been infected with SARS-CoV-2, with or without
accompanying symptoms, and who have developed antiviral immunity.
This allows for additional analyses of strength and durability of
immunity across general populations.
Commercially available
COVID-19 tests currently fall into two major categories. The first
category includes molecular assays for detection of SARS-CoV-2 viral
RNA using polymerase chain reaction (PCR)-based techniques or nucleic
acid hybridization-related strategies. The second category includes
serological and immunological assays that largely rely on detecting
antibodies produced by individuals as a result of exposure to the
virus or on detection of antigenic proteins in infected individuals.
It is important to reemphasize that these two categories of tests
serve overlapping purposes in management of the COVID-19 pandemic.
Testing for SARS-CoV-2 viral RNA identifies SARS-CoV-2-infected individuals
during the acute phase of infection. Serological testing subsequently
identifies individuals who have developed antibodies to the virus
and could be potential convalescent plasma donors. It also furthers
the ability to conduct contact tracing and monitor the immune status
of individuals and groups over time.
3
Timely diagnosis, effective treatment, and future prevention are
key to management of COVID-19. The current race to develop cost-effective
point-of-contact test kits and efficient laboratory techniques for
confirmation of SARS-CoV-2 infection has fueled a new frontier of
diagnostic innovation. In order to assist ongoing innovation, we developed
this report to provide an overview of current COVID-19 diagnostic
trends and strategies based on conventional and novel methodologies,
including CRISPR. It includes current information on test kits and
developers as well as data on COVID-19 diagnostic trends based on
journal publication information extracted from the CAS content collections
and MEDLINE.
2
Molecular Assays for Detection
of Viral Nucleic Acids
SARS-CoV-2 is a single-stranded, positive-sense RNA virus, and since its entire genetic
sequence was uploaded
to the Global Initiative on Sharing All Influenza Data (GISAID) platform
on January 10, 2020, companies and research groups in a matter of
weeks have developed a range of diagnostic kits for COVID-19. The
availability of sequence data has facilitated the design of primers
and probes needed for the development of SARS-CoV-2-specific testing.
4
2.1
Reverse Transcription-Polymerase
Chain Reaction (RT-PCR)
RT-PCR relies on its ability to amplify
a tiny amount of viral genetic material in a sample and is considered
to be the gold standard for identification of SARS-CoV-2 virus. Currently,
RT-PCR tests for COVID-19 generally use samples collected from the
upper respiratory system using swabs. In addition, a few studies have
also been done using serum, stool, or ocular secretions.
5−7
Recently, the Rutgers Clinical Genomics Laboratory developed an
RT-PCR assay (TaqPath COVID-19 Combo kit) that uses self-collected
saliva samples, which is quicker and less painful than other sample
collection methods, lowers the risks to healthcare providers, and
may enable higher volume testing.
8,9
As illustrated
in Figure 1
, RT-PCR
starts with laboratory conversion of viral genomic RNA into DNA by
RNA-dependent DNA polymerase (reverse transcriptase). This reaction
relies on small DNA sequence primers designed to specifically recognize
complementary sequences on the RNA viral genome and the reverse transcriptase
to generate a short complementary DNA copy (cDNA) of the viral RNA.
In real-time RT-PCR, the amplification of DNA is monitored in real
time as the PCR reaction progresses. This is done using a fluorescent
dye or a sequence-specific DNA probe labeled with a fluorescent molecule
and a quencher molecule, as in the case of TaqMan assays. An automated
system then repeats the amplification process for about 40 cycles
until the viral cDNA can be detected, usually by a fluorescent or
electrical signal.
10
Figure 1
Reverse transcription-polymerase
chain reaction (RT-PCR). The RT-PCR creates a cDNA copy of a specific
segment of the viral RNA, which is converted to dsDNA that is exponentially
amplified.
RT-PCR has traditionally been
carried out as a one-step or a two-step procedure. One-step real-time
RT-PCR uses a single tube containing the necessary primers to run
the entire RT-PCR reaction. Two-step real-time RT-PCR involves more
than one tube to run the separate reverse transcription and amplification
reactions, but offers greater flexibility and higher sensitivity than
the one-step procedure. It requires less starting material and allows
for the ability to stock cDNA for quantification of multiple targets.
11
The one-step procedure is generally the preferred
approach for detection of SARS-CoV-2 because it is quick to set up
and involves limited sample handling and reduced bench time, decreasing
chances for pipetting errors and cross-contamination between the RT
and real-time PCR steps.
To date, the majority of molecular
diagnostic tests have utilized the real-time RT-PCR technology targeting
different SARS-CoV-2 genomic regions, including the ORF1b or ORF8
regions, and the nucleocapsid (N), spike (S) protein, RNA-dependent
RNA polymerase (RdRP), or envelope (E) genes (see Table 1
in section 2.5
and Supporting Information Table S1).
12−15
Table 1
Examples of Molecular
Diagnostic Tests Used to Detect Viral Genetic Material in SARS-CoV-2
test name
test type
manufacturer/organization name
sample source
gene or
region detected
test result time/additional
information
throughput information
EUAb
country of approval
ID NOW COVID-19
isothermal nucleic
acid amplification technology
Abbott Diagnostics Scarborough,
Inc.
nasal, nasopharyngeal and throat swabs
RdRP gene
positive results <5 min and negative results in 13 min
1 sample/run
US
FDA 3/27/2020
United
States
iAMP COVID-19 detection kit
real-time RT isothermal amplification test
Atila
BioSystems, Inc. D11
nasal, nasopharyngeal, and oropharyngeal swabs
ORF1ab and/or N gene
results <1.5 h
high throughputa
US FDA 4/10/2020
United States
BioFire COVID-19 test
multiplex real-time RT-PCR
BioFire Defense, LLC
nasopharyngeal swabs
ORF1ab and ORF8
results in ∼45 min
94 samples/run
US FDA 3/23/2020
United States
CDC 2019- Novel
Coronavirus Real-Time RT-PCR Diagnostic Panel
real-time
RT-PCR
CDC-US
nasopharyngeal or oropharyngeal
aspirates/washes/swabs and bronchoalveolar lavage fluid, tracheal
aspirates, sputum
N gene
human RNase P gene used as control
264
samples/day
US FDA 2/4/2020
United States
Xpert Xpress SARS-CoV-2 test
real-time RT-PCR
Cepheid
nasopharyngeal, nasal, and midturbinate
swabs
N2 and E genes
results in ∼45 min with <1 min of hands-on time
high throughputa
US FDA 3/20/2020
Australia,
Canada, Singapore, United States
CRISPR-based
tests for SARS-CoV-2
CRISPR-based lateral flow assay isothermal amplification
Cepheid Sherlock Biosciences
respiratory samples
viral RNA
combines Sherlock’s Cas12 and
Cas13 enzymes for nucleic acid detection with Cepheid’s GeneXpert
test-processing instruments
US FDA 3/20/2020
United States
VitaPCR SARS-CoV-2 assay
real-time PCR
Credo Diagnostics Biomedical Pte
Ltd.
nasal and oropharyngeal swabs
viral
RNA
results in 20 min with 1 min of hands-on time
2000 samples/day
CE mark 3/2020
Singapore
LYRA SARS-CoV-2 assay
real-time RT-PCR
Diagnostic Hybrids, Inc. Quidel Corporation
nasopharyngeal
and oropharyngeal swabs
pp1ab
results
in <75 min after extraction
US FDA 3/17/2020
Canada
SARS-CoV-2 assay
real-time RT-PCR
Diagnostic Molecular
Laboratory – Northwestern Medicine
nasopharyngeal,
oropharyngeal, nasal, and midturbinate nasal swabs and bronchoalveolar
lavage fluid
N1 and RdRP genes
results
in <1 h without manual RNA extraction
US FDA 4/2/2020
United States
Simplexa COVID-19
Direct
real-time RT-PCR
DiaSorin Molecular
LLC
nasopharyngeal swabs
OFR1ab and S
gene
results in ∼1 h with no RNA extraction
high throughputa
US FDA 3/19/2020
United States
ePlex SARS-CoV-2 test
RT-PCR
GenMark Diagnostics, Inc.
nasopharyngeal swabs
RNA
<2 min hands-on
time and results in ∼2 h
US FDA 3/19/2020
United States
Panther Fusion SARS-CoV-2 assay (Panther Fusion System)
real-time transcription-mediated amplification
Hologic
Inc.
nasopharyngeal and oropharyngeal swabs
ORF1ab regions 1 and 2
each Panther Fusion system can provide
results in <3 h and process up to
1150 coronavirus tests in 24-h period
1 sample/run
Australia 3/20/2020, US FDA 3/16/2020
Australia, United States
COVID-19 RT-PCR test
real-time RT-PCR
LabCorp Laboratory Corporation of America
nasopharyngeal
and oropharyngeal swabs/washes/aspirates and sputum, bronchoalveolar
lavage fluid
N gene
results in 2–4 days
24 samples/run
US FDA 3/16/2020
United States
ARIES SARS-CoV-2 assay
real-time RT-PCR
Luminex Corporation
nasopharyngeal swabs
ORF1ab and N gene
minimal hands-on time and an
automated workflow delivers results in ∼2 h
high throughputa
US FDA 4/3/2020
United States
SARS-CoV-2 DETECTR
CRISPR-based lateral
flow assay isothermal amplification
Mammoth Biosciences
respiratory samples
E and N genes
CRISPR Cas12a-based lateral flow assay results
in 30–40 min
high throughputa
filed for US FDA 4/16/2020
United States
Accula SARS-CoV-2 test
PCR and lateral flow
technologies
Mesa Biotech Inc.
throat
and nasal swabs
N gene
results in 30 min, the palm-sized device can be used
in physician office or patients’ home
144 tests/day
US FDA 3/23/2020
United States
MiRXES FORTITUDE KIT 2.0
real-time RT-PCR
MiRXES Pte Ltd.
nasopharyngeal swabs
viral RNA genes less prone to
mutation
results in 90 min, produces 100,000 test kits/wk
96 samples in 1 run
Singapore HSA 3/2020
Singapore
QIAstat-Dx Respiratory SARS-CoV-2 panel
multiplex real-time
RT-PCR
Qiagen GmbH
nasopharyngeal swabs
E and RdRP genes
results in ∼1 h, by differentiating
novel coronavirus from 21 other bacterial and viral respiratory pathogens
1 sample/run
US
FDA 3/30/2020
United
States
cobas SARS-CoV-2
real-time RT-PCR
Roche Molecular Systems,
Inc.
nasopharyngeal and oropharyngeal swabs
viral RNA
results in ∼3.5 h, instruments can process up to 384 results (cobas 6800 System) and
1056 results (cobas 8800 System) in 8 h
high throughputa
Australia 2/20/20, CE mark 2020, US FDA 3/12/20
Australia, Brazil, Canada, Japan, Singapore, United
States
TaqPath COVID-19 combo kit
multiplex real-time RT-PCR
Rutgers Clinical Genomics
Laboratory ThermoFisher-Applied Biosystems
oropharyngeal,
nasopharyngeal, anterior nasal, midturbinate nasal swabs and saliva
specimens
ORF1b and N and S genes
high throughputa
US FDA 3/13/2020
United States
Novel Coronavirus
(2019-nCoV) Nucleic Acid diagnostic kit (PCR-fluorescence probing)
real-time RT-PCR
Sansure Biotech Inc.
nasopharyngeal and oropharyngeal swab, serum, blood and feces
ORF1ab and N gene
results in 30 min
China NMPA 4/2020
China
STANDARD M nCoV RT detection kit
real-Time
RT-PCR
SD BIOSENSOR
oropharyngeal swabs
E and RdRP genes
results within 90 min
Korea MFDS 2/27/2020
South Korea
Allplex 2019-nCoV
assay
multiplex real-time RT-PCR
Seegene
nasopharyngeal, oropharyngeal, or anterior nasal swabs, midturbinate
and sputum specimens
E, N, and RdRP genes
results in <2 h after extraction
CE mark 2/2020, Korea MFDS 2/12/2020, US FDA 4/21/2020
Australia,
South Korea, Singapore, United States
Viracor SARS-CoV-2 assay
real-time RT-PCR
Viracor Eurofins Clinical Diagnostics
nasopharyngeal,
nasal, oropharyngeal washes/swabs and bronchoalveolar lavage fluid
N gene
results
the same day, 12–18 h from receipt
of specimen
US FDA 4/6/2020
United States
a
Information from
FDA https://www.fda.gov/media/136702/download.
b
Emergency Use Authorization
by U.S. FDA or other drug regulatory authorities.
The earliest COVID-19 RT-PCR diagnostic tests to come
on the scene included (1) COVID-19 RT-PCR (LabCorp),
16
(2) 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic
Panel [U.S. Centers for Disease Control and Prevention (CDC)],
17
(3) TaqPath COVID-19 Combo kit (ThermoFisher-Applied
Biosystems),
18
(4) Allplex 2019-nCoV Assay
(Seegene),
19
and (5) cobas SARS-CoV-2 (Roche).
20
Detailed information about these tests is provided
in Table 1
in section 2.5
and Supporting Information I.
RT-PCR tests
are constantly evolving with improved detection methods and more automated
procedures. For example, the ePlex SARS-CoV-2 test developed by GenMark
Diagnostics, Inc.
21
uses “The True
Sample-to-Answer Solution” ePlex instrument to detect SARS-CoV-2
in nasopharyngeal swabs. Each test cartridge contains reagents for
magnetic solid-phase extraction of viral RNA, cDNA amplification,
and detection combining electrowetting and GenMark’s eSensor
technology. Target DNA is mixed with ferrocene-labeled signal probes
complementary to specific targets. The target DNA hybridizes to the signal and capture
probes which are both bound to gold-plated
electrodes. The presence of a target is determined using voltammetry
which generates specific electrical signals from the ferrocene-labeled
signal probe.
Although RT-PCR is the most widely used method
for detecting SARS-CoV-2 infections, it has the disadvantage
of requiring expensive laboratory instrumentation highly skilled
laboratory personnel, and can take days to generate results. As a result,
a number of companies and laboratories around the globe are working
to further improve the efficiency and timeliness of the RT-PCR technologies
and develop various other techniques.
2.2
Isothermal
Nucleic Acid Amplification
RT-PCR requires multiple temperature
changes for each cycle, involving sophisticated thermal cycling equipment.
22
Isothermal nucleic acid amplification is an
alternative strategy that allows amplification at a constant temperature
and eliminates the need for a thermal cycler. Therefore, several methods
based on this principle have been developed.
2.2.1
Reverse
Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)
RT-LAMP has been developed as a rapid and cost-effective testing
alternative for SARS-CoV-2. As shown in Figure 2
, RT-LAMP requires a set of four primers
specific for the target gene/region to enhance the sensitivity and
combines LAMP with a reverse transcription step to allow for the detection
of RNA. The amplification product can be detected via photometry,
measuring the turbidity caused by magnesium pyrophosphate precipitate
in solution as a byproduct of amplification. The reaction can be followed
in real time either by measuring the turbidity or by fluorescence
using intercalating dyes. Since real-time RT-LAMP diagnostic testing
requires only heating and visual inspection, its simplicity and sensitivity
make it a promising candidate for virus detection.
23
Figure 2
Reverse transcription loop-mediated isothermal amplification (RT-LAMP).
Step 1: At the 3′-end of the viral RNA, reverse transcriptase
and BIP primer initiate conversion of RNA to cDNA. Step 2: At the
same end, DNA polymerase and B3 primer continue to generate the second
cDNA strand to displace and release the first cDNA strand. Step 3:
The FIP primer binds to the released cDNA strand and DNA polymerase
generates the complementary strand. Step 4: F3 primer binds to the
3′ end, and DNA polymerase then generates a new strand while
displacing the old strand. LAMP cycling produces various sized double-stranded
looped DNA structures containing alternately inverted repeats of the
target sequence as detected by a DNA indicator dye. Reagents*: Primers
and master mix containing reverse transcriptase, DNA polymerase with
strand displacing activity, dNTPs, and buffers.
As shown in Table 1
in section 2.5
,
a few of the currently available molecular assays for detecting SARS-CoV-2
utilize real-time RT-LAMP technology, such as the ID NOW COVID-19
test from Abbott Diagnostics (see Supporting Information I for details). This point-of-care
test is rapid (13 min or
less) and is used to detect SARS-CoV-2 viral RNA in upper respiratory
swabs, but is limited to one sample per run.
24,25
The RT-LAMP test prepared by Zhang et al. uses reverse transcriptase
(WarmStart RTx from BioLabs) to convert the viral RNA to cDNA, which
is subsequently amplified by the DNA-dependent DNA polymerase (Bst2.0
Warmstart) for rapid colorimetric detection with a DNA-binding dye
(SYTO-9, ThermoFisher S34854).
26
The enzyme
is a unique in silico designed RNA-directed DNA polymerase coupled
with a reversibly bound aptamer that inhibits RTx activity below 40
°C. It is particularly well-suited for use in LAMP. The colorimetric
LAMP has been shown to be effective at detecting viral RNA in cell
lysates at levels of approximately 480 RNA copies, without interference,
providing an alternative to RT-PCR for rapid and simple detection
of SARS-CoV-2 RNA.
2.2.2
Transcription-Mediated
Amplification (TMA)
TMA is a patented single tube, isothermal
amplification technology modeled after retroviral replication which
can be used to amplify specific regions of either RNA or DNA much
more efficiently than RT-PCR.
27
It uses
a retroviral reverse transcriptase and T7 RNA polymerase and has been
used for detection of nucleic acids from multiple pathogens. On the
basis of this principle, Hologic’s Panther Fusion SARS-CoV-2
test was developed, validated, and issued Emergency Use Authorization
(EUA) by the U.S. Food and Drug Administration (FDA).
28
The Panther fusion platform is distinctive because of its
high testing throughput (up to 1000 tests in 24 h) and its capability
to simultaneously screen for other common respiratory viruses whose
symptoms overlap with COVID-19 using the same patient sample and collection
vial.
The initial step involves hybridization of the viral RNA
target to a specific capture probe and an additional oligonucleotide
containing a T7 promoter primer, which are captured onto magnetic
microparticles by application of a magnetic field. Then, the captured
RNA target hybridized to the T7 promoter primer is reverse transcribed
into a complementary cDNA. The RNase H activity of the reverse transcriptase
subsequently degrades the target RNA strand from the hybrid RNA–cDNA
duplex, leaving a single-stranded cDNA, which includes the T7 promoter.
An additional primer is used to generate a double-stranded DNA, which
is subsequently transcribed into RNA amplicons by T7 RNA polymerase.
These new RNA amplicons then reenter the TMA process allowing this
exponential amplification process to generate billions of RNA amplicons
in less than 1 h. The detection process involves the use of single-stranded
nucleic acid torches that hybridize specifically to the RNA amplicon
in real time. Each torch is conjugated to a fluorophore and a quencher.
When the torch hybridizes to the RNA amplicon, the fluorophore is
able to emit a signal upon excitation.
2.2.3
CRISPR-Based
Assays
Clustered Regularly Interspaced Short Palindromic
Repeats (CRISPR) represents a family of nucleic acid sequences found
in prokaryotic organisms, such as bacteria. These sequences can be
recognized and cut by a set of bacterial enzymes, called CRISPR-associated
enzymes, exemplified by Cas9, Cas12, and Cas13. Certain enzymes in
the Cas12 and Cas13 families can be programmed to target and cut viral
RNA sequences.
29
Two companies, Mammoth
Biosciences and Sherlock Biosciences, established by the CRISPR pioneer
scientists, are independently exploring the possibility of using the
gene-editing CRISPR methodology for detection of SARS-CoV-2. The SHERLOCK
method developed by Sherlock Biosciences uses Cas13 that is capable
of excising reporter RNA sequences in response to activation by SARS-CoV-2-specific
guide RNA.
30
The DETECTR assay by Mammoth
Biosciences relies on the cleavage of reporter RNA by Cas12a to specifically
detect viral RNA sequences of the E and N genes, followed by isothermal
amplification of the target, resulting in a visual readout with a
fluorophore.
31
These CRISPR-based methods,
as depicted in Figure 3
, do not require complex instrumentation and can be read using paper
strips to detect the presence of the SARS-CoV-2 virus without loss
of sensitivity or specificity. These tests are both low-cost and can
be performed in as little as 1 h. These tests have great potential
for point-of-care diagnosis.
4,32,33
Figure 3
Two alternative
CRISPR methods for detecting viral RNA. Method A (SHERLOCK assay
30
):
RT-RPA (recombinase polymerase amplification) converts viral RNA to
dsDNA. T7 transcription generates complementary RNA from the dsDNA
template. The Cas13–tracrRNA complex binds to the target sequence,
which activates the general nuclease enzyme activity of Cas13 to cleave
the target sequence and the fluorescent RNA reporter. Method B (DETECTR
assay
31
): RT-RPA (recombinase polymerase amplification) converts viral
RNA to dsDNA. The Cas12a–tracrRNA complex binds to the target
sequence, which activates the general nuclease enzyme activity of
Cas12a to cleave the target sequence and the fluorescent RNA reporter.
2.2.4
Rolling Circle Amplification
An alternative method of isothermal nucleic acid amplification
known as rolling circle amplification (RCA) has attracted considerable
attention for nucleic acid detection, since in isothermal conditions,
it is capable of 109-fold signal amplification of each
circle within 90 min. RCA is advantageous in that it can be performed
under isothermal conditions with minimal reagents and avoids the generation
of false-positive results frequently encountered in PCR-based assays.
An efficient assay for the detection of SARS-CoV by RCA was previously
performed in both liquid and solid phases and used to test clinical
respiratory specimens.
34
This method, however,
has not been deployed for detection of SARS-CoV-2 at this point.
2.3
Nucleic Acid Hybridization Using Microarray
Microarray assays have been used for rapid high-throughput detection
of SARS-CoV nucleic acids. As shown in Figure 4
, they rely on the generation of cDNA from
viral RNA using reverse transcription and subsequent labeling of cDNA
with specific probes. The labeled cDNAs are loaded into the wells
of microarray trays containing solid-phase oligonucleotides fixed
onto their surfaces. If they hybridize, they will remain bound after
washing away the unbound DNA, thus signaling the presence of virus-specific
nucleic acid.
35
The microarray assay has
proven useful in identifying mutations associated with SARS-CoV and
has been used to detect up to 24 single nucleotide polymorphisms (SNP)
associated with mutations in the spike (S) gene of SARS-CoV with 100%
accuracy.
36
Figure 4
Nucleic acid hybridization
using microarray. Viral cDNA and reference cDNA with different fluorescent
labels are mixed and applied to the microarray wells coated with specific
DNA probes.
The ability to detect different
emergent strains of SARS-CoV-2 may become necessary as the COVID-19
pandemic evolves, and microarray assays provide a platform for rapid
detection of those strains as a result of mutational variation. Although
one of the drawbacks of microarray testing has been the high cost
generally associated with it, a nonfluorescent, low-cost, low-density
oligonucleotide array test has been developed to detect multiple coronavirus
strains with a sensitivity equal to that of individual real-time RT-PCR.
37
In addition, a portable diagnostic platform
based on the microarray chip has been used to identify nucleic acids
specific to the MERS coronavirus as well as to influenza and respiratory
syncytial viruses.
38
2.4
Amplicon-Based
Metagenomic Sequencing
This diagnostic technique for identification
of SARS-CoV-2 relies on a dual approach involving the use of amplicon-based
sequencing in addition to metagenomics sequencing. Metagenomics sequencing
is used primarily to address the background microbiome of infected
individuals. It allows for the ability to rapidly identify both the
SARS-CoV-2 virus and other pathogens contributing to secondary infections
influencing the severity of COVID-19 symptoms. Amplicon-based sequencing
of SARS-CoV-2 allows for potential contact tracing, molecular epidemiology,
and studies of viral evolution. Metagenomics approaches such as sequence-independent
single primer amplification (SISPA) provide additional checks on sequence
divergence. This dual technique is particularly relevant to SARS-CoV-2
in assessment of its rate of mutation and to detect its possible recombination
with other human coronaviruses, both of which have implications for
vaccine development and antiviral efficacy.
Amplicon and metagenomics
MinION based sequencing were used by Moore et al. (2020) to rapidly
(within 8 h) sequence the genome of SARS-CoV-2 and the other microbiome
in nasopharyngeal swabs obtained from patients with COVID-19 by the
ISARIC 4C consortium.
39
For the amplicon-based
system, the group chose 16 primer binding sites from conserved regions
in the SARS-CoV-2 genome to sequentially amplify roughly 1000 bp fragments
with an approximately 200 bp overlapping region. These primer sets
were then used to generate 30 amplicons from the cDNA, which were
subsequently sequenced using MinION.
A next-generation shotgun
metagenomics sequencing platform has been developed by Illumina with
the ability not only to detect the presence of multiple strains of
coronaviruses but also to comprehensively examine multiple pathogenic
organisms present in a complex sample. The Illumina metagenomics workflow
involves sample preparation using their TruSeq Ribo-Zero Gold rRNA
depletion kit, library preparation using TruSeq stranded total RNA,
sequencing using the Illumina benchtop sequencing system, and final
data analysis using their LRM Resequencing module or IDbyDNA Explify
Platform.
40
2.5
High-Level
Overview of Current Molecular Genetic Assays on SARS-CoV-2 Detection
While the scientific literature has enumerated many different molecular
genetic assays that have been used to detect viral nucleic acids over
the years, most recent assays developed to detect the SARS-CoV-2 virus
rely on RT-PCR and isothermal nucleic acid amplification technologies.
Because of the urgent need for assays for COVID-19 diagnosis, manufacturers,
commercial laboratories, and molecular-based laboratories can request
EUA for their tests from the FDA or other regulatory agencies for
diagnostic purposes. New tests approved by the FDA for emergency use
are based on analytic validity and developed under idealized conditions
using verified positive samples and negative controls without a requirement
for demonstrated clinical validity.
41
EUA
approval for tests from manufacturers and commercial laboratories
limits the testing to be done in clinical laboratories that have been
certified under the Clinical Laboratory Improvement Amendments of
1988 (CLIA). On the other hand, the EUA approval of tests from molecular-based
laboratories (laboratory developed tests) limits the testing to be
done at the single laboratory that developed the test and is certified
under CLIA.
Of 112 currently available molecular assays for
detecting SARS-CoV-2 (detailed in Supporting Information Table S1), 90% utilize PCR
or RT-PCR technologies, 6% utilize
isothermal amplification technologies, 2% utilize hybridization technologies,
and 2% utilize CRISPR-based technologies. Some of these tests use
high-throughput platforms or have short time to results, and therefore
are in high demand. Table 1
provides an overview of over 20 diagnostic tests that employ
molecular genetic assays and includes information on test type, developer/manufacturer,
throughput, time to results, and other related features. More molecular
genetic assay-based tests are listed in Supporting Information Table S1.
3
Serological
and Immunological Assays
While RT-PCR-based viral RNA detection
has been widely used in diagnosis of COVID-19, it cannot be used to
monitor the progress of the disease stages and cannot be applied to
broad identification of past infection and immunity.
Serological
testing is defined as an analysis of blood serum or plasma and has
been operationally expanded to include testing of saliva, sputum,
and other biological fluids for the presence of immunoglobulin M (IgM)
and immunoglobulin G (IgG) antibodies. This test plays an important
role in epidemiology and vaccine development, providing an assessment
of both short-term (days to weeks) and long-term (years or permanence)
trajectories of antibody response, as well as antibody abundance and
diversity. IgM first becomes detectable in serum after a few days
and lasts a couple of weeks upon infection and is followed by a switch
to IgG. Thus, IgM can be an indicator of early stage infection, and
IgG can be an indicator of current or prior infection. IgG may also
be used to suggest the presence of post-infection immunity. In recent
years, the sophistication and sensitivity of immunological assays
have increased not only for the detection of antibodies themselves
but also for the application of antibodies (primarily monoclonal antibodies)
to the detection of pathogen-derived antigens. These tests have a
huge potential for the epidemiology of COVID-19,
32,42−45
but test results can be impacted
by at least three situations: (1) a subset of subjects with a positive
result from molecular genetic assays for SARS-CoV-2 infection are
seronegative due to the lag in antibody production following infection,
(2) the subjects may be seropositive yet negative for molecular genetic
assay results reflecting clearance of an earlier, milder infection,
and (3) limitation in sensitivity and specificity of the assays. The
last issue is particularly important because even a small percentage
of false positive results due to low specificity (cross reaction)
may lead to misleading predictive antibody prevalence among a given
population, which may have undesirable impact on the socioeconomic
decisions and overall public confidence in the results.
46,47
The determination of SARS-CoV-2 exposure relies largely on
the detection of either IgM or IgG antibodies that are specific for
various viral antigens including, but not exclusively, the spike glycoprotein
(S1 and S2 subunits, receptor-binding domain) and nucleocapsid protein.
The methodology for these determinations includes the traditional
enzyme-linked immunosorbent assay (ELISA), immunochromatographic lateral
flow assay, neutralization bioassay, and specific chemosensors. Each
of these formats brings advantages (speed, multiplexing, automation)
and disadvantages (trained personnel, dedicated laboratories). Complementary to these
antibody-detecting methods are the rapid
antigen tests wherein antibodies are used to detect the presence of
viral antigen(s) in serological samples. Development of high-throughput
serology tests is a current focus of major diagnostic companies.
45
The FDA granted EUA status to the first serology
test, qSARS-CoV-2 IgG/IgM Rapid Test, manufactured by Cellex Inc.,
on April 1, 2020,
48
but continues to allow
clinical laboratories and commercial manufacturers to launch serology
tests without an EUA.
3.1
Enzyme-Linked Immunosorbent
Assay (ELISA)
ELISA is a microwell, plate-based assay technique
designed for detecting and quantifying substances such as peptides,
proteins, antibodies, and hormones. The test can be qualitative or
quantitative, and the time to results is typically 1–5 h. In
the case of SARS-CoV-2 as shown in Figure 5
A, the plate wells are typically coated with
a viral protein. If present, antiviral antibodies in the patient samples
will bind specifically, and the bound antibody–protein complex
can be detected with an additional tracer antibody to produce a colorimetric
or fluorescent-based readout. ELISA is speedy, has the ability to
test multiple samples, and is adaptable to automation for increased
throughput but can be variable in sensitivity and is suitable
for point-of-care determinations.
Figure 5
ELISA assays detecting antibodies (A)
or antigens (B).
3.2
Lateral
Flow Immunoassay
This test is typically a qualitative (positive
or negative) chromatographic assay that is small, portable, and used
at the point-of-care. The test is a type of rapid diagnostic test
(RDT) as the result can be obtained in 10–30 min. In practice,
fluid samples are applied to a substrate material that allows the
sample to flow past a band of immobilized viral antigen. If present,
anti-CoV antibodies are collected at the band, where, along with co-collected
tracer antibodies, a color develops to indicate the results as shown
in Figure 6
. The test
is inexpensive and requires no trained personnel, but provides only
qualitative results. When used in conjunction with symptomology, a
diagnosis of infection may be feasible. Rapid antigen tests (section 3.6
), where anti-CoV
antibodies are used in place of immobilized viral antigen, allow for
a more direct assessment of ongoing infection.
Figure 6
Lateral flow immunoassay
for detection of anti-SARS-CoV-2 antibodies. Samples move via capillary
flow on the nitrocellulose membrane. When anti-SARS-CoV-2 antibodies
are present, they bind to the labeled antigen and continue to move
until they are captured by the immobilized antihuman antibodies. The
presence of the captured antibody–antigen complex is visualized
as a colored test band. The labeled control antibodies comigrate until
they are captured at the control band.
3.3
Neutralization Assay
Neutralization assays
determine the ability of an antibody to inhibit virus infection of
cultured cells and the resulting cytopathic effects of viral replication.
For this assay, patient samples of whole blood, serum, or plasma are
diluted and added at decreasing concentrations to the cell cultures.
If neutralizing antibodies are present, their levels can be measured
by determining the threshold at which they are able to prevent viral
replication in the infected cell cultures. The time to results for
neutralization assays is typically 3–5 days, but recent advances
have reduced this to hours.
49,50
This type of testing
requires cell culture facilities, and in the case of SARS coronavirus,
Biosafety Level 3 (BSL3) laboratories. Despite these limitations,
determination of neutralizing antibodies is important in the short
term for the therapeutic application of convalescent plasma and, in
the long term, for vaccine development.
3.4
Luminescent
Immunoassay
Luminescent immunoassays comprise methods that
lower the limits of detection for antibody-based reagents. Generally
they involve chemiluminescence and fluorescence. Cai et al. have developed a peptide-based
magnetic chemiluminescence
enzyme immunoassay for diagnosis of COVID-19, and Diazyme Laboratories,
Inc. (San Diego, California) announced the availability of two new
fully automated serological tests for SARS-CoV-2 that are run on the
fully automated Diazyme DZ-lite 3000 Plus chemiluminescence analyzer.
51,52
3.5
Biosensor Test
Biosensor tests rely on
converting the specific interaction of biomolecules into a measurable
readout via optical, electrical, enzymatic, and other methods. Surface
plasmon resonance (SPR) is a technique that measures interference
with incident light at a solid boundary due to local disturbances
such as the adsorption of antibody or antigen. An SPR-based biosensor
was developed for the diagnosis of SARS using coronaviral surface
antigen (SCVme) anchored onto a gold substrate.
53
The SPR chip had a lower limit of detection of 200 ng/mL
for anti-SCVme antibodies within 10 min. Most recently, PathSensors
Inc. announced a CANARY biosensor to detect the novel SARS coronavirus.
This platform utilizes a cell-based immunosensor that couples capture
of the virus with signal amplification to provide a result in 3–5
min. The biosensor is slated to be available for research purposes
in May 2020.
54
3.6
Rapid
Antigen Test
Complementary to molecular genetic assays are
the rapid antigen tests that allow detection of viral antigens.
55,56
As shown in Figure 5
B, these tests rely on specific monoclonal antibodies to provide
a mechanism for the capture of viral antigens from an analytical sample.
These assays are not restricted to a particular format. Examples include
a colorimetric enzyme immunoassay for SARS-CoV in 2004,
57
an enhanced chemiluminescent immunoassay for
SARS-CoV in 2005,
58
and more recently a
fluorescence lateral flow assay
59
for the
detection of SARS-CoV-2 nucleocapsid protein.
3.7
High-Level
Overview of Current Serological and Immunological Assays for COVID-19
Diagnosis
Table 2
provides a collection of current available serological and
immunological assays for COVID-19 diagnosis, and a complete list of
similar assays is provided in Supporting Information Table S2.
12−15
Finally, while serological and immunological tests have a huge potential
for tracing the SARS-CoV-2 virus, most of these tests are still in
the development phase. Serological testing has been used to a limited
extent to determine infection status (in combination with molecular
genetic assays), seroprevalence, and immune protection status for
healthcare workers. This testing is impacted by the fact that
only a subset of patients with positive molecular genetic assay results
for SARS-CoV-2 infections are seropositive, due to the delay in antibody
production. There is currently no clear or strong evidence correlating
seropositivity with immune protection.
Table 2
Examples
of Serological and Immunological Tests Used to Detect Viral Protein
or Antibodies to SARS-CoV-2 Virus
test name
test type
manufacturer/organization name
sample source
Ig or protein
detected
test result time/additional
information
EUAa
country
of approval
m2000 SARS-CoV-2 assay
chemiluminescent microparticle
immunoassay
Abbott Core Laboratory
serum/plasma/whole
blood
IgG
runs up to 100–200 tests/h
United States
COVID-19 IgG/IgM LF
lateral flow immunoassay
Advagen Biotech
serum/plasma/whole blood
IgG/IgM
results in 10 min
Brazil
COVID-19 IgG/IgM Point of Care
Rapid test
lateral flow immunoassay
Aytu
Biosciences/Orient Gene Biotech
serum/plasma/whole blood
IgG/IgM
results in 2–10 min
China, United States
COVID-19 IgM/IgG rapid test
lateral flow
immunoassay
BioMedomics
serum/plasma/whole
blood
IgG/IgM
results in 15 min
United States
IgG antibody test kit for novel coronavirus 2019-nCoV
magnetic particle-based chemiluminescence immunoassay
Bioscience (Chongqing) Diagnostic Technology Co., Ltd.
serum
IgG
NMPA
China
One-Step COVID-2019 test
lateral flow immunoassay
Celer Biotechnologia
serum/plasma/whole blood
IgG/IgM
results in 15 min
Brazil
qSARS-CoV-2 IgG/IgM rapid test
lateral flow immunoassay
Cellex Inc.
serum/plasma/whole blood
IgG/IgM
results in 15–20 min, antibodies specific for N protein
Australia 3/31/2020, US FDA 4/01/2020
Australia, United States
COVID-19 Ag Respi-Strip
lateral flow immunoassay (dipstick)
Coris Bioconcept
nasal mucus swabs
viral antigen
results in 15 min
Belgium
DPP
COVID-19 IgM/IgG system
lateral flow immunoassay
Chembio Diagnostics
serum/plasma/whole blood
IgG/IgM
results in 15 min
US FDA 4/14/2020
Brazil
DEIASL019/020 SARS-CoV-2 IgG ELISA kit
ELISA
Creative Diagnostics
serum/plasma
IgG/IgM
IgG specific for N protein
United States
OnSite COVID-19 IgG/IgM
rapid test
lateral flow immunoassay
CTK
Biotech Inc. (USA)
serum/plasma/whole blood
IgG/IgM
results in 10 min
Australia
Diazyme DZ-Lite SARS-CoV-2 IgG/IgM
test
luminescent immunoassay
Diazyme Laboratories
blood sample
IgG/IgM
EUA not required
United States
KT-1033 EDI Novel Coronavirus COVID-19 ELISA kit
ELISA
Epitope Diagnostics
serum
IgG/IgM
United States
VivaDiag COVID-19 IgM/IgG rapid test
lateral
flow immunoassay
Everest Links Pte Ltd.
serum/plasma/whole blood
IgG/IgM
results
in 15 min
Singapore
COVID-19 IgG/IgM rapid test cassette
lateral flow immunoassay
Hangzhou Biotest Biotech
Co. Ltd.
serum/whole blood
IgG/IgM
results in 15–20 min
Australia 4/4/2020
Australia
VITROS-Immunodiagnostics Products
Anti-SARS-CoV-2 total reagent pack
ELISA
Ortho-Clinical Diagnostics
blood serum/plasma
IgG/IgM
cannot distinguish between IgG/IgM
US FDA 4/14/2020
United States
SARS-CoV-2 rapid test
lateral flow immunoassay
PharmACT
whole blood/serum
IgG/IgM
results in 20 min, N protein, S1 and S2 subunits used as antigens
Germany
Standard Q
COVID-19 IgM/IgG Duo
lateral flow immunoassay
SD Biosensor
serum/plasma/whole blood
IgG/IgM
results in 10 min
EUA not required
South Korea
Standard Q COVID-19 Ag
chromatographic immunoassay
SD Biosensor
nasopharyngeal swabs
viral antigen
results in 30 min
South Korea
iFLASH-SARS-CoV-2-IgG/IgM
immunoassay
Shenzhen Yhlo Biotech Company
serum/plasma/whole blood
IgG/IgM
China
MAGLUMI IgG/IgM
de 2019-nCoV (CLIA)
chemiluminescence immunoassay
Snibe Diagnostic (China)
blood serum/plasma
IgG/IgM
results in 30 min
CE mark 2/2020
Brazil
a
Emergency Use Authorization by US
FDA or other drug regulatory authorities.
4
Literature Trends and Notable
Journal Articles Related to Diagnostics for COVID-19
4.1
Trends in Literature Publications Related to Diagnostic Tests
for COVID-19
A significant research effort has been devoted
to the development of new COVID-19 diagnostic tests that are faster
and more reliable. Since the outbreak of the COVID-19 crisis, over
500 journal articles related to laboratory testing have been published. Figure 7
shows that the number
of journal articles focused on COVID-19 diagnostics has significantly
increased over the past few months, indicating a continuous research
and development effort in this area.
Figure 7
Monthly trend of journal publications
related to COVID-19 diagnostics in 2020.
Table 3
lists 10 notable
journal articles published from January 31, 2020 through April 10,
2020. These articles that highlight a variety of COVID-19 diagnostic
methods were selected based on a number of factors, including journal
impact factor and number of citations/downloads.
Table 3
Ten Notable Journal Articles Published from January 31, 2020 through
April 10, 2020 Related to COVID-19 Diagnostic Testing
title
source
organization
type of test
digital
object identifier
Correlation
of Chest CT and RT-PCR Testing in Coronavirus Disease 2019 (COVID-19)
in China: A Report of 1014 Cases
Radiology (February 26, 2020)
Department of Radiology, Tongji
Hospital, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, Hubei, China
chest CT and RT-PCR
https://dx.doi. org/10.1148/radiol.2020200642
Development and Clinical Application of a Rapid IgM-IgG
Combined Antibody Test for SARS-CoV-2 Infection Diagnosis
Journal of Medical Virology (February 27, 2020)
State Key Laboratory of Respiratory Disease, National Clinical Research
Center for Respiratory Disease, Guangzhou Institute of Respiratory
Health, The First Affiliated Hospital of Guangzhou Medical University,
Guangzhou, China
lateral flow immunoassay, POC
https://dx.doi.
org/10.1002/jmv.25727
Fecal Specimen
Diagnosis 2019 Novel Coronavirus-Infected Pneumonia
Journal of Medical Virology (March 3, 2020)
Department of Respiration,
Jinhua Municipal Central Hospital, Jinhua Hospital of Zhejiang University,
China
RT-PCR
https://dx.doi. org/10.1002/jmv.25742
Improved Molecular Diagnosis of COVID-19
by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time
Reverse Transcription-Polymerase Chain Reaction Assay Validated in
Vitro and with Clinical Specimens
Journal of
Clinical Microbiology (March 4, 2020)
The University of Hong Kong-Shenzhen Hospital,
Shenzhen, Guangdong, The University of Hong Kong, Pokfulam, Hong Kong,
and Hainan Medical University, Haikou, Hainan, all in China
real-time RT-PCR
https://dx.doi. org/10.1128/JCM.00310-20
Molecular Diagnosis of a Novel Coronavirus
(2019-nCoV) Causing an Outbreak of Pneumonia
Clinical Chemistry (April 1, 2020), Vol. 66 (4), pp. 549–555
School of Public Health,
LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China
real-time RT-PCR
https://dx.doi. org/10.1093/clinchem/hvaa029
Comparative Performance of SARS-CoV-2 Detection Assays Using Seven Different
Primer/Probe Sets and One Assay Kit
Journal
of Clinical Microbiology (April 8, 2020)
Department of Laboratory Medicine, University
of Washington, Seattle, WA, and Vaccine and Infectious Diseases Division,
Fred Hutchinson Cancer Research Center, Seattle, WA, USA
real-time RT-PCR
https://dx.doi. org/10.1128/JCM.00557-20
Analytical Sensitivity and Efficiency Comparisons
of SARS-CoV-2 qRT-PCR Assays
medRxiv (2020), 1–18
Department of Epidemiology of Microbial Diseases,
Yale School of Public Health, New Haven, CT, USA
qRT-PCR
https://dx.doi. org/10.1101/2020.03.30.20048108
ddPCR: A More Sensitive and Accurate Tool for SARS-CoV-2 Detection in Low Viral Load
Specimens
medRxiv (2020), 1–24
State Key Laboratory of Virology, Modern Virology
Research Center, College of Life Sciences, Wuhan University, Wuhan,
China
droplet digital PCR
https://dx.doi.
org/10.1101/2020.02.29.20029439
Evaluating the Accuracy of Different Respiratory Specimens in the
Laboratory Diagnosis and Monitoring the Viral Shedding of 2019-nCoV
Infections
medRxiv (2020) 1–17
Shenzhen Key Laboratory
of Pathogen and Immunity, National Clinical Research Center for Infectious
Disease, State Key Discipline of Infectious Disease, Shenzhen Third
People’s Hospital, Second Hospital Affiliated to Southern University
of Science and Technology, Shenzhen, China
qRT-PCR
https://dx.doi. org/10.1101/2020.02.11.20021493
Rapid Detection of 2019 Novel Coronavirus SARS-CoV-2 Using a CRISPR-Based DETECTR
Lateral Flow
Assay
medRxiv (2020), 1–27
Mammoth Biosciences, Inc.;
University of California, San Francisco, California, USA
CRISPR, lateral flow, RT-LAMP
https://dx.doi.
org/10.1101/2020.03.06.20032334
Many of the diagnostic methods discussed are based on patented technologies developed
over the past 20
years and scientific journal publications related to diagnosis of
viral infections. SARS-CoV-2 belongs to the genus Betacoronavirus
that also includes SARS-CoV and MERS-CoV. Therefore, diagnostic studies
on those closely related coronaviruses may shed light on the ongoing
efforts to develop effective diagnostic assays for SARS-CoV-2 infection.
More information about patents related to detection of SARS-CoV and
MERS-CoV is provided in Supporting Information II.
4.2
SARS-CoV-2 Sequences in
Journal Publications with Application for Diagnosis
Nucleic
acid and amino acid sequences in CAS REGISTRY obtained from SARS-CoV-2-related
journals were analyzed for potential applications in diagnostics. Table 4
lists selected journal
articles and corresponding numbers of sequences related to SARS-CoV-2.
These sequence records include primers and probes, gene and genomic
sequences, protein domains and epitopes, and viral protein sequences
with potential use for strain differentiation. Additional sequence
information from SARS-CoV-related patents is provided in the Supporting Information
Table S3.
Table 4
Journal Articles with SARS-CoV-2-Related Sequences for Potential Applications
in Diagnostics
publication date
title
journal
nucleic
acids
proteins
2020
Nanopore Target Sequencing for Accurate
and Comprehensive Detection of SARS-CoV-2 and Other Respiratory Viruses
medRxiv
40 primers
2020
A Single and Two-Stage,
Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19)
at Home, Clinic, and Points of Entry
ChemRxiv
6 COVID-19 LAMP primers
2020
Transmission and
Clinical Characteristics of Coronavirus Disease 2019 in 104-Outside-Wuhan
Patients, China
medRxiv
6 primers and probes
2020
A Pneumonia Outbreak
Associated with a New Coronavirus of Probable Bat Origin
Nature
4
50
2020
A New Coronavirus Associated with Human
Respiratory Disease in China
Nature
1
10
2020
A Sequence Homology and Bioinformatic Approach Can Predict
Candidate Targets for Immune Responses to SARS-CoV-2
Cell Host & Microbe
51
2020
Comparative
Analysis of Primer-Probe Sets for the Laboratory Confirmation of SARS-CoV-2
bioRxiv
20 primers, 10 probes
2020
Spike Protein Binding Prediction with Neutralizing
Antibodies of SARS-CoV-2
bioRxiv
3
2020
SARS-CoV-2 Proteome Microarray for Mapping COVID-19 Antibody Interactions at
Amino Acid Resolution
bioRxiv
11
2020
Evaluation of Recombinant Nucleocapsid and Spike Proteins for Serological
Diagnosis of Novel Coronavirus Disease 2019 (COVID-19)
medRxiv
12 primers
2020
RBD Mutations from Circulating SARS-CoV-2 Strains Enhance the Structure Stability
and Infectivity of the Spike Protein
bioRxiv
8
2020
Teicoplanin Potently Blocks the Cell Entry of 2019-nCoV
bioRxiv
14
134
2020
Differential Antibody Recognition
by SARS-CoV-2 and SARS-CoV Spike Protein
Receptor Binding Domains: Mechanistic Insights and Implications for
the Design of Diagnostics and Therapeutics
bioRxiv
7
2020
A Proposal of an Alternative Primer for the ARTIC Network’s
Multiplex PCR to Improve Coverage of SARS-CoV-2 Genome Sequencing
bioRxiv
2
2020
First 12 Patients with Coronavirus Disease 2019 (COVID-19) in the
United States
medRxiv
12
109
5
Summary and Perspectives
While the past few months have witnessed rapid progress in diagnostic
kit development for COVID-19, the race continues to develop even more
efficient laboratory techniques and cost-effective, point-of-care
test kits that can be deployed in mass quantities. This report provides
a broad survey of molecular genetic assays, and serological and immunological tests
for
identification of COVID-19 infection. While RT-PCR has been the dominant
technique for detection of viral RNA, other nucleic acid assays including
isothermal amplification assays, hybridization microarray assays,
amplicon-based metagenomics sequencing, and the cutting-edge CRISPR-related
technologies are also under development or have resulted in approved
tests.
60
The efficiency of such testing
has also been significantly improved. This report also provides trend
analysis of journal articles related to COVID-19 diagnostic techniques.
The number of FDA EUA-approved tests available for COVID-19 diagnosis
keeps growing, but the many new tests are still in various
stages of development. Ultrarapid test kits and point-of-care tests
are a major focus of development in order to speed up the response
time for treatment and eliminate the need for elaborate laboratory
equipment and waiting time involved with testing in approved laboratories.
The urgent need for accurate and rapid diagnosis of SARS-CoV-2
infection remains critical as global healthcare systems continue to
operate during the course of the COVID-19 pandemic. In particular,
serological and immunological testing of infected asymptomatic and
symptomatic individuals, and their close contacts, is expected to
be in high demand. In addition to its role complementary to molecular
genetic testing to confirm suspected cases, this type of testing would
provide valuable information about the course and degree of immune
response as well as the durability of immunity in both infected individuals
and participants in vaccine clinical trials.
The results from these tests may assist epidemiological assessment
and can be used to manage the return to normal activities. However,
many questions regarding serological tests remain to be addressed,
including their degree of sensitivity and specificity. Finally, it
remains to be confirmed that the presence of antibodies against SARS-CoV-2
indeed correlates with immunity to the virus.
In summary, significant
progress has been made in the development of diagnostic tests despite
all the remaining questions and challenges. Ongoing global efforts
are working to communicate and facilitate new diagnostic assay development
and worldwide test kit delivery. To promote more accurate and faster
diagnostic solutions, a number of organizations are supporting these
efforts by inviting assay developers to submit their test products
for independent evaluation or by providing huge investments for greater
collaboration. As similar initiatives and knowledge sharing become
available, including collaborative technological advancements, it
is likely that the COVID-19 diagnostic market will continue to thrive
well into the future.