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American Journal of  
Life Science and Innovation (AJLSI)

Laboratory Diagnosis of  Novel Human Coronavirus (SARS-CoV-2) Infections-A Review
Aswathy C Ashok1*, R. Harish1

Volume 1 Issue 2, Year 2022
ISSN: 2833-1397 (Online)

DOI: https://doi.org/10.54536/ajlsi.v1i2.1012
https://journals.e-palli.com/home/index.php/ajlsi

Article Information ABSTRACT

Received: November 26, 2022

Accepted: November 29, 2022

Published: December 03, 2022

COVID-19 is a pandemic, highly contagious infectious disease caused by the Severe Acute 
Respiratory Syndrome Corona virus-2 (SARS-CoV-2). The World Health Organization has 
declared the ongoing outbreak a global public health emergency. This disease has spread 
rapidly and affected millions of  people worldwide. Currently, there are no specific clinical 
signs or symptoms of  SARS that can be used to differentiate it from other causes of  com-
munity- or hospital-acquired viral pneumonia. Accurate diagnosis of  cases holds the key to 
managing any pandemic through identification, isolation, and treatment of  patients while 
defining the epidemiology of  the pathogen. Because an increasing number of  asymptom-
atic symptomatic individuals must be tested for COVID-19, a safe and efficient screening 
system is required. The diagnosis of  suspected cases is presently confirmed by nucleic acid 
assays with real-time PCR using respiratory samples. On the other side, serological tests are 
comparatively easier to perform, but their utility may be limited by their ease of  performance 
and the fact that antibodies appear later in the disease course. This review is aimed at sum-
marizing the currently available information on different methods used for screening and 
diagnosing COVID-19 infections.

Keywords

SARS-CoV2, RTPCR, 
Immunological Rapid Assay

INTRODUCTION
Severe acute respiratory syndrome coronavirus (SARS-
CoV-2) is a novel virus that caused the first major 
pandemic disease in the family Coronaviridae. Many 
coronavirus infections in the past 20 years were not 
regarded as highly pathogenic to human beings until the 
outbreaks of  SARS (Severe Acute Respiratory Syndrome) 
and MERS (Middle East Respiratory Syndrome) (Zhong 
et al., 2003; Drosten et al., 2003; Fouchier et al., 2003). At 
the end of  2019, the China Office of  the WHO (World 
Health Organization) reported a cluster of  pneumonia 
cases in Wuhan City, China, and the causative pathogen 
was identified as Novel Coronavirus (nCoV 2019); the 
WHO named this disease COVID-19 (Wu et al., 2020; 
Zhou et al., 2020; Wang et al., 2020).
COVID-19 is an extremely infectious disease. 
Coronaviruses are enveloped RNA viruses belonging 
to the Coronaviridae family and the order Nidovirales, 
which contain approximately 27–32 kilobytes of  positive-
sense single-stranded RNA. This subfamily consists 
of  four genera: Alpha coronavirus, Beta coronavirus, 
Gamma coronavirus, and Delta coronavirus, on the 
basis of  their phylogenetic relationships and genomic 
structures. These subfamilies are broadly distributed 
and cause infections in humans and other mammals. 
Alpha and beta coronaviruses infect only mammals. 
Gamma and delta coronaviruses infect birds, but some 
of  them can also infect mammals. Although the source 
of  the beta coronavirus 2019 SARS Cov-2 is unknown, 
initial cases have been linked to the South Hunan 
seafood market. The infected people may have severe 
symptoms in the respiratory and digestive organs. Like 
other coronaviruses, the SARS-CoV-2 has at least six 

open reading frames (ORFs) and many other accessory 
genes. There are two open reading frames (ORFs) at 
the 5’ terminal two-thirds of  the genome, ORFs1 and 
ORF2. These ORF encode two polyproteins, namely 
pp1a and pp1ab, which are further cleaved into 11 and 
16 proteins, respectively. Nucleocapsid (N), membrane 
protein (M), envelope protein (E), and spike (S) are 
among the structural proteins found at 3’ terminals. In 
the case of  COVID-19, the spike protein appears to be 
the primary protein interacting with host cells. Hence, 
the spike protein is likely the protein to which antibodies 
are raised, but this is not clear at this time. These viruses 
also contain some accessory proteins, which aid in 
virus replication. The S gene aids SARS-Cov-2 in host 
specificity and receptor binding, and some virion may 
also contain hemagglutinin esterase (HE) protein. (Cui, 
J., et al., 2019; Huang C et al.,2020 and Miller et al.,2016) 
Clinical manifestations of  COVID-19 infection include 
fever and cough as primary clinical manifestations, as well 
as shortness of  breath and myalgia. Some patients have 
serious complications such as acute respiratory distress 
syndrome (ARDS) and cytokine storm, which may lead to 
death. (Malik et al., 2020). It has the capacity for human-
to-human transmission. The lack of  awareness in hospital 
infection control and international air travel facilitated the 
rapid global dissemination of  this agent.
The collection of  appropriate specimens is very crucial 
for the detection of  most of  the infected cases of  
COVID-19. Nasopharyngeal swabs are typically collected, 
but we may miss the detection in some cases; therefore, 
lower respiratory tract specimens such as sputum and 
bronchoalveolar lavage (BAL) may be an alternative 
choice. Therefore, there is an urgent need to have an 

1 Department of  Biosciences Mar Thoma College, Thiruvalla, Kerala, India
* Corresponding author’s e-mail: aswathycashok5@gmail.com

https://doi.org/10.54536/ajlsi.v1i2.1012
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accurate, rapid, readily available, and reliable diagnostic 
test for SARS-CoV-2 infection. Various immunological, 
nucleic acid, and amplification diagnostic tests have 
been developed and are widely available to date. Various 
integrated point-of-care molecular devices are currently 
under development, and some are available to provide 
accurate and fast diagnostic services for SARS-CoV-2 
infections. (Loeffelholz et al., 2020).
In view of  the present crisis of  the COVID-19 pandemic, 
fast and reliable testing strategies are imperative. In this 
review, we attempt to learn more about the current 
diagnostic methods for SARS and Cov-2 infections. 
The different methods used for screening and sample 
collection will also be discussed.

Screening and Specimen collection of  patients with 
COVID-19 pneumonia.
Accurate diagnoses of  cases hold the key to managing 
any pandemic through identification, isolation, and 
treatment of  patients while defining the epidemiology 
of  the pathogen. Because an increasing number of  
asymptomatic symptomatic individuals must be tested 
for COVID-19, a safe and efficient screening system is 
required. Presently, no specific clinical signs or symptoms 
of  COVID-19 can be used to differentiate it from other 
causes of  community- or hospital-acquired pneumonia. 
In order to decide if  a patient should be tested, 
WHO published case definitions for surveillance but 
encouraged countries to adapt these depending on their 
local epidemiological situation and other factors. (WHO 
2020). A suspect case is defined as (i) a patient with acute 
respiratory illness (fever and at least one sign or symptom 
of  respiratory disease, such as cough or shortness of  
breath) and a history of  travel to or residence in a location 
reporting community transmission of  COVID-19 disease 
within 14 days of  the onset of  symptoms (ii) a patient 
with severe acute respiratory illness (fever and at least 
one sign or symptom of  respiratory disease, e.g., cough, 
shortness of  breath, and requiring hospitalization) and in 
the absence of  an alternative diagnosis that fully explains 
the clinical presentation. As per the WHO guidelines, a 
“probable case” is a suspect case for whom testing for 
the SARS-CoV-2 is inconclusive or for whom testing 
could not be performed for any reason. WHO further 
defines a “contact” as a person who experienced any 
one of  the following exposures during the 2 days 
before and the 14 days after the onset of  symptoms of  
a probable or confirmed case: (i) face-to-face contact 
with a probable or confirmed case within 1 meter and 
for more than 15 min. (ii) direct physical contact with 
a probable or confirmed case. (iii) direct care for a 
patient with probable or confirmed COVID-19 disease 
without using proper personal protective equipment. For 
confirmed asymptomatic cases, the period of  contact is 
measured as the 2 days before through the 14 days after 
the date on which the sample was taken, which led to 
confirmation. (Venter, M., & Richter, 2020). Specific 
and real-time diagnostic tests should be performed not 

only for the identification of  potential cases but also 
for contacts who need to be quarantined and guided 
on epidemiological questions around the infection. 
Selection of  the relevant specimen and knowledge of  
the incubation period, viremia, and shedding period 
are important criteria in diagnosing individual cases 
and defining transmissibility to inform the extent of  
isolation periods for patients. Nucleic acid testing (reverse 
transcriptase PCR) is recommended for the diagnosis 
of  acute cases. Serological assays have an important 
role in answering epidemiological questions, including 
determining the exposure rate and accessing community 
spreads, but are not relevant for accurate diagnoses of  
acute cases. Only laboratory confirmation can be used to 
make an etiological diagnosis and differentiate atypical 
pneumonia from other causes. Proper collection of  
samples is the most important step in the laboratory 
diagnosis of  infectious diseases. A specimen that is not 
collected correctly may lead to a negative result.
As per the Center for Disease Control and Prevention 
(CDC) recommendations, the upper respiratory 
specimen should be collected for RT-PCR-based testing 
of  COVID-19, and especially the nasopharyngeal 
exudate is the preferred choice (CDC, 2020). Within 
a week from the onset of  symptoms, patients with 
COVID-19 usually possess high viral loads in their 
upper and lower respiratory tracts. (Zhou et al., 2020). 
A nasopharyngeal swab and/or an oropharyngeal swab 
are often recommended for screening or diagnosing 
early infections. Nasopharyngeal swabs usually reach 
the correct area to be tested in the nasal cavity (Wang et 
al., 2020) reported that oropharyngeal swabs were used 
much more frequently than nasal swabs in China during 
the current COVID-19 outbreak; however, the SARS-
CoV-2 RNA was detected in only 32% of  oropharyngeal 
swabs, which was significantly lower than the level in 
nasal swabs (63%). for the late detection and monitoring 
of  patients with severe COVID-19 pneumonia. Ideally, 
sputum sampling or bronchoalveolar lavage (BAL) has 
been used for collecting lower respiratory tract specimens 
as they have yielded the highest viral loads for the 
diagnosis of  COVID-19 (Li et al., 2020; Yu et al., 2020). 
A recent study revealed that samples of  bronchoalveolar 
lavage fluid yielded the highest SARS-CoV-2 RNA rate, 
although this study did not compare or evaluate results 
from nasopharyngeal swabs (Wang et al., 2020). Patients 
who present with severe pneumonia and acute respiratory 
distress syndrome may require emergent intubation as 
well as respiratory isolation in a negative-pressure room. 
If  possible, a lower respiratory tract sputum specimen 
should be collected during the intubation procedure. 
Alternatively, sputum and/or bronchoalveolar lavage fluid 
specimens may be collected after intubation (Pan et al., 
2020). Collecting a respiratory swab specimen may carry a 
theoretical risk of  transmitting SARS-CoV-2, particularly 
if  chances of  airborne transmission are demonstrated. 
Currently, biosafety level (BSL)-2 conditions are 
recommended for the handling of  specimens for 

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molecular testing. While collecting specimens, health care 
professionals should follow WHO infection prevention 
and control guidelines and wear personal protective 
equipment (PPE) such as gloves, gowns, eye protection, 
and N95 masks (WHO 2020). All specimens collected 
for laboratory investigations are regarded as potentially 
infectious.
Testing of  specimens from multiple sites (e.g., upper and 
lower respiratory tracts) may improve the sensitivity of  the 
RT-PCR and reduce false-negative test results, especially 
during the second week of  illness. For asymptomatic 
patients and patients with mild symptoms, the collection 
of  both nasopharyngeal swabs and oropharyngeal swabs 
is recommended; these should be placed together in 
the same viral transport medium (VTM) to increase the 
sensitivity (WHO 2020, CDC 2020). Additional clinical 
specimens can be collected as the COVID-19 virus has 
been detected in blood, urine, and stool. In the case of  
deceased patients, the collection of  autopsy material, 
including lung tissue, is also considered. (Kaijin et al., 2020). 
As serological assays become available retrospectively in 
recovered patients, paired serum (acute and convalescent) 
can be useful in defining cases (Zhang et al., 2020; ShiX et 
al., 2005; Ding Y. et al., 2004). Several studies reported the 
detection of  higher viral loads in older patients. 
However, the viral load did not positively correlate 
with disease severity (Tsang et al., 2020; Pung et al., 
2020; Wang et al., 2020). Since SARS-CoV-2 and most 
other respiratory viruses are RNA-based, care should 
be taken to select extraction and inactivation protocols 
that will not damage RNA. A recent study suggests that 
heat inactivation at 56 °C for 30 min may result in false 
negatives for samples with low viral loads (Pan et al., 
2020). Specimens for virus detection should reach the 
laboratory as soon as possible after collection. Correct 
handling of  specimens during transportation is essential. 

Specimens that can be delivered to the laboratory can be 
stored and shipped at 2–8 °C. When there is likely to be 
a delay in specimens reaching the laboratory, the use of  
viral transport medium (VTM) is strongly recommended 
in the laboratory biosafety guidance related to the novel 
coronavirus (2019-nCoV). (Guidance on regulations for 
the transport of  infectious substances, 2019–2020) The 
primary container should be sealed with a screw cap. The 
container should be made of  plastic that has a low risk 
of  breakage. Patient information should be recorded on 
the specimen container using two or more identifiers 
(e.g., name, patient number), along with the necessary 
information for testing requests. 
The outer surface of  the primary container should 
be disinfected using appropriate disinfectants, such 
as 70% ethanol. The container should be packed in a 
zipper bag and placed in a secondary container before 
transportation. The secondary container should be sealed 
and shock-resistant, and it should be labelled to indicate 
that it contains infectious substances. When transporting 
specimens to a laboratory within the same institution, 
the specimens should be transported in person; the 
pneumatic tube system should not be used. A separate 
route should be used for transportation. 
The personnel transporting the specimen should be 
trained in spill decontamination procedures in case of  
specimen leakage. Specimens may be frozen to -20°C or, 
ideally, -70°C and shipped on dry ice if  further delays are 
expected. It is important to avoid repeated freezing and 
thawing of  specimens (Kwon et al., 2020; Korea Center 
for Disease Control and Prevention, 2020; CDC, 2020). 
WHO documents a summary of  the optimum sample 
collection procedures and storage, which are similar to 
those for influenza. The specimen collection and storage 
temperature protocol to investigate emerging acute 
respiratory diseases is summarized in Table 1.

Table 1: The specimen collection and storage protocol to investigate non-seasonal influenza and other emerging 
acute respiratory diseases
Specimen types Collection material Storage temperature 
Nasopharyngeal and oropharyngeal swabs Dacron or polyester flocked swabs      2-8°C 
Sputum  Sterile container      2-8°C 
Bronchoalveolar lavage Sterile container      2-8°C 
Nasopharyngeal or nasal aspirates or washes Sterile container      2-8°C 
Tissue from biopsy or autopsy, including lung tissue Sterile container with saline or VTM      2-8°C
Serum Serum separator tubes      2-8°C 
Whole blood  Collection tube      2-8°C 
Stool Stool container       2-8°C 
Urine Urine collection container      2-8°C 

Surveillance and Sampling Strategies
Individual case definition mechanisms (2.1), as 
mentioned above, help choose whether a patient should 
be tested or not. However, a pandemic like COVID-19 
necessitates robust surveillance systems at local and 
global levels, which are required to strengthen the 

effective implementation of  control measures. The aim 
of  these surveillance systems is to limit the spread of  
disease, enable public health authorities to manage the 
risk, and thereby enable economic and social activity 
to resume to the extent possible. Surveillance is also 
necessary to predict the longer-term trends of  COVID-19 

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transmission and the changes ahead (WHO 2020). A 
“case sample” is considered a subset of  individuals 
from a larger population. Sampling is simply stated as 
selecting a portion of  the infected population that will 
actually collect evidence from the research area under 
surveillance. Samples are used to make inferences about 
populations. (Landreneau, K. J., et al., 2009).
There are two types of  sampling methods employed in 
epidemiological surveillance:

Probability Sampling
This includes some form of  random selection in choosing 
the elements. Greater confidence can be placed in the 
representativeness of  probability samples. This type 
of  sampling involves a selection process in which each 
element in the population has an equal and independent 
chance of  being selected.

• Simple random sampling: in a simple random sample, 
every member of  the population has an   equal chance of  
being selected.

• Stratified random sampling: this sampling method is 
appropriate when the population has mixed characteristics 
(like age, demography, etc.) and you want to ensure that 
every characteristic is proportionally represented in the 
sample.

• Cluster sampling: cluster sampling also involves 
dividing the population into subgroups, but each 
subgroup should have similar characteristics to the whole 
sample. Instead of  sampling    individuals from each 
subgroup, entire subgroups are randomly selected.

• Systematic Sampling: This is similar to simple random 
sampling, but it is usually slightly easier to conduct. Every 
member of  the population is listed with a number, but 
instead of  randomly generating numbers, individuals are 
chosen at regular intervals for sampling.

Non-probability Sampling
The elements that make up the sample are selected by 
nonrandom methods. This type of  sampling is less likely 
than probability sampling to produce representative 
samples. Even though this is true, researchers can and 
do use non-probability samples. The three main methods 
are:

• Convenience Sampling: A convenience sample 
includes the individuals who happen to be   most 
accessible to the researcher.

• Quota sampling is primarily motivated by ease of  
access. Instead of  the researcher choosing participants 
and directly contacting them, people volunteer themselves 
(e.g., by responding to a public online survey).

• Purposive Sampling: This type of  sampling involves 
the researcher using their judgement to select a sample 
that is most useful for the purposes of  the research.
Any COVID-19 surveillance system placed should be 
geographically comprehensive and include all people 
and communities at risk. Surveillance for vulnerable 
or high-risk populations should be enhanced. This will 
require a combination of  surveillance systems, including 

contact tracing, in the entire health care system, at the 
community level, as well as in closed residential settings 
and for vulnerable groups. Surveillance at the primary 
care level is needed to detect cases and clusters in the 
community. Where possible, testing facilities are made 
available at primary care clinics. A complementary 
option is to establish dedicated COVID-19 community 
testing facilities. Patients with probable and confirmed 
COVID-19 cases are notified within 24 hours of  
identification. Fast data reporting and analysis are critical 
to detecting new cases and clusters. Therefore, only the 
minimum number of  data variables are to be collected 
(e.g., age, sex, date of  illness onset, date of  sample taken, 
test result, location of  testing site, etc.). Data reporting 
to local or national public health authorities is done 
on a daily basis. Patients with probable or confirmed 
COVID-19 diagnoses in hospitals are notified within 
24 hours of  identification. All COVID-19 deaths are 
reported within 24 hours of  the death. The minimum 
essential data from hospital settings includes: age, sex/
gender, and place of  residence; date of  onset; date of  
sample collection; date of  admission; laboratory test 
result; severity on admission: admitted to the intensive 
care unit (ICU); treated with ventilation, if  the case is a 
health care worker; outcome (discharge or death); etc. 
Existing sentinel surveillance of  influenza-like illness (ILI) 
or acute respiratory infections (ARI) is useful to monitor 
trends in community transmission of  the COVID-19 
virus and to ensure that other priority respiratory diseases 
are being detected. Integration of  COVID-19 with the 
Global Influenza Surveillance and Response System 
(GISRS) is described in operational considerations for 
COVID-19 surveillance using GISRS. Virologic sentinel 
surveillance of  COVID-19 is conducted using clinical 
specimens obtained through sentinel surveillance of  ILI, 
ARI, and SARI (Severe Acute Respiratory Infection). 
Integrated epidemiological and virological surveillance 
will play a significant role in monitoring the spread 
and evolution of  COVID-19 virus, understanding the 
cocirculation of  COVID-19 virus with influenza and 
other respiratory viruses, and subsequent interpretation 
of  respiratory epidemiological and disease observations 
in relation to COVID-19, as well as supporting the update 
of  diagnostic tests. Infections in health workers should, at 
a minimum, be systematically integrated into the national 
surveillance system. Dedicated enhanced surveillance for 
some high-risk groups is necessary to ensure the prompt 
detection of  cases and clusters, faster than through 
primary care or hospital-based surveillance. People who 
live in closed environments, such as prisons, or residential 
facilities, such as retirement communities or care homes 
for persons with disabilities, can be especially vulnerable 
because they may not be able to seek help themselves. 
Vulnerable groups may also live in settings where the 
probability of  transmission is higher than in the general 
population or have health conditions or predisposing 
factors that increase their risk of  severe illness. Enhanced 
surveillance includes the use of  active case finding, such as 

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through daily screening of  signs and symptoms, including 
daily temperature monitoring, and daily zero reporting 
for all individuals in high-risk groups under surveillance.
While surveillance systems will typically capture the 
number of  COVID-19 cases, it is also important to collect 
information on the total number of  laboratory tests 
conducted for the COVID-19 virus. Data on the number 
of  tests conducted for SARS-CoV-2 are collected from all 
relevant laboratories. Knowing the testing denominator 
can indicate the level of  surveillance activity, and the 
proportion of  positive tests can indicate the intensity of  
transmission among symptomatic individuals. Presently, 
reverse transcriptase polymerase chain reaction (RT-
PCR) testing (explained in 2.3) is the most common and 
reliable laboratory diagnostic method. If  other diagnostic 
methods are used, the number of  tests conducted and 
cases confirmed by different laboratory diagnostic 
methods need to be recorded.
It is widely accepted that a large fraction of  COVID-19 
cases goes undetected. However, this is subject to 
significant ascertainment bias because tests are typically 
ordered only from symptomatic cases, whereas a large 
proportion of  infected people may show little to no 
symptoms (Mizumoto et al., 2020). Non-symptomatic 
infections can still shed the SARS-CoV-2 virus and 
are therefore detectable by RT-PCR-based tests. It is 
therefore possible to test randomly selected individuals 
to estimate the true disease prevalence in a population. 
Recent technical advances have enabled high-throughput 
PCR, in which multiple samples are pooled into one 
tube. Combining probes from several individuals and 
testing them together reduces the total amount of  testing 
needed. This method is known as “pooling” or “group 
testing.” The main idea is that when samples from several 
people are mixed together and tested, the test will report 
negative when everyone is healthy and positive when at 
least one is positive (Abdalhamid et al., 2020). The other 
ideas include using ten-fold fewer tests (Verdun et al., 
2021) and clearing 20 times the number of  people from 
isolation with the same number of  tests (Gollier and 
Gossner, 2020). 
However, their efficiency is highly dependent on the 
frequency of  positive samples, which varies significantly 
across regions and even within regions as testing criteria 
and conditions change. Two possible optimized pooling 
strategies are currently employed for diagnostic SARS-
CoV-2 testing on large scales; both address dynamic 
conditions. In the first, an estimate of  the target frequency 
determines the initial pool size, and any subsequent pools 
found positive are re-pooled at half  size and tested again. 
The second method is a simpler approach of  optimized 
one-time pooling followed by individual tests on positive 
pools. These strategies are convenient, and they offer 
a significant reduction in the number of  materials, 
equipment, and time needed to test large numbers of  
samples. (Shani et al., 2020). On the other hand, a pool 
testing strategy could potentially increase worldwide 
testing capacity many times over, thus boosting a 

country’s capacity to test mildly to asymptomatically 
affected individuals. This strategy proposes that instead 
of  individually testing patients with low clinical suspicion 
of  SARS-CoV-2 infections, samples are pooled together 
in what is called a “minipool” and then tested together, 
running a single RT-PCR for all the unified samples. 
Preliminary results showed that there is no dilution and 
no decrease in test sensitivity when minipools of  five 
samples each are used. Since the RT-PCR looks directly 
at the viral RNA, a negative result in a pool test is reliable. 
Thus, the infection was discarded in all the patients 
included in the pooled sample.

Nucleic Acid Amplification Assay
SARS-CoV-2 is a single-stranded, positive-sense RNA 
virus. The availability of  sequence data has facilitated the 
design of  primers and probes needed for the development 
of  SARS-CoV-2-specific testing. Routine confirmation 
of  cases of  COVID-19 is based on the detection of  
unique sequences of  virus RNA by real-time reverse 
transcription polymerase chain reaction (RT-PCR), with 
confirmation by nucleic acid sequencing when necessary. 
The majority of  molecular diagnostic tests have utilized 
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. 
The most widely used method of  COVID-19 diagnostics 
is a reverse transcription quantitative polymerase chain 
reaction (RT-qPCR) assay to detect the presence of  SARS-
CoV-2 RNA in patient samples, typically nasopharyngeal 
swabs. RNA extraction is a major bottleneck in current 
COVID-19 testing. (Shen et al., 2020; Smyrlaki et al., 
2020). Because of  its high sensitivity and specificity, 
polymerase chain reaction (PCR) is regarded as the gold 
standard test for the molecular diagnosis of  viral and 
bacterial infections. Isothermal nucleic acid amplification, 
including transcription-mediated amplification and 
CRISPR-based methodologies, is considered a promising 
alternative assay due to its fundamental advantage in 
quick procedure time at constant temperature without 
thermocycler operations. As such, real-time reverse 
transcriptase-PCR (RT-PCR) is of  great interest today 
for the detection of  SARS-CoV-2 due to its benefits as 
a specific and simple qualitative assay. Furthermore, real-
time RT-PCR has sufficient sensitivity to aid us in early 
infection diagnosis. Therefore, the “criterion-referenced” 
real-time RT-PCR assay can be considered as the main 
method to be applied to detect the causative agent of  
COVID-19, i.e., SARS-CoV-2 (Carter et al., 2020; Shen et 
al., 2020; Wan et al., 2016; Noh et al., 2017; etc.).

Reverse Transcription-Polymerase Chain Reaction 
(RT-PCR)
In acute respiratory infections, RT-PCR is routinely used 
to detect causative viruses from respiratory secretions 
in nucleic acid testing assays. The real-time reverse 

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transcription (PCR) method is one of  the best and most 
accurate laboratory methods for detecting, tracking, 
and studying the coronavirus. Real-time RT-PCR is a 
method by which the presence of  specific target genetic 
material can be detected (Sethuraman et al., 2020). This 
reaction relies on small DNA sequence primers designed 
to specifically recognize complementary sequences on 
the viral RNA 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 labelled with a fluorescent 
molecule until the viral cDNA can be detected. 
Coronaviruses have a number of  molecular targets within 
their positive-sense, single-stranded RNA genome that 
can be used for PCR assays (Corman et al., 2020). 
These include genes encoding structural proteins, 
including envelope glycoproteins spike (S), envelope (E), 
transmembrane (M), helicase (Hel), and nucleocapsid (N) 
(Chan et al., 2020). In addition to the genes that encode 
structural proteins, there are species-specific accessory 
genes that are required for viral replication, like RNA-
dependent RNA polymerase (RdRp), hemagglutinin 
esterase (HE), and open reading frames ORF1a and 
ORF1b (Corman et al., 2020; Lan et al., 2020). The assay 
includes at least two molecular targets to avoid potential 
cross-reaction with other endemic coronaviruses as well 
as potential genetic drift of  SARS-CoV-2. In the United 
States, the CDC recommends two nucleocapsid protein 
targets (N1 and N2) (Holshue et al., 2020), while the 
WHO recommends first-line screening with an E gene 
assay followed by a confirmatory assay using the RdRp 
gene (Corman et al., 2020). RT-PCR has traditionally been 
carried out as a one-step or 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. (VanGuilder, H. D., et al., 
2008; Wong et al., 2005). 
Positive test results from a single sample must be 
confirmed by a repeat test detecting a different region 
of  the SARS-CoV-2 genome on the same sample. If  
possible, another repeat sample should also be tested to 
exclude false positive results due to amplicon carryover. 
Since the viral load in nasopharyngeal aspirate usually 
peaks on the 10th day after the onset of  symptoms, 
suspected SARS cases must have the tests repeated as 
the disease evolves to avoid false-negative results. Stool 
specimens should be sent for testing on a regular basis, 
as a high percentage of  patients develop diarrhoea and 
shed virus during the second week of  illness. Viral load 
determination of  nasopharyngeal specimens or serum 
upon presentation might have clinical value as it is an 
important prognostic factor. Any treatment regimen 
would benefit from long-term monitoring of  viral load. 

(Cheng et al., 2004; Peiris et al., 2003; Chan K. et al., 
2004; Chu et al., 2020; etc.). Apart from sensitivity issues, 
RT-PCR has some other drawbacks, such as possible 
biological safety hazards that may occur during transport 
and sample processing, nucleic acid extraction, and the 
requirement of  sophisticated laboratory equipment like 
biosafety cabinets. Technical expertise, along with sample 
transportation, which is inevitable, makes the overall 
process time-consuming. All these drawbacks could make 
the process less useful in case of  a health emergency or 
global outbreak situation. Moreover, in PCR, we are able 
to detect not only the target virus, but it can also perform 
codetection of  several other respiratory viruses, which 
leads to an increase in false positive or negative results 
(Cho et al., 2014).

Isothermal nucleic acid amplification
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. Isothermal amplification techniques 
are conducted at a single temperature and do not need 
specialized laboratory equipment to provide similar 
analytical sensitivities to PCR. isothermal amplification 
techniques can be multiplexed during the amplification 
or readout stage. 
This is done by using polymeric beads encoded with 
unique optical signatures like organic fluorescent 
molecules for each gene. Multiplexing increases the 
amount of  information gained from a single test and 
improves clinical sensitivity and specificity. Loop-
Mediated Isothermal Amplification (LAMP) is a 
relatively new molecular amplification point-of-care 
technique that is widely used for COVID-19 diagnosis. 
The technique is based on the synthesis of  target DNA 
at a constant temperature of  60–650C using a specially 
designed primer and DNA polymerase that has strand 
displacement activity instead of  heat denaturation as in 
other PCR techniques. 
This novel technique can amplify any genomic material 
with high efficiency and in a shorter amount of  time. RT-
LAMP has been developed as a rapid and cost-effective 
testing alternative for SARS-CoV-2. It requires a set 
of  four primers specific for the target gene or region 
to enhance the sensitivity and combines LAMP with a 
reverse transcription step to allow for the detection of  
RNA. Photometry can be used to detect the amplification 
product by measuring the turbidity caused by magnesium 
pyrophosphate precipitate in the 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. The RT-LAMP test uses reverse 
transcriptase to convert the viral RNA to cDNA, which 

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is subsequently amplified by the DNA-dependent DNA 
polymerase for rapid colorimetric detection with a DNA-
binding dye.
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. (Notomi et al., 2000; Thai et al., 2004). Transcription-
Mediated Amplification (TMA) is a patented single-tube, 
isothermal amplification technology modelled after 
retroviral replication that can be used to amplify specific 
regions of  either RNA or DNA much more efficiently 
than RT-PCR. It uses a retroviral reverse transcriptase and 
T7 RNA polymerase and has been used for the detection 
of  nucleic acids from multiple pathogens. 
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. Then, the 
captured RNA target hybridized to the T7 promoter 
primer is reverse transcribed into a complementary 
cDNA. The RNase 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 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 to generate billions of  RNA amplicons. 
The detection process involves the use of  single-stranded 
nucleic acid pyrotechnics that hybridize specifically to the 
RNA amplicon in real time. Each torch is conjugated to 
a fluorophore and a quencher. When the torch hybridizes 
with the RNA amplicon, the fluorophore is able to emit 
a signal upon excitation. (Kacian et al., 1999) 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. Certain 
enzymes in these families can be programmed to target 
and cut viral RNA sequences. 
The CRISPR-based methods 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 an hour. 
These tests have great potential for point-of-care 
diagnosis. (Zhang et al., 2020; Broughton et al., 2020). The 
rolling circle amplification (RCA) method has attracted 
considerable attention in nucleic acid determination. 
In isothermal conditions, RCA is capable of  a 109-fold 
signal amplification of  each circle within 90 minutes. An 
efficient assay for the detection of  SARS-CoV-2 by RCA 
has been set up in both liquid and solid phases and has 
yielded preliminary results on a small number of  clinical 
respiratory specimens. The main advantage of  RCA is 

that it can be performed under isothermal conditions 
with minimal reagents and avoids the generation of  
false-positive results, which are frequently encountered 
in PCR-based assays. (Chapin et al., 2011, Xu et al., 2019, 
Wang et al., 2005, etc.).

Cartridge Based Nucleic Acid Amplification test 
(CB-NAAT) and True NAAT
Unlike traditional RT-PCR tests, the sample preparation in 
CB-NAAT tests is automated, and the results are available 
within half  an hour. The test uses nose- or throat-swab 
samples, which are collected from patients and dipped in 
a solution that inactivates the virus. A few drops of  the 
solution are then placed on a cartridge. On inserting this 
cartridge into a machine, a preprogrammed reaction is 
initiated, which extracts the nucleic acids or the genetic 
material from the samples. This has to be followed by RT-
PCR. The purified nucleic acid is added into a microtube 
containing freeze-dried RT-PCR reagents and allowed 
to stand for a minute, then applied to a microchip and 
then inserted into another machine, where the reverse 
transcription and PCR take place. The advantage of  this 
test is that it is quick and portable. This allows the easy 
setup of  mobile testing centers or kiosks in containment 
zones instead of  having to transport samples to labs. 
True NAT is an indigenously developed, portable version 
of  CB-NAAT, also known as the GeneXpert test. Both 
of  these tests were originally designed to screen for 
tuberculosis.

IMMUNOLOGICAL ASSAYS 
Immunological tests measure the antibodies generated 
by the host body’s immune response against the virus 
infection (antibody test) or the proteins of  COVID-19 
virus present in the respiratory specimens (antigen test). 
When a virus enters the human body, it triggers an 
immune response that results in the production of  an 
antibody against the virus; detecting such an antibody 
in an infected person is extremely useful regardless of  
whether the person has symptoms. Antibody tests are 
blood-based tests that can be used to identify whether 
people have been exposed to a particular pathogen. The 
serum includes antibodies to specific components of  
pathogens, called antigens. 
These antigens are recognized by the immune system as 
foreign and are targeted by the immune response. These 
types of  tests are often used in viral infections to see if  
the patient has an immune response to a pathogen of  
interest, such as SARS-CoV-2. The role of  serological 
assays to detect IgG, IgA, or IgM anti-SARSCoV-2 
antibodies in serum, plasma, or capillary blood provides 
a clear picture of  the outbreak size in each country and 
helps to assess the degree of  immunization. (Okba et al., 
2020). Serological testing for COVID-19 is particularly 
attractive because of  the relatively short time to diagnosis 
and the ability to test for an active immune response 
against the virus. Serological tests have variable sensitivity 
and specificity. Research has demonstrated that the spike 

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(S) and nucleocapsid (N) proteins are the primary viral 
antigens against which antibodies are raised. (Chan et al., 
2009; Kumar et al., 2020). These antigens are the most 
commonly used in serological tests. During infection, 
several types of  antibodies are raised against the virus. 
IgM antibodies emerge first, after 5 days of  post-
symptom onset. IgG antibodies typically emerge after 10 
days of  post-symptom onset. Many serology tests detect 
both IgG and IgM simultaneously, which increases the 
specificity of  the test. IgA antibodies may also increase 
during infection and are typically found in mucous. While 
serological tests are now widely available, the correlates 
of  immunity are still poorly understood. 
The presence of  antibodies only indicates a previous 
SARS-CoV-2 infection. The results of  serological tests 
can then be used to estimate the true spread of  the 
virus through a population, even if  individuals were 
asymptomatic or were never diagnosed. The presence of  
antibodies does not indicate that an individual is protected 
from reinfection since there is limited understanding of  
the levels and persistence of  antibodies necessary for 
protective immunity. Therefore, serological tests cannot 
inform an individual of  their immunity to reinfection 
(Yu et al., 2020). However, the test results may also help 
in choosing convalescent plasma, which can be used as 
a promising treatment option for COVID-19-infected 
individuals.

Antibody Tests
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 the 
spike glycoprotein (S1 and S2 subunits) and nucleocapsid 
protein. The methodology for these determinations 
includes the traditional enzyme-linked immunosorbent 
assay (ELISA), immunochromatographic lateral flow 
assay, neutralization bioassay, and specific luminescent 
immunoassays. Each of  these formats brings advantages 
(speed, multiplexing, automation) and disadvantages 
(trained personnel, dedicated laboratory requirements, 
etc.). Rapid antigen tests, which use antibodies to detect 
the presence of  viral antigen(s) in serological samples, 
are complementary to these. Major diagnostic companies 
are currently focusing on the development of  high-
throughput serology tests.

• Enzyme-linked immunosorbent assays (ELISA): 
a faster serological test performed in a laboratory that 
provides a readout of  antigen-antibody interactions.
Essentially, patient antibodies are “sandwiched” between 
the viral protein of  interest and reporter antibodies so that 
any active patient antibodies are detected. A serological 
assay was performed using an ELISA kit that was 
developed for detecting IgM or IgG antibodies against the 
N proteins of  SARS-CoV-2. For IgM detection, ELISA 
plates were previously coated with mouse and anti-human 
antibodies. This test can be qualitative or quantitative and 
is generally a lab-based test that is obtainable within a few 
hours. These tests usually use whole blood, plasma, or 

serum samples from patients. The test relies on a plate 
that is coated with a viral protein of  interest, such as 
spike protein. Patient samples are then incubated with 
the protein, and if  the patient has antibodies to the viral 
protein, they bind together. The bound antibody-protein 
complex can then be detected with another wash of  
antibodies that produce a colored or fluorescent-based 
readout. (Liu et al., 2020). 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.

• Lateral flow assays (LFAs): Lateral flow assays 
(LFAs), also called rapid diagnostic tests (RDTs), display 
a colorimetric, qualitative readout of  the presence of  
antibodies. These are often used in point-of-care settings. 
The patient sample is passed through a membrane on 
which the target antigen is anchored. If  the sample 
contains antibodies specific to that antigen, they form a 
complex that results in a colored band on the strip. These 
are similar to pregnancy tests. This is facilitated by a 
recombinant antigen present on immunochromatographic 
paper, on which the test sera are applied, and antigen 
antibody binding is detected visually by a color change on 
a membrane. The results can be obtained within 15–20 
minutes. The test is inexpensive and requires no trained 
personnel, but it provides only qualitative results. When 
used in conjunction with rapid antigen tests, where anti-
SARS-CoV2 antibodies are used in place of  immobilized 
viral antigen, they allow for a more direct assessment of  
ongoing infection.

• Neutralization assays: determine an antibody’s 
ability to prevent virus infection of  cultured cells and 
the 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. This type of  testing 
requires cell culture facilities, and in the case of  the SARS 
coronavirus, Biosafety Level 3 (BSL3) laboratories are 
required. Despite these limitations, the 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.

• Luminescent immunoassay: This test shows 
whether a patient has antibodies to a pathogen by 
displaying a fluorescent signal when patient antibodies 
interact with virus proteins. Luminescent immunoassays 
comprise methods that lower the limits of  detection 
for antibody-based reagents. Generally, they involve 
chemiluminescence and fluorescence. For SARS-
CoV-2, two-step chemiluminescent immunoassays for 
the detection of  IgG and IgM SARS-CoV-2 antibodies 
in human serum or plasma have been demonstrated. 
Samples react with paramagnetic microparticles coated 

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with SARS-CoV-2-specific antigens (recombinant N- 
and S-proteins), and alkaline phosphate-labeled anti-
human IgG or IgM monoclonal antibodies are added 
to the reaction mixture, resulting in a chemiluminescent 
emission, measured as relative light units (RLU) by a 
photomultiplier built into the system. After 25 minutes, 
the first results were generated (Nuccetelli et al., 2020)

Antigen Detection Assays
Antigen detection with monoclonal antibodies or 
monospecific polyclonal antibodies against the protein 
was found to be a sensitive and specific test for the 
diagnosis of  SARS (Kumar et al., 2020). In a large study 
with sera collected from 317 SARS patients at different 
time points of  illness, the detection of  SARS-N antigen 
was performed using a panel of  three monoclonal 
antibodies. Over 80% of  SARS cases were discovered 
within the first 7 days of  illness. As serum antibody levels 
started to rise at day 7, the sensitivity of  the serum antigen 
assay progressively decreased to 0% at day 21 (Chan et al., 
2004, Hsueh et al., 2003).

RAPID ASSAY METHODS
Rapid tests are non-automated, primarily qualitative (but 
also quantitative in some cases), and used for in   vitro 
diagnostics. These tests can provide results within 10–30 
min, so their results are considered instant as compared to 
the molecular tests, which generally take 4–6 h. Moreover, 
these tests are user-friendly, so they won’t require any 
extensive training or expertise to operate and can be used 
either in a hospital environment or in the field without any 
difficulty. The manifestation of  the COVID-19 infection 
is highly nonspecific, including respiratory symptoms 
such as fever, cough, dyspnea, and viral pneumonia 
(Huang et al., 2020). Thus, extensive diagnostic tests 
specific to this infection are urgently required to confirm 
suspected cases, screen patients, and conduct virus 
surveillance. Rapid tests are used to diagnose patients 
without sending samples to centralized facilities, thereby 
enabling communities without laboratory infrastructure 
to detect infected patients. In this, a point-of-care (PoC) 
device, i.e., a rapid, robust, and cost-efficient device that 
can be used onsite and, in the field, and which does 
not necessarily require a trained technician to operate 
(Nguyen et al., 2018),
LAMP assays (explained in 2.3.2) in PoC devices have 
high specificity and sensitivity and are simple to perform; 
hence, soon after their initial development, they became 
an enormously popular isothermal amplification method 
in molecular biology, with applications in pathogen 
detection. LAMP uses strand-displacement polymerases 
instead of  heat denaturation to generate a single-stranded 
template; hence, it has the advantage. LAMP technology is 
proven to be more stable and more sensitive in detection 
compared to PCR (Francois et al., 2011). Lateral flow 
antigen detection (explained in 2.4.1) for SARS-CoV-2 
is another point-of-care approach under development 
for diagnosing COVID-19. In commercial lateral flow 

assays, a paper-like membrane strip is coated with gold 
nanoparticle-antibody conjugate, and capture antibodies 
are used. These assays have previously demonstrated 
reliable clinical sensitivity (57%), specificity (100%), and 
accuracy (69) for IgM and 81%, 100%, and 86% for 
IgG, respectively. A test that detected both IgM and IgG 
yielded a clinical sensitivity of  82% (Xiang et al., 2020). 
However, lateral flow assays do not directly confirm 
virus presence; instead, they provide serological evidence 
of  recent infections (Li et al., 2020). Microarray assays 
have been used for rapid, high-throughput detection of  
SARS-CoV-2 nucleic acids. They rely on the generation 
of  cDNA from viral RNA using reverse transcription and 
the subsequent labelling of  cDNA with specific probes 
loaded into the wells of  microarray trays. The microarray 
assay has proven useful in identifying mutations associated 
with SARS-CoV-2 and has been used to detect up to 24 
single nucleotide polymorphisms (SNP) associated with 
mutations in the spike (S) gene of  SARS-CoV-2 with 
100% accuracy. 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 variations. 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.
Chest CT images from patients with COVID-19 typically 
demonstrate bilateral, peripheral ground glass opacities. 
Because this chest CT imaging pattern is nonspecific 
and overlaps with other infections, the diagnostic 
value of  chest CT imaging for COVID-19 may be low 
and dependent upon other interpretations. Given the 
variability in chest imaging findings, a chest radiograph 
or CT alone is not recommended for the diagnosis of  
COVID-19. The American College of  Radiology also 
does not recommend CT for screening or as a first-line 
test for the diagnosis of  COVID-19. Lymphopenia is the 
most common laboratory finding in COVID-19 and is 
found in as many as 83% of  hospitalized patients. (Huang 
et al., 2020) Lymphopenia, neutrophilia, elevated serum 
alanine aminotransferase and aspartate aminotransferase 
levels, elevated lactate dehydrogenase, high CRP, and 
high ferritin levels may be associated with greater illness 
severity (Chen et al., 2020). Patients with critical illness had 
high plasma levels of  inflammatory markers, suggesting 
potential immune dysregulation (Wang et al., 2020). 
This review summarizes various diagnostic methods used 
for the identification of  COVID-19 infections. 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. 
A comparative account of  different diagnostic methods 

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Table 1: A comparative account of  different diagnostic methods used for detection of  COVID-19 
Method Working Principle Advantage Time 

Required
Disadvantage

Next 
generation 
sequencing 
(NGS) 

Wholegenome 
sequencing,shotgun 
metagenomics etc. 

Highly sensitive and specific, 
could provide all related 
information; can identify 
novel strain. Helps to examine 
multiple pathogenic organisms 
present in a complex sample.  

1-2 days Require high expertise 
& cost. Highly 
sophisticated Lab 
required. 

 RT-PCR Specific  primer-probe 
based detection 

Fast results, higher sensitivity, 
well established methodology in 
viral diagnostics.

3-4 hrs. High cost due to 
expensive consumables. 
Expensive lab 
equipment. RNA 
extraction is highly 
tedious and sensitive. 

Isothermal 
nucleic acid 
amplification 

Synthesis of  target 
DNA at constant  
temperature of  60 
-650C using specially 
designed primer

No thermal cycler required. 
Can be easily detected by color 
change or turbidity

1-2 hrs. Too sensitive, highly 
prone to false positive 
results due to carry-over 
or cross contamination 

LAMP More than two sets of  
specific primers pair-
based detection.

Highly repeatable and accurate. 
No thermal cycler required

1 hr. Primer designing is 
complex 

CB NAAT/
True NAAT

Cartridge Based Nucleic 
Acid Amplification and 
detection

Automated sample preparation, 
pre-programmed reaction. Can 
be used in a PoC devices 

15–30 
min

CRISPR  based 
methods

Enzymes programmed 
to target and cut viral 
RNA sequences

low-cost, do not require 
complex instrumentation, can 
be read using paper strips. 

15–30 
min

Not properly 
standardized for SARS 
Cov2 detection

Serological 
(traditional) 

Antigen/Antibodies 
IgG/ IgM/ELISA etc. 

Sensitive and specific. useful 
for choosing the convalescent 
plasma therapy.  

4-6 hrs Samples taken after 3-4 
days of  infection. 

Rapid
Serological 
(traditional) 

Antigen/Antibodies 
IgG/IgM 

Convenient, can be used in a 
PoC devices 

15-30 min Samples taken after 3-4 
days of  infection. 

Lateral flow 
assay 

colorimetric, qualitative 
detection of  the 
presence of  antibodies 

Inexpensive and convenient, 
can be used in a PoC devices 

10-15 min Nature of  the sample 
affects capillary action, 
(e.g. blood clotting) pre-
treat meant is required.  

Luminescent 
immune assay

Chemiluminescence or 
fluorescencelabelled 
antibody or antigen 

Highly sensitive, Quantitative 1-2 hrs. Closed analytical 
systems required for 
detection

Microarray rapid high-throughput 
detection of  viral RNA 
using cDNA 

Highly sensitive, longer target 
sequences can be detected

1 hr. Fabrication of  cDNA 
micro array is labor 
intensive. 

CT scan Computerized 
tomography of  Chest 
images

Enhance sensitivity of  detection 
if  findings combined with 
RT-PCR results 

1 hr. Indistinguishability 
from other viral 
pneumonia other chest 
complications 

Biochemical 
tests 

Blood counts, 
Lymphopenia, elevated 
serum enzyme levels, 
high 

Could support routine 
symptomatic treatment regimens 
of  COVID 19

1-24 hrs Not conclusive 
evidence for COVID 19 
infections

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available for COVID-19 is given in table 1. 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.

CONCLUSION
The COVID-19 pandemic, caused by the SARS-CoV-2, 
has resulted in over 17 million confirmed cases and over 7 
lakh deaths worldwide in less than six months. 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 
widespread availability of  accurate and rapid testing 
procedures is extremely valuable in unravelling the 
complex dynamics involved in SARS-CoV-2 infection 
and immunity. One of  the many challenges in containing 
the spread of  COVID-19 is the inability to identify 
asymptomatic cases that result in the virus spreading to 
close contacts. Thus, the global outbreak of  COVID-19 
has emphasized the importance of  the laboratory 
diagnosis of  human coronavirus infections in order 
to limit the spread as well as appropriately treat those 
patients who have serious complications. WHO has 
published a uniform case definition for surveillance and 
testing for COVID-19 infections. However, the focus on 
implementing the most reliable diagnostic tools varies in 
different places. Since COVID-19 is a new nosological 
entity, there are no data as of  yet that would enable the 
determination of  standards for the interpretation of  
specific diagnostic tests. As with any other infectious 
disease, the accuracy of  each method depends on the 
method of  collecting the material, the quality of  the 
sample, and the equipment applied. Although RT-PCR 
testing plays a crucial role in accurately detecting SARS-
CoV-2 on a case-by-case basis, it also has inherent 
problems that limit its utility. Current obstacles to the 
widespread use of  RT-PCR testing include a shortage 
of  testing kits and an extended processing period of  
several hours before results are obtained. Moreover, the 
results of  real-time RT-PCR tests must be cautiously 
interpreted. A combination of  real-time RT-PCR and 
clinical features, especially CT images, could facilitate 
better disease management. Proper sampling procedures, 
good laboratory practice standards, and using high-
quality extraction and a real-time RT-PCR kit could 
improve the approach and reduce inaccurate results. 
Loop-Mediated Isothermal Amplification (LAMP) is a 
relatively new, convenient molecular amplification point-
of-care technique that is widely used for COVID-19 
diagnosis. The technique does require sophisticated 
laboratory equipment to provide similar analytical 
sensitivity to RT-PCR. Serological testing for COVID-19 
is particularly attractive because of  the relatively short 
time to diagnosis and the ability to test for an active 

immune response against the virus. A lot remains to be 
understood regarding the value of  serological testing 
in COVID-19 diagnosis and monitoring. The results 
of  serological tests can then be used to estimate the 
true spread of  the virus through a population, even if  
individuals were asymptomatic or were never diagnosed. 
More comprehensive evaluations of  the performance of  
serology tests are rapidly under way. Considerations for 
the use of  serology methods for COVID-19 require the 
correct and appropriate interpretation of  the results and 
understanding the strengths and limitations of  such tests.

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