Table 3.

Summary of diagnostic methods used to detect SARS-CoV-2 (171–204).

	Name of the method	Salient features/advantages	Disadvantages	Time taken for detection from the time of sample collection	Feasible for pooled testing?	References
1. Detection of RNA	RT-PCR	Gold standard method for screening and diagnosis in the early phase of infection. Advantages include automation, high-throughput analysis and relatively reliability. Also determines relatively viral load.	Higher occurrence of false positives and false negatives, requires thermocyclers, laboratory set up and expertise to carryout the test and the analysis. Lack of symptoms in positive individuals; Highly vulnerable for errors due to cross contamination during sample collection, processing and while performing the test. High chance of laboratory error during sampling Need of skilled personnel	~ 4-6 hours	Yes	(32, 176, 188, 205)
	RT-LAMP	Performed using isothermal amplification. Advantages: It does not require specialized laboratory equipment (e.g. thermocyclers). Can be performed at a wide range of pH and temperature. Does not require thermocyclers, faster test results, easy to use, cost-effective, sensitivity comparable to RT-PCR Colorimetric visualization of results; Non-processed samples can be assayed.	Requires more number of primers and thereby increases chances of primer dimer formation contributing to false positives. For the same reason, primer designing is challenging. Multiplexing could be problemative. Less versatile than PCR .	<1 h	Yes	(172)
	RT-RPA	Performed using isothermal amplification. Advantages: It does not require specialized laboratory equipment (e.g. thermocyclers). Can be performed at a wider range of temperatures. Does not require thermocyclers, faster test results, easy to use, cost-effective, sensitivity comparable to RT-PCR Colorimetric visualization of results; Non-processed samples can be assayed.	Primer and probe design for RPA are less established; Less sensitive than RT-PCR and RT-LAMP. Requires higher concentrations of dNTPs.	<1h	Yes	(198)
	RNA NASBA	Employs reverse transcription followed by amplification of RNA by T7 RNA polymerase followed by detection of the amplified RNA using fluorescent molecular beacon DNA probe. It is an isothermal reaction (41°C) and has faster amplification kinetics compared to RT-PCR and RT-LAMP; Does not require thermocyclers. Compatible for multiplexing and high throughput analsysis.	Low temperature of the reaction conditions increasese the chances of non-specific primer interactions. Except one, NASBA enzymes are heat-labile, requiring the addition of these enzymes after the melting step. Since the primers used are not incorporated in the amplicon and therefore labeled primers can't be used for detection.	90 minutes	Yes	(179, 185)
	SHERLOCK	Viral RNA is reverse transcribed first to produce cDNA following which fluorescently-labeled single-stranded DNA (ssDNA) reported probes are indiscriminately cleaved either through FnCas9- or Cas12a-sgRNA complex. Alternatively synthesized cDNA can be used for in vitro transcription and the RNA thus produced will acticate nuclease activity of Cas13a, resulting similar cleavage of fluorescently-labeled ssDNA reported probesor Cas12based. All components of SHERLOCK can be freeze-dried; Highly sensitive and specific. Capable of detecting single target RNA/DNA molecule. Amenable for multiplexing and is one of the rapid nucleic acid detection method.	Multi-step detection, protocol optimization is challenging. Cas13a depends on intact in vitro-synthesized RNA and therefore proned to challenges associated with RNA degradation.	~ 40-90 minutes	?	(186)
2. Detection of viral antigen	Rapid antigen test	Antibodies specific to vrial antigens are used to detect the virus. One of the most rapid methods, easy-to-perform and interpret, requiring no specialized equipment or expertise. Most suitable for point-of-care diagnostics.	It takes weeks or months to produce high-titer antibodies and usually they are not as sensitive and specific as nucleic acid-based approaches. Often, confirmation of the negative results by rapid antigen test requires RT-PCR to rule out infection.	~15 minutes	No	(195)
3. Detection of antibodies generated in response to SARS-CoV-2 infection	Lateral flow assays	Fast detection of IgM/IgG antibodies specific for SARS-CoV-2 proteins. Direct detection from plasma, serum, whole blood or fingertip blood and requires less amount of sample. Instruments, specialized expertise not required. Amenable for self-testing at home as well as for point-of-care testing centres. Long shelf -life.	Often, confirmation of the negative results by rapid antigen test requires RT-PCR to rule out infection. Requires highly purified antigens for accuracy.	~15 minutes	No	(175, 187, 190, 193)
	ELISA (IgA, IgM and IgG)	One of the most commonly used methods to detect both antigen/antibodies. Hihgly sensitivity in detecting antiviral antibodies. Allows highthroughput analysis.	Clinical performance data are scarce; Cross-reactivity has been reported; Cost per test is high as compared to other tests	2-3 h	Yes	(181)
4. Detection of Symptoms	Chest computed tomography (CT) Scan	Chest CT scans help if identifying characteristic lung pathology associated with the disease such as ground glass opacity, bilateral consolidation, interlobular septal thickening and pleural effusion. Low false-negative rate. Can be used to monitor the progression of the disease/recoverey.	Requires expensive equipment, proper lab facility and techincal expertise to carry out the test and analyze the results. It requires physical presence of the suspected individuals/patients. Diagnosis is not specific to the pathogen. Imaging protocols vary across locations	~1h	No	(206, 207)
	Radiography (X-ray)	Radiography (X-ray) identifies unilateral and bilateral infilterate. Rapid, low false-negative rate. Can be used to monitor the progression of the disease/recoverey. The cost is lower than CT but at the same less-sensitive and is more useful in monitoring the disease during later stages.	It is less sensistive and demostrates limited pathological features associated with the disease. Generally it cannot detect early stages of the disease. Requires expensive equipment, proper lab facility and techincal expertise to carry out the test and analyze the results. It requires physical presence of the suspected individuals/patients. Diagnosis is not specific to the pathogen.	~ 45 minutes	No	(208)