Table 1.

Advantages and limitations of nucleic acid-based amplification techniques.

Detection methods	Advantages	Limitations	References
Conventional PCR	(i) Sensitive and specific	(i) High risk of contamination	[28, 33–35]
	(ii) Widely employed nucleic acid-based detection format	(ii) Prone to inhibitors
	(iii) Multiplex detection potential	(iii) Time-consuming and labor-intensive
		(iv) Qualitative
		(v) Requires thermal cycler and gel documentation apparatus
Conventional RT-PCR	(i) Sensitive and specific	(i) RNA handling might be difficult	[10, 14, 36–40]
	(ii) Multiplex detection potential	(ii) High risk of contamination
		(iii) Time-consuming and cumbersome
		(iv) Relatively expensive
		(v) Prone to inhibitors
		(vi) Mutation within PCR primer regions may occur in some RNA viruses which have high mutation rates, leading to reduced sensitivity
Real-time PCR/RT-qPCR	(i) Highly sensitive and specific	(i) Requires expensive laboratory equipment and fluorescent probe	[33, 34, 39–41]
	(ii) Lower cross-contamination risk due to closed tube operation	(ii) Designing of TaqMan probes requires almost complete information of the target nucleic acid sequence
	(iii) Rapid and less labor-intensive	(iii) Primer dimer artifact is a problem in case of SYBR green method
	(iv) Multiplex detection	(iv) Prone to inhibitors
	(v) Genotyping
	(vi) Determination of the viral load (quantitative)
TMA + NASBA	(i) Sensitive and specific	(i) RNA handling might be difficult	[36, 42–45]
	(ii) Simple and rapid (fewer cycles are required)	(ii) Requirement of three enzymes in case of NASBA
	(iii) Multiplexing potential	(iii) Use of enzymes that are not thermostable
	(iv) Quantification,	(iv) Nonspecific interactions of the primers may increase as the amplification process occurs at a lower temperature (41°C)
	(v) Genotyping
	(vi) Does not require thermal cycler as the reaction takes place isothermally at 41°C
LAMP/LAMP-RT	(i) Highly sensitive and specific	(i) Requirement of six primers	[35, 46–49]
	(ii) Easy to perform	(ii) High risk of carryover contamination
	(iii) Does not require expensive thermal cycler	(iii) Limitation for multiplexing
	(iv) Rapid (results in <1 h)	(iv) Visual detection using naked eye alone is subjective since it depends on observer's perception of color
	(v) Quantitative
	(vi) Genotyping
	(vii) Simple detection systems (using naked eye)
	(viii) Relatively resistant to inhibitors present in the sample