|
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 | |
|