Amplification methods | LAMP | NASBA | RCA | HDA | RPA/RAA |
---|---|---|---|---|---|
Template | DNA and RNA | RNA | DNA and RNA | DNA and RNA | DNA and RNA |
Reaction temperature | 65°C | 37-42°C | 37°C | 65°C | 37-42°C |
Amplification time | 60 min | 90 min | 30-240 min | 60-120 min | 10-30 min |
Enzymes for amplification | Bst DNA polymerase | Reverse transcriptase, RNase H, and T7 RNA polymerase | DNA ligase and DNA polymerase | Helicase, single-stranded DNA-binding protein, and DNA polymerase | Recombinase, Bst DNA polymerase, and single-stranded DNA-binding protein |
Number of primers | 4-6 | 2 | 1 | 2 | 2 |
Detection methods for the amplified products | Double-stranded chimeric dye, turbidimetry, indicator lateral flow chromatography, gel electrophoresis, and ELISA | Molecular beacon probes, gel electrophoresis, and ELISA | Fluorescent probes and gel electrophoresis | Gel electrophoresis and fluorescent probes | Fluorescent probes, lateral flow chromatography, and gel electrophoresis |
Advantages | Simple reaction system, multiple detection methods, and product detection with naked eyes | Direct detection of RNAs and prevention of contamination | High specificity and low risk of contamination | Low and constant temperature and high sensitivity | Rapid detection, low and constant temperature |
Disadvantages | False positivity, complex primer design, and being prone to nonspecific amplification | A necessary preheating step and failure in achieving a real constant temperature | More operating procedures and longer reaction duration | Longer reaction duration | Longer primer probes and being easy to form dimers |