Other Target Amplification Methods
Molecular Diagnostics
(MDx) Review 559
METHOD EXPLANATION
PCR modifications
Nested PCR
Real-time PCR
Multiplex PCR
Reverse transcriptase PCR (RT-PCR)
Transcription-based amplification systems (TAS)
Self-sustaining sequence replication (3SR)
Transcription-mediated amplification (TMA)
Nucleic acid sequence-based amplification (NASBA)
Uses 2 pairs of primers & 2nd round of amplification to ↑sensitivity &
specificity.Simultaneous amplification & detection (fluorescence) in sealed tube.
Less subject to contamination. Eliminates need for analysis of product
by gel electrophoresis. Quantitative. Commonly used for detection of
microorganisms.Uses more than 1 set of primers so multiple targets can be amplified in
same tube.Method to amplify RNA (mRNA or microbial RNA). Same procedure as
PCR except RNA is 1st converted to cDNA by reverse transcriptase. Used to
detect HIV & HCV, measure viral loads in HIV & HCV infections, measure
gene expression.Non-PCR methods to amplify target RNA. Similar methods, different
manufacturers. cDNA is formed from target RNA. Millions of copies of RNA
produced by transcription of cDNA. Assays to detect RNA viruses, Mycobac-
terium tuberculosis, Chlamydia trachomatis. TMA & NASBA are isothermal
rxn. Don’t require thermocycler.