- DNA extraction – essential first step. Quantity of DNA obtained can be assayed spectrophotometrically.
- Reverse transcriptase step – for detection of RNA viruses
- Nucleic acid amplification – know how PCR works! This PCR animation is easy to understand. Many other methods now that are more complex and are isothermal – do not require cyclers to change the temperature of the reaction.
- Detection of product – most assays rely on closed tube ‘real-time’ detection of product. There are a variety of nucleic acid probe methods described. Have a look here for a short video instruction. Quantification becomes possible and is used widely.
- DNA sequencing – this is a basic technique these days (see below)
Excellent background materials from ICPMR, NSW ( circa 2009):
Molecular-diagnosis-in-microbiology-ferguson-2016: overview presentation.
- pre-analytical and assay factors that affect sensitivity and specificity
- quality control (see above presentation)
- amplification product quantification and its usefulness
N.B. visit your local molecular lab if there is one and examine and understand all of the assays in use – for commercial assays, obtain the product inserts to read.
- Next generation sequencing methods: basically a way of doing massive parallel sequencing of short multiple sequences in the same sample
- GenXpert and similar methods : of great relevance for Nepal, PNG and elsewhere
- Multiplex commercial molecular platforms – e.g. Biofire filmarray
- Whole genome analyses