Basics

1. DNA extraction – essential first step. Quantity of DNA obtained can be assayed spectrophotometrically.
2. Reverse transcriptase step – for detection of RNA viruses
3. 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.
4. 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.
5. DNA sequencing – this is a basic technique these days (see below)

Excellent background materials from ICPMR, NSW ( circa 2009): 

• molecular-diagnostic-techniques
• molecular-specimen-processing
• quality-control-in-molecular-microbiology
• typing-techniques

Molecular-diagnosis-in-microbiology-ferguson-2016: overview presentation.

Important concepts

• 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.  

Modern methods

• 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