Rapid sequencing and analysis for pathogen confirmation

Abstract

Genetic amplification methods and especially those based on the polymerase chain reaction (PCR) are revolutionising diagnostic microbiology. Genetic amplification is rapidly becoming the method of choice to detect viruses, ‘single pathogen’ infections such as Chlamydia and Gonorrhoea, slow growing or difficult-to-grow bacteria such as mycobacteria and legionella, or toxigenic bacteria such as Shigatoxigenic Escherichia coli and Clostridium difficile. This trend has been accelerated with the use of fluorescent, probe-based, real-time PCR, high-throughput thermocyclers and the use of robotics. Traditional biochemistry-based identification is adequate for many bacterial isolates, as is the specificity afforded by real-time probes for direct PCR detection. However, the sequencing of amplified products (amplicons) is also often performed where increased resolution is required, even though longer amplicons than those generated by real-time PCR are required. Two benefits are clear. Firstly, sequence is constructed solely from the four nucleotides so can be likened to a digital signal. In comparison, biochemical identifications are more like an analogue signal, where weak or equivocal reactions can create ambiguity. Secondly, the resolution afforded by biochemical reactions (less than 100 character states i.e. 48 reactions that can be positive or negative) is easily exceeded by sequencing (2000 character states for a 500 bp sequence i.e. 500 sites which can contain one of four nucleotides). Further, the sequence is reproducible and not dependent on variables such as the bacterial growth conditions and easily lends itself to computer-based analysis.