The predominant strain of influenza virus circulating last winter was not as expected, and resulted in more severe illness. Here, Sally Taylor and Bob Jones assess a new multiplex PCR assay that provides results in hours rather than days, and offers patients prompt diagnoses.
Image: CDC/National Institute of Allergy and Infectious Diseases (NIAID)
Recent reports show that seasonal influenza A H3N2 viruses – a particularly harmful strain – have been the most commonly seen causes of influenza cases in the 2014/2015 season. These viruses are genetically different from the strains used to make this season's vaccines and often cause more severe illness. They can also lead to hospitalisation and are potentially fatal. It is therefore crucial that clinical testing laboratories have the tools needed to make rapid, accurate diagnoses. The tools used to make such diagnoses have come a long way in recent years. Rather than spending days or weeks growing and testing a culture, polymerase chain reaction (PCR) assays for various pathogens can be conducted in a few hours. This allows clinicians to decide on treatment plans and begin treatment within the day, which can only be beneficial to patient welfare.
Real-time PCR in diagnostic testing
Real-time PCR-based systems involve transcribing RNA into complementary DNA (cDNA) using a specific primer-mediated reverse transcription step, followed immediately in the same tube by PCR. Detection of products is via a dual-labelled molecular probe for each virus and bacteria of the multiplex PCR. The presence of specific viral and bacterial sequences in the reaction is distinguished by an increase in fluorescence observed from the relevant dual-labelled probe, and is reported as a cycle threshold (ct) by the real-time thermocycler.
This technique allows clinical laboratories to detect the agents of disease directly from clinical samples, without the need to wait for a culture to grow. This is particularly valuable in the fast identification of fastidious microorganisms, along with those that cannot be grown in the laboratory. It is therefore unsurprising that it has been picked up by a great many clinical laboratories, and that it has been the focus of a great deal of development over recent years, allowing the number of microbial agents detectable by real-time PCR to increase.
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