Simultaneous detection of RNA and DNA from pathogens with hyperplex PCR

Rapid and reliable identification of pathogens is of great importance in healthcare settings and in the control of agonistic and antagonistic disease outbreaks. Broad-range methods for molecular detection of parasites, bacteria and viruses in samples with unknown contents are thus highly desirable. Hyperplex PCR (hpPCR) is a recently invented analysis technique that allows for parallel detection of up to 100 DNA and RNA targets. We present the development and evaluation of a proof-of-concept 16-plex hpPCR method targeting RNA viruses, bacteria and a parasite to enable detection of various gastrointestinal pathogens relevant in food or waterborne disease outbreak scenarios. The panel also includes highly pathogenic microbes such as Bacillus anthracis and Yersinia pestis. The hpPCR method showed high selectivity with detection of all expected targets. None of the tested bacteria (11), parasites (1) or viruses (4) gave false positive signals or cross-reactions for any other targets in the panel. The limit of detection was comparable to single-plex qPCR/RT-qPCR assays targeting the same pathogens. HpPCR tolerated high amounts of humic acid, with somewhat better results for the bacteria compared to the viruses. The 16-plex hpPCR method may be supplemented with additional assays to further widen the group of targeted pathogens. A hpPCR method simultaneously targeting a multitude of microbes including bacteria, parasites and viruses may thus provide timely and selective detection and identification of pathogens in an outbreak situation.