Molecular Assay Detects Pathogenic Bacteria Concurrently

A real-time multiplex polymerase chain reaction (PCR) has been developed that identifies common diarrhea-causing bacteria in fecal samples.

Sets of primers and probes were designed that were specific for Campylobacter jejuni, Salmonella, Shigella/enteroinvasive Escherichia coli EIEC, and Yersinia species, suitable for use in a one-tube PCR assay.

Scientists at the Bernhard Nocht Institute (Hamburg, Germany) tested a total of 393 stool samples, submitted for routine culture of enteric pathogens that had been collected from 2007 to December 2008 from various institutions. The samples were obtained from patients with different qualities of diarrhea (watery and bloody), but none of them had a recent travel history. All samples were examined by conventional microbiological methods and subsequently by real-time multiplex PCR in a blinded manner. DNA was extracted from the stools using QIAamp DNA Stool Mini Kit and the real-time multiplex PCR was carried out on a RotorGene6000 Cycler, both obtained from Qiagen (Hilden, Germany).

The PCR yielded results within three hours including DNA purification and no false-positive signals or cross-reactions were observed. Compared with culture, PCR detected 79 out of 81 C. jejuni (97.5%), 71 out of 74 Salmonella (96%), 8 out of 8 Shigella (100%), and 10 out of 10 Yersinia-positive (100%) clinical samples. In the 192 culture-negative samples, PCR additionally detected two Shigella, one Salmonella, and five C. jejuni infections. The authors concluded that real-time multiplex PCR provides reliable results within a short time and might be useful as an additional diagnostic tool whenever time is important in the diagnosis of enteropathogenic bacteria.

The approach presented in the study could be complemented with combinations of multiplex PCRs for the detection of other bacterial, parasitic, and viral infections to cover the entire spectrum of possible diarrhea-causing infectious agents. Integrated into clinical microbiology, these molecular tools may facilitate, accelerate, and optimize the diagnostic workflow. The study was available online on August 19, 2011, in the International Journal of Medical Microbiology.

Related Links:
Bernhard Nocht Institute
Qiagen