Multiplex Real-Time PCR for the Detection of Gastrointestinal Bacterial Pathogens: A Comparison of Two Commercial Assays



Multiplexed molecular panels allow rapid detection of multiple stool pathogens, reducing workload and turn around time compared to conventional methods. This study evaluated the performance of Seegene Allplex™ GI-Bacteria (I) and (II) panels and Roche LightMix® gastrointestinal bacterial assays.


A total of 233 frozen, pre-characterized clinical or simulated Copan FecalSwab® specimens were tested on Allplex™ panels, while 219 were tested on LightMix® panels. Fecal swab fluid (190uL) was combined with 10uL of each Allplex™ GI-BP internal control and LightMix® phocine herpesvirus extraction control and extracted on the Roche MagNA Pure Compact. PCR was performed in parallel on the Bio-Rad CFX96 and LightCycler 480II, using reaction parameters specified by the manufacturers. Campylobacter spp, Salmonella spp, Shigella spp, Y. enterocolitica, Aeromonas spp and Shiga toxin 1/2 genes were compared between both assays, with C. difficile, Vibrio spp and E. coli O157 only evaluated on Allplex™ and P. shigelloides only evaluated on LightMix®. Sensitivity and specificity were calculated using a modified gold standard, composed of at least 2 positive between routine stool culture, Allplex™ and LightMix® PCR.


Allplex™ panels provided accurate results in 192/198 (97%) clinical specimens while LightMix® was accurate in 183/189 (96.8%). When both clinical and simulated specimens were considered, Allplex™ had excellent performance for all targets, apart from Campylobacter spp (87.5% sensitivity) and Vibrio spp (66.7% sensitivity), since more unusual species (eg: C. upsaliensis, V. fluvialis) were not detected (Table 1). LightMix® performed well with >90% sensitivity and >99% specificity for all targets.


Both assays performed well in detecting clinically relevant infections, compared to conventional methods.