Evaluation of an Assay Based On Multiple Detection Temperature Technique for Simultaneous Detection of Viral Gastroenteritis-Causing Pathogens

Multiple detection temperature (MuDT) technique is an advanced method for the analysis of multiple Ct (cycle threshold) values in a single channel..
The advantage of this method has been shown only in DNA samples, restricting its diagnostic applicability. This technique was evaluated in this study for its efficacy in the analysis of target RNA..
Allplex GI-virus assay was developed to detect pathogens causing viral gastroenteritis, one of the major diseases caused by RNA viruses. This one-step multiplex real-time polymerase chain reaction (PCR) based on the MuDT technique permits simultaneous amplification and detection of target nucleic acids of norovirus GI, norovirus GII, rotavirus A, adenovirus F, astrovirus, and sapovirus genogroups. The assay was tested for analytical sensitivity, cross-reactivity, repeatability, and applicability to clinical samples..
The analytical performance was validated for each target. The assay demonstrated high analytical sensitivity and no cross-reactivity, and the repeatability tests showed excellent performance with high accuracy. Analytical performance validation indicated high positive agreement and negative agreement for this method. In the analyses comparing Allplex GI-virus Assay and commercial Seeplex Diarrhea-V ACE Detection using clinical specimens, the positive and negative agreements between the test results were found to be 94.9% and 98.8%, respectively. Statistical analysis showed that there was no difference in the performance between the two products..
The Allplex GI-virus Assay can rapidly detect six viruses in a single tube without the complementary DNA synthesis step, and this assay was shown to represent an improved molecular diagnostic tool for the simultaneous detection of several RNA viruses. Therefore, our results suggest that the MuDT technique may represent a new molecular diagnostic method for the detection of RNA viruses..