Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of XXXX .

@entity294 @entity1902 @entity325 @entity23493 @entity5163
BACKGROUND: Several viruses can cause @entity5163 , a leading cause of morbidity and mortality worldwide. Existing diagnostic methods include ELISA and nucleic acid amplification, usually performed individually. OBJECTIVES: (1) To develop a multiplexed assay for simultaneous detection of major enteric viral pathogens. (2) Quantitation of viral load by normalizing with an extrinsic control. STUDY DESIGN: A simple protocol combining a one-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) with microsphere-based fluorescence detection was developed for norovirus GI and GII, rotavirus, astrovirus, sapovirus, and adenovirus. An extrinsic control, bacteriophage @entity23493 , was spiked into each fecal sample before nucleic acid extraction to normalize between samples for the efficiency of nucleic acid extraction and amplification. RESULTS: The fluorescent results were quantitative and nearly as sensitive as the corresponding singleplex real time RT-PCR (qRT-PCR) assay on analytic samples. Upon testing 229 fecal samples from inpatients with @entity294 in Tanzania the assay yielded between 88% and 100% sensitivity and specificity for all analytes. The difference in fluorescence intensities of @entity23493 between samples indicated variable extraction efficiency and was used to better refine the viral load of each specimen. CONCLUSIONS: This one-step nucleic acid-based assay enables rapid, sensitive and specific detection of the major viral causes of @entity1902 . The quantitation yielded by the assay is informative for clinical research particularly in the context of @entity325 .
