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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
immunology and microbiology
Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis
Journal of Clinical Virology, Volume 50, No. 4, Year 2011
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Description
Background: Several viruses can cause diarrheal disease, 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 MS2, 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 diarrhea in Tanzania the assay yielded between 88% and 100% sensitivity and specificity for all analytes. The difference in fluorescence intensities of MS2 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 gastroenteritis. The quantitation yielded by the assay is informative for clinical research particularly in the context of mixed infections. © 2011 Elsevier B.V.
Authors & Co-Authors
Liu, Jie
United States, Charlottesville
University of Virginia
Kibiki, Gibson S.
Tanzania, Moshi
Kilimanjaro Christian Medical Centre
Maro, Venance Phillip
Tanzania, Moshi
Kilimanjaro Christian Medical Centre
Maro, Athanasia
Tanzania, Moshi
Kilimanjaro Christian Medical Centre
Kumburu, Happiness Houka
Tanzania, Moshi
Kilimanjaro Christian Medical Centre
Swai, Ndealilia S.
Tanzania, Moshi
Kilimanjaro Christian Medical Centre
Taniuchi, Mami
United States, Charlottesville
University of Virginia
Gratz, Jean C.
United States, Charlottesville
University of Virginia
Toney, Denise M.
United States, Richmond
Division of Consolidated Laboratory Services
Kang, Gagandeep
India, Vellore
Christian Medical College, Vellore
Houpt, Eric R.
United States, Charlottesville
University of Virginia
Statistics
Citations: 118
Authors: 11
Affiliations: 4
Identifiers
Doi:
10.1016/j.jcv.2010.12.009
ISSN:
13866532
Research Areas
Genetics And Genomics
Infectious Diseases
Study Approach
Quantitative
Study Locations
Tanzania