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PLoS One. 2016 Jun 23;11(6):e0158199. doi: 10.1371/journal.pone.0158199. eCollection 2016.

Optimization of Quantitative PCR Methods for Enteropathogen Detection.

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Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, United States of America.
Haydom Global Health Institute, Haydom, Tanzania.
Kilimanjaro Clinical Research Institute, Moshi, Tanzania.
Department of Pediatrics, University of Virginia, Charlottesville, Virginia, United States of America.
Department of Pathology, University of Virginia, Charlottesville, Virginia, United States of America.
Department of Pediatrics and Child Health, Aga Khan University, Karachi, Pakistan.
Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.
International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh.
Christian Medical College, Vellore, Tamil Nadu, India.


Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen's extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.

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