Journal Article

Optimization of Quantitative PCR Methods for Enteropathogen Detection

Haque, Rashidul, Silapong, Sasikorn, Walongo, Thomas, Kang, Gagandeep, Houpt, Eric R., Kabir, Furqan, Nataro, James, Jeamwattanalert, Pimmada, Mason, Carl, Begum, Sharmin, Bodhidatta, Ladaporn, Amour, Caroline, Liu, Jie, Maro, Athanasia, Haverstick, Doris M., Gratz, Jean, Boisen, Nadia, Praharaj, Ira, Mduma, Esto, Platts-Mills, James, Nshama, Rosemary, Lertsethtakarn, Paphavee

2016

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Acids / Amplification / Analysis / Armed forces / Biology and Life Sciences / Campylobacter / Children / Children & youth / Childrens health / Collection / Deoxyribonucleic acid / Diarrhea / Diarrhea - diagnosis / Diarrhea - etiology / DNA / Etiology / Feces - microbiology / Feces - parasitology / Feces - virology / Gene Dosage / Genetic aspects / Humans / Identification and classification / Infections / Infectious diseases / Medicine and Health Sciences / Methods / Molecular diagnostic techniques / Nucleic acids / Optimization / Pathogenic microorganisms / Pathogens / Pediatrics / Polymerase chain reaction / Reagent Kits, Diagnostic / Real-Time Polymerase Chain Reaction - methods / Real-Time Polymerase Chain Reaction - standards / Rectum / Reproducibility of Results / Research and analysis methods / Ribonucleic acid / RNA / Salmonella / Sensitivity and Specificity / Shigella / Specimen Handling / Viruses