PMID- 30284505 OWN - NLM STAT- MEDLINE DCOM- 20190131 LR - 20200225 IS - 1943-4936 (Electronic) IS - 1040-6387 (Print) IS - 1040-6387 (Linking) VI - 30 IP - 6 DP - 2018 Nov TI - Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR. PG - 807-812 LID - 10.1177/1040638718805534 [doi] AB - We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4 degrees C or 25 degrees C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate-phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4 degrees C or 25 degrees C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4 degrees C or 25 degrees C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4 degrees C or 25 degrees C); samples thawed at 4 degrees C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001). FAU - Weiser, Ashley C AU - Weiser AC AD - Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman), Iowa State University, Ames, IA. AD - Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA. FAU - Poonsuk, Korakrit AU - Poonsuk K AD - Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman), Iowa State University, Ames, IA. AD - Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA. FAU - Bade, Sarah A AU - Bade SA AD - Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman), Iowa State University, Ames, IA. AD - Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA. FAU - Gauger, Phillip C AU - Gauger PC AD - Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman), Iowa State University, Ames, IA. AD - Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA. FAU - Rotolo, Marisa AU - Rotolo M AD - Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman), Iowa State University, Ames, IA. AD - Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA. FAU - Harmon, Karen AU - Harmon K AD - Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman), Iowa State University, Ames, IA. AD - Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA. FAU - Gonzalez, Wendy M AU - Gonzalez WM AD - Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman), Iowa State University, Ames, IA. AD - Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA. FAU - Wang, Chong AU - Wang C AD - Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman), Iowa State University, Ames, IA. AD - Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA. FAU - Main, Rodger AU - Main R AD - Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman), Iowa State University, Ames, IA. AD - Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA. FAU - Zimmerman, Jeffrey J AU - Zimmerman JJ AD - Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman), Iowa State University, Ames, IA. AD - Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA. LA - eng PT - Journal Article DEP - 20181004 PL - United States TA - J Vet Diagn Invest JT - Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc JID - 9011490 SB - IM MH - Animals MH - Linear Models MH - Logistic Models MH - Porcine Reproductive and Respiratory Syndrome/*diagnosis MH - Porcine respiratory and reproductive syndrome virus/*isolation & purification MH - Real-Time Polymerase Chain Reaction/methods/veterinary MH - Reverse Transcriptase Polymerase Chain Reaction/methods/veterinary MH - Saliva/*virology MH - Specimen Handling/methods/*veterinary MH - Swine PMC - PMC6505852 OTO - NOTNLM OT - Oral fluid OT - RT-rtPCR OT - porcine reproductive and respiratory syndrome virus COIS- Declaration of conflicting interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. EDAT- 2018/10/05 06:00 MHDA- 2019/02/01 06:00 PMCR- 2019/10/04 CRDT- 2018/10/05 06:00 PHST- 2018/10/05 06:00 [pubmed] PHST- 2019/02/01 06:00 [medline] PHST- 2018/10/05 06:00 [entrez] PHST- 2019/10/04 00:00 [pmc-release] AID - 10.1177_1040638718805534 [pii] AID - 10.1177/1040638718805534 [doi] PST - ppublish SO - J Vet Diagn Invest. 2018 Nov;30(6):807-812. doi: 10.1177/1040638718805534. Epub 2018 Oct 4.