Effects of sample handling on PRRSv detection in oral fluids by PCR

J Vet Diagn Invest. 2018 Oct 4:1040638718805534. doi: 10.1177/1040638718805534. [Epub ahead of print]

Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR.

Weiser AC1,2, Poonsuk K1,2, Bade SA1,2, Gauger PC1,2, Rotolo M1,2, Harmon K1,2, Gonzalez WM1,2, Wang C1,2, Main R1,2, Zimmerman JJ1,2.

Author information

1

Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine (Weiser, Poonsuk, Bade, Gauger, Rotolo, Harmon, Gonzalez, Main, Zimmerman).

2

Department of Statistics, College of Liberal Arts and Sciences (Wang), Iowa State University, Ames, IA.

Abstract

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°C or 25°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°C or 25°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°C or 25°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°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).

KEYWORDS: 

Oral fluid; RT-rtPCR; porcine reproductive and respiratory syndrome virus

PMID:

 

30284505

 

DOI:

 

10.1177/1040638718805534