Comparison of commercial real-time reverse transcription-PCR assays for reliable, early, and rapid detection of heterologous strains of porcine reproductive and respiratory syndrome virus in experimentally infected or noninfected boars by use of different sample types

Abstract

The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) real-time reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days -2, 1, 3, 5, 7, 14, and 21 postinoculation (p.i.) and tested by one of three commercially available real-time RT-PCR assays (VetMax from Applied Biosystems, Foster City, CA [abbreviated AB]; VetAlert from Tetracore, Rockville, MD [TC]; and AcuPig from AnDiaTec GmbH, Kornwestheim, Germany [AD]). At day 1 p.i., all assays detected at least one positive sample in each group. The highest detection rates were on days 3 and 5 p.i. Between days 1 and 7 p.i., serum samples had the highest detection rate (90%) with 100% agreement between tests, followed by blood swabs (kappa value of 0.97) and semen (kappa value of 0.80). Oral fluids had the lowest detection rates (AB, 55%; TC, 41%; AD, 46%) and the highest disagreement between kits (kappa value of 0.63). Pools of five samples did not reduce the detection rates if there was one positive sample with a large amount (cycle threshold, <30) of viral RNA in the pool. Serum and blood swab samples had shorter turnaround times for RNA extraction. The AB assay had a 1.6-times-shorter PCR time. In summary, serum and blood swabs had the best performance with highest detection rates and agreement between assays and the shortest turnaround times.

Fig 1

Detection rates for PRRSV RNA in serum (A), blood swab (B), oral fluid (C), and semen (D) samples of 15 experimentally infected boars on days 1, 3, 5, 7, 14, and 21 postinfection by three real-time RT-PCR tests (AB, TC, and AD). Asterisks indicate differences between assays (McNemar's test, P < 0.05).

Fig 1

Detection rates for PRRSV RNA in serum (A), blood swab (B), oral fluid (C), and semen (D) samples of 15 experimentally infected boars on days 1, 3, 5, 7, 14, and 21 postinfection by three real-time RT-PCR tests (AB, TC, and AD). Asterisks indicate differences between assays (McNemar's test, P < 0.05).

Fig 2

Cumulative detection rates of PRRSV RNA in serum, blood swab, oral fluids and semen of experimentally infected boars with PRRSV strains VR2385, SDSU73, JA142, 2010011381, and FL12 on days postinfection 1 to 21 by three real-time RT-PCR tests (AB, TC, and AD). Asterisks indicate differences between assays (McNemar's test, P < 0.05).

Fig 3

Effects of sample pool size on detection of a single positive sample in the pool of serum (A) and blood swab (B) samples by RT-PCR. A positive sample was diluted in negative-control samples so as to simulate pool sizes of 1, 2, 3, 5, or 10 samples. Three positive ranges were used for each pool representing high-positive (cycle threshold [C_T] of < 29.5), moderate-positive (C_T of 30 to 34.5), and low-positive (C_T of 35 to 37) samples. The horizontal line at C_T 37 represents the cutoff for negative samples (C_T of >37). The box-and-whiskers plots show cumulative results of one tested sample of each strain (VR2385, SDSU73, JA142, 2010011381, and FL12) tested by AB, TC, and AD assays.