Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a devastating disease in swine. The presence and transmission of PRRSV by boar semen has been demonstrated by using a swine bioassay. In this assay, 4-to 8-week-old pigs were inoculated intraperitoneally with semen from PRRSV-infected boars. Seroconversion of these piglets indicated the presence of PRRSV in semen. Seroconversion in gilts has also been demonstrated following artificial insemination with semen from PRRSV-infected boars. These methods of detecting PRRSV in boar semen are time-consuming, laborious, and expensive. The objective of this study was to develop a reliable and sensitive PCR assay to directly detect PRRSV in boar semen. Primers from open reading frames 1b and 7 of the PRRSV genome were used in nested PCRs. Virus was detected at concentrations as low as 10 infectious virions per ml in PRRSV-spiked semen. Specificity was confirmed by using a nested PCR and a 32 P-labeled oligonucleotide probe. The primers did not react with related arteriviruses or other swine viruses. The PCR assay showed good correlation with the swine bioassay, and both methods were superior to virus isolation. To consistently identify PRRSV in boar semen, the cell fraction was separated by centrifugation at 600 ؋ g for 20 min, a lysis buffer without a reducing agent (2-mercaptoethanol) was used, and nondiluted and 1:20-diluted cell fractions were evaluated by PCR. PRRSV was not reliably detected in the seminal plasma fraction of boar semen. RESULTSOptimization of PCR amplification conditions. To improve the specificity and sensitivity of the PCR, various MgCl 2 concentrations (1.6 to 5 mM), primer concentrations (0.2 to 0.4 M), annealing (50 to 58ЊC) and denaturing (94, 95, or 98ЊC) temperatures, and numbers of cycles (30, 40, or 45) were tested. The optimum product yield was achieved with 5 mM MgCl 2 , 0.4 M primer, annealing and denaturing temperatures of 58 and 95ЊC, respectively, and 30 cycles.

Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes

“…This situation requires that diagnostic tests be able to detect as well as precisely type the PRRSV virus. Virus isolation on PPAM and MARC monolayers is currently the gold standard of virus detection; yet, some PRRSV-containing materials (such as semen) contain too high levels of cytotoxic compounds to be reliably screened by tissue culture methods (Christopher-Hennings et al, 1995b;Nielsen et al, 1997;Shin et al, 1997). Typing PRRSV isolates with EU-type specific and US-type-specific MAbs is a rapid and accurate method; however, finer sub-typing of virus is not currently possible with this method, excluding detailed epidemiological studies.…”

“…By optimizing RNA extraction as well as crucial steps of the RT-PCR procedure, we routinely detected PRRSV diluted in clinically relevant materials such as serum and boar semen at a concentration of 1±10 TCID 50 ml À1 . In previous studies, detection of PRRSV at these low levels in boar semen required fractionation of semen and nested PCR (Christopher-Hennings et al, 1995b;Shin et al, 1997), making the test cumbersome for routine use, or was accompanied by a very small PCR amplicon size (Legeay et al, 1997), making the test of limited value for virus-typing/sequencing. Thus, the ability to sensitively amplify PRRSV sequences of up to 820 bp from`difficult' material such as boar semen represents a significant and non-trivial technical advantage of our assay.…”

“…It follows from the above that a RT-PCR test should be able to sensitively detect as well as type PRRSV from relevant biological material, and provide the maximal amount of sequence information, preferably by amplification of whole viral open reading frames. None of the published RT-PCR tests seems to completely fulfil this combination of requirements (Mardassi et al, 1994;Sua Ârez et al, 1994;Van Woensel et al, 1994;Christopher-Hennings et al, 1995b;Kono et al, 1996;Gilbert et al, 1997;Larochelle and Magar, 1997;Legeay et al, 1997;Shin et al, 1997). In the present study, we report the development and characterization of a RT-PCR test which meets the above criteria, and thus should provide a valuable tool for the epidemiological surveillance of emerging PRRSV-types.…”

Rapid detection of a highly virulent Chinese-type isolate of Porcine Reproductive and Respiratory Syndrome virus by real-time reverse transcriptase PCR

“…Because of its economic significance, a great deal of resource has been invested in developing effective prevention and control strategies. But protocols providing consistent success have been elusive due to the high rate of genetic change and antigenic variability (Christopher-Hennings et al, 1995;Kapur et al, 1996;Halbur et al, 1996;Meng, 2000). A diagnosis of PRRSV infection is based mainly on typical clinical signs, seroconversion, characteristic light microscopic lesions and the demonstration of PRRSV by virus isolation, fluorescent antibody (FA) examination, immunohistochemistry, in situ hybridization, or PCR (Rossow et al, 1996;Rossow, 1998).…”

Reverse transcription loop-mediated isothermal amplification for the detection of highly pathogenic porcine reproductive and respiratory syndrome virus

“…All the strains tested by RT-LAMP were also identified by nested RT-PCR and sequenced. The details of primers and condition for nested RT-PCR assay for the detection of PRRSV have been previously described (Christopher-Hennings et al, 1995), with minor modifications. The outer sense and antisense primers were N1F and N1R and the nested sense and antisense primers were N2F and N2R, respectively (Table 3).…”