DETECTION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS IN BOAR SEMEN BY PCR
DETECTION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS IN BOAR SEMEN BY PCR
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DOI：
10.1128/jcm.33.7.1730-1734.1995
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发表时间：
1995-07-01
影响因子：
9.4
通讯作者：
BENFIELD, DA
中科院分区：
文献类型：
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作者：
CHRISTOPHERHENNINGS, J;NELSON, EA;BENFIELD, DA
猪繁殖与呼吸综合征病毒（Porcine Reproductive and Respiratory Syndrome Virus，PRRSV）是一种严重危害猪健康的病毒。猪繁殖与呼吸综合征病毒的存在和传播的公猪精液已证明通过使用猪的生物测定。在本试验中，4- 8周龄的猪腹腔内接种来自PRRSV感染公猪的精液。这些仔猪的血清转化表明精液中存在PRRSV。用PRRSV感染公猪的精液进行人工授精后，也证明了后备母猪的血清转化。这些检测猪精液中PRRSV的方法费时、费力且昂贵。本研究的目的是建立一种可靠、灵敏的猪繁殖与呼吸综合征病毒（PRRSV）的PCR检测方法。在巢式PCR中使用来自PRRSV基因组的开放阅读框1b和7的引物。在PRRSV加标精液中检测到的病毒浓度低至每毫升10个感染性病毒粒子。通过使用巢式PCR和P-32标记的寡核苷酸探针确认特异性。引物与相关动脉炎病毒或其它猪病毒均不发生反应。结果表明，PCR检测与猪源病毒生物学检测具有良好的相关性，且两种方法均上级病毒分离。为了一致地鉴定公猪精液中的PRRSV，通过以600 x g离心20 min分离细胞组分，使用不含还原剂（2-巯基乙醇）的裂解缓冲液，并通过PCR评价未稀释和1：20稀释的细胞组分。猪精液的精浆组分中没有可靠地检测到PRRSV。
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 mi in PRRSV-spiked semen. Specificity was confirmed by using a nested PCR and a P-32-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 x 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.