Barlough J, East N, Rowe J D, Van Hoosear K, DeRock E, Bigornia L, Rimstad E
Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis 95616.
J Virol Methods. 1994 Dec;50(1-3):101-13. doi: 10.1016/0166-0934(94)90167-8.
A nested polymerase chain reaction (PCR) for detecting proviral DNA of caprine arthritis-encephalitis virus (CAEV) in biological samples was developed. Primers for both gag and pol sequences of the CAEV genome were included in a single tube for simultaneous amplification ('double' PCR), and the resulting bands were resolved visually in ethidium bromide-stained agarose gels. Internal gag and pol probes were used to verify the identity of the amplified products by non-radioactive Southern hybridization. Final confirmation of the identity of representative PCR bands was provided by DNA sequence analysis. A comparison between the PCR and an antibody ELISA (with recombinant CAEV p28 as target) using 141 caprine blood samples indicated very strong agreement between the two assays (kappa = 0.912). Four of 7 goats with indeterminate ELISA results were PCR-positive as were 5 of 40 (12.5%) seronegative goats, most probably indicating delayed seroconversion. Eleven of 27 goats (41%) PCR-positive on blood had detectable CAEV proviral DNA in milk. Proviral DNA was also detected in lung, mesenteric lymph node, bone marrow, synovial membrane, and mammary gland of a seropositive, clinically affected goat, but not in equivalent tissues of a healthy seronegative goat.
开发了一种用于检测生物样品中山羊关节炎-脑炎病毒（CAEV）前病毒DNA的巢式聚合酶链反应（PCR）。CAEV基因组的gag和pol序列的引物包含在单个管中用于同时扩增（“双重”PCR），并且在溴化乙锭染色的琼脂糖凝胶中目视分辨得到的条带。内部gag和pol探针用于通过非放射性Southern杂交验证扩增产物的身份。通过DNA序列分析对代表性PCR条带的身份进行最终确认。使用141份山羊血样对PCR和抗体ELISA（以重组CAEV p28为靶标）进行比较，结果表明两种检测方法之间具有很强的一致性（kappa = 0.912）。ELISA结果不确定的7只山羊中有4只PCR呈阳性，40只（12.5%）血清阴性山羊中有5只PCR呈阳性，这很可能表明血清转化延迟。血样PCR呈阳性的27只山羊中有11只（41%）在乳汁中可检测到CAEV前病毒DNA。在一只血清阳性、临床上受感染的山羊的肺、肠系膜淋巴结、骨髓、滑膜和乳腺中也检测到了前病毒DNA，但在健康血清阴性山羊的相应组织中未检测到。
