Brinkhof J M A, van Maanen C, Wigger R, Peterson K, Houwers D J
Animal Health Service Ltd., Arnsbergstraat 7, Deventer, The Netherlands.
J Virol Methods. 2008 Feb;147(2):338-44. doi: 10.1016/j.jviromet.2007.10.013. Epub 2007 Dec 11.
Real-time polymerase chain reaction (RT-PCR) detection of proviral nucleic acid sequences of small ruminant lentiviruses (SRLV) in blood samples was developed and evaluated. Priming oligonucleotides were designed on the highly conserved 5' untranslated leader-gag region while those on the long terminal repeat (LTR) assay were derived from literature. DNA was extracted from the buffycoat interlayer of centrifuged blood samples. Real-time PCR was performed by means of LightCycler technology (Roche Applied Science) using melting temperature analysis (SYBR Green I) for detection. Results were compared with those of serology using samples from Dutch sheep and goat flocks with known SRLV statuses, with sequential samples from a natural transmission experiment and samples from different regions in Norway, France, Spain and Italy. Real-time PCR testing, especially the application of oligonucleotides for priming the leader-gag region appeared promising in detecting SRLV specific proviral DNA in blood samples from both sheep and goats.
我们开发并评估了用于检测血液样本中小反刍兽慢病毒（SRLV）前病毒核酸序列的实时聚合酶链反应（RT-PCR）方法。引物寡核苷酸是根据高度保守的5'非翻译前导区- gag区域设计的，而长末端重复序列（LTR）检测的引物则来源于文献。从离心后的血液样本的血沉棕黄层中间层提取DNA。使用罗氏应用科学公司的LightCycler技术通过熔解温度分析（SYBR Green I）进行实时PCR检测。将结果与来自荷兰已知SRLV感染状况的绵羊和山羊群的样本、自然传播实验的连续样本以及挪威、法国、西班牙和意大利不同地区的样本的血清学检测结果进行比较。实时PCR检测，尤其是用于扩增前导区- gag区域的寡核苷酸的应用，在检测绵羊和山羊血液样本中的SRLV特异性前病毒DNA方面似乎很有前景。
