一种用于定量有蹄类细小病毒1的TaqMan定量聚合酶链反应在多种基质中得到验证。
A TaqMan qPCR for quantitation of Ungulate protoparvovirus 1 validated in several matrices.
作者信息
Streck André Felipe, Hergemöller Francine, Rüster Dana, Speck Stephanie, Truyen Uwe
机构信息
Institute for Animal Hygiene and Veterinary Public Health, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany; Laboratory of Virology, Faculty of Veterinary Medicine, Rio Grande do Sul Federal University, Avenida Bento Gonçalves 9090, 91540-000 Porto Alegre, Brazil.
Institute for Animal Hygiene and Veterinary Public Health, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany.
出版信息
J Virol Methods. 2015 Jun 15;218:46-50. doi: 10.1016/j.jviromet.2015.03.003. Epub 2015 Mar 14.
Ungulate protoparvovirus 1 (UPV1) is one of the major causes of reproductive disorders in swine. Recently, the rapid viral evolution of UPV1 and its viral persistence in several tissues has been described. Based upon this, a real-time qPCR method using upgraded primers targeting VP1 and applying the TaqMan technology was developed in this study for UPV1, and it was validated in feces, serum and tissue. Within the results, the limit of detection of the qPCR was 100copies of the viral genome per reaction of serum and feces and 1000copies of the viral genome per reaction of the grinded tissue (pre-inoculated matrices with diluted serially viruses). No cross reactivity was observed with other viruses associated with reproductive disorders. The assay was specific and reproducible, presenting low intra- and inter-assay variation (0.93% and 1.06%, respectively). In 50 clinical samples, the method was found to be more sensitive than immunofluorescence and a SYBR Green PCR. In conclusion, this qPCR represents an upgraded and useful tool to quantify UPV1 in different sample matrices for diagnostic and research purposes.
有蹄类动物细小病毒1（UPV1）是猪繁殖障碍的主要病因之一。最近，已经描述了UPV1的快速病毒进化及其在多个组织中的病毒持续性。基于此，本研究开发了一种使用靶向VP1的升级引物并应用TaqMan技术的实时定量PCR方法用于检测UPV1，并在粪便、血清和组织中进行了验证。结果显示，定量PCR的检测限为血清和粪便每次反应100个病毒基因组拷贝，研磨组织（预先接种了系列稀释病毒的基质）每次反应1000个病毒基因组拷贝。未观察到与其他与繁殖障碍相关病毒的交叉反应。该检测方法具有特异性和可重复性，分析内和分析间变异低（分别为0.93%和1.06%）。在50份临床样本中，该方法比免疫荧光和SYBR Green PCR更灵敏。总之，这种定量PCR是一种升级的有用工具，可用于诊断和研究目的，对不同样本基质中的UPV1进行定量。
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