Department of Biotechnology, Tajen University, Pingtung, Taiwan.
J Microbiol Immunol Infect. 2010 Apr;43(2):85-92. doi: 10.1016/S1684-1182(10)60014-X.
BACKGROUND/PURPOSE: The postweaning multisystemic wasting syndrome, caused by the porcine circovirus type 2 (PCV-2), is a major disease that poses a significant threat to the global swine industry. The purpose of this study was to establish a real-time polymerase chain reaction (PCR) method for the quantification of PCV-2 and to enable the rapid differentiation of porcine circoviruses type 1 and 2 (PCV-1 and PCV-2). Such a method would significantly speed up the process of clinical diagnosis, and could also be used to study the pathogenic mechanisms of diseases associated with PCV-2.
Multiplex real-time PCR, together with LightCycler PCR data analysis software, was used for the quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2. A 263-bp DNA fragment was amplified from the 3' end of the open reading frame-2 of PCV-2 by nested PCR, and its DNA sequence was verified as having 100% identity with a PCV-2 standard (NCBI accession number: AF055394). The 263-bp DNA fragment was cloned into the pGEM-T easy vector, and the recombinant plasmid was serially diluted and quantified using real-time PCR. A standard curve was then constructed for quantification of the PCV-2 levels in field samples. The differentiation of PCV-1 and PCV-2 was carried out by analyzing the melting temperatures of the genotype-specific PCR products.
To quantify the PCV-2 levels in field samples, a standard curve (1 x 10(2) -1 x 10(9) copies/microL) was constructed. PCV-2 concentrations as low as 1 x 10(2) copies/microL could be detected in specimens taken from the lymph nodes or infected tissues in samples of PCV-2-infected pigs. The diagnosis of PCV-1 and PCV-2 infections and the quantification of the viral load in the field samples could be completed within 45 minutes after extracting the viral DNA using a commercial extraction kit.
This study demonstrate that real-time PCR is a clinically feasible method for the accurate quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2.
背景/目的：由猪圆环病毒 2 型（PCV-2）引起的断奶后多系统消耗综合征是一种对全球养猪业构成重大威胁的主要疾病。本研究旨在建立一种实时聚合酶链反应（PCR）方法来定量 PCV-2，并能够快速区分猪圆环病毒 1 型和 2 型（PCV-1 和 PCV-2）。这种方法将大大加快临床诊断过程，也可用于研究与 PCV-2 相关疾病的发病机制。
采用多重实时 PCR 结合 LightCycler PCR 数据分析软件对 PCV-2 进行定量，并对 PCV-1 和 PCV-2 进行快速区分。通过巢式 PCR 从 PCV-2 开放阅读框 2 的 3'末端扩增出 263bp 的 DNA 片段，其 DNA 序列与 PCV-2 标准品（NCBI 登录号：AF055394）完全一致。将 263bp 的 DNA 片段克隆到 pGEM-T easy 载体中，用实时 PCR 对重组质粒进行连续稀释和定量，然后构建标准曲线，用于定量田间样本中的 PCV-2 水平。通过分析基因型特异性 PCR 产物的熔点来区分 PCV-1 和 PCV-2。
为了定量田间样本中的 PCV-2 水平，构建了一个标准曲线（1x10(2)-1x10(9)拷贝/μL）。在从 PCV-2 感染猪的淋巴结或感染组织样本中提取的标本中，可检测到低至 1x10(2)拷贝/μL 的 PCV-2 浓度。使用商业提取试剂盒提取病毒 DNA 后，可在 45 分钟内完成对 PCV-1 和 PCV-2 感染的诊断和田间样本中病毒载量的定量。
本研究表明，实时 PCR 是一种准确定量 PCV-2 和快速区分 PCV-1 和 PCV-2 的临床可行方法。
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