用于检测猪圆环病毒 2 型的 LUX 实时 PCR 检测法。

LUX real-time PCR assay for the detection of porcine circovirus type 2.

机构信息

University of Veterinary Medicine and Pharmacy, Dept. of Infectious Diseases and Parasitology, SK-041 81 Kosice, Slovakia.

出版信息

J Virol Methods. 2010 May;165(2):216-21. doi: 10.1016/j.jviromet.2010.01.023. Epub 2010 Feb 6.

DOI:10.1016/j.jviromet.2010.01.023

PMID:20138916

Abstract

Light Upon eXtension real-time PCR (LUX real-time PCR) assay was developed for the detection of porcine circovirus type 2 (PCV2). The primers flanking a 114 bp fragment were selected from ORF1. The optimized assay could detect 20 viral copies of pBluescript SK+ plasmid containing inserted PCV2 DNA. The dynamic range of quantitative analysis covered a 7-order interval ranging from 20 to 2 x 10(8) genome equivalents per assay with the best results in the range from 2 x 10(2) to 2 x 10(7) viral copies. The LUX real-time PCR assay had a high specificity since it detected PCV2 but not PCV1, CSFV, PRRSV or negative samples. There was good agreement between the LUX real-time PCR and the conventional PCR when lymph nodes from PCV2 infected animals were tested. A comparison of the LUX real-time PCR with the TaqMan PCR and SYBR Green PCR indicated that the amount of viral copies determined using linear calibration curve differed from assay to assay but not more than an order. LUX real-time PCR, similar to the TaqMan PCR, was more specific for generation of fluorogenic signal than SYBR Green PCR.

摘要

LUX 实时 PCR（LUX real-time PCR）检测方法是为了检测猪圆环病毒 2 型（PCV2）而开发的。该引物位于 ORF1 侧翼，可检测到含有插入 PCV2 DNA 的 pBluescript SK+质粒的 20 个病毒拷贝。定量分析的动态范围涵盖了 7 个数量级，从每个检测的 20 到 2 x 10(8)基因组当量，最佳结果在 2 x 10(2)到 2 x 10(7)病毒拷贝范围内。由于该检测方法仅检测 PCV2，而不检测 PCV1、CSFV、PRRSV 或阴性样本，因此具有很高的特异性。当检测感染 PCV2 的动物的淋巴结时，LUX 实时 PCR 与常规 PCR 之间具有良好的一致性。与 TaqMan PCR 和 SYBR Green PCR 相比，LUX 实时 PCR 表明使用线性校准曲线确定的病毒拷贝数量因检测而异，但不超过一个数量级。与 SYBR Green PCR 相比，LUX 实时 PCR 与 TaqMan PCR 一样，在产生荧光信号方面更具特异性。