中国人兽共患病学报2012，Vol.28Issue(9)：922-926,5.DOI:10.3969/cjz.j.issn.1002-2694.2012.09.013
猪嵴病毒实时荧光定量RT-PCR方法的建立
Establishment of a real-time RT-PCR method based on SYBR GreenⅠfor diagnosis of porcine kobuvirus
摘要
Abstract
A pair of specific primers targeted to the 3D gene of porcine kobuvirus was designed and a real-time reverse-transcription polymerase chain reaction (RRT-PCR) based on SYBR Green I fluorescent was developed for quantization of the porcine kobuvirus infection. The standard curve generated a wide dynamic range of 6. 42 × 102 -6. 42 × 108 DNA copies/μL with a linear correlation (R2) of 0. 999 and efficiency of 100% between the Ct value and the logarithm of the plasmid copy number. The melting curve analysis using SYBR Green I dye showed one specific peaked with a melting temperature (Tm) of (84. 94± 0. 24) ℃ with no primer-dimer peak represent. No amplificon was detected from unrelated DNA samples by this method, such as porcine transmissible gastroenteritis virus, porcine rotavirus, porcine circovirus, porcine reproductive and respiratory syndrome, pseudorabies virus, and classical swine fever virus. Kxcellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0. 26% - 1. 14% % and inter-assay of 0. 63% - 1. 79%. A real-time RT-PCR method established in this study may be used for earlier diagnosis and quantitative analysis of the infection status and the target organs infected with porcine kobuvirus.关键词
猪嵴病毒/SYBR GreenⅠ/实时荧光定量RT-PCRKey words
porcine kobuvirus/SYBR Green I/real time reverse-transcription polymerase chain reaction分类
农业科技引用本文复制引用
修金生,陈小权,王斌,李涛..猪嵴病毒实时荧光定量RT-PCR方法的建立[J].中国人兽共患病学报,2012,28(9):922-926,5.基金项目
福建省农业科学技术项目(2009-02)(2009-03) （2009-02）
