猪细小病毒重组酶聚合酶扩增快速检测方法的建立

doi:10.16431/j.cnki.1671-7236.2017.11.030

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (11): 3320-3326.doi: 10.16431/j.cnki.1671-7236.2017.11.030

猪细小病毒重组酶聚合酶扩增快速检测方法的建立

刘立兵^1,2, 王建昌^1,2, 经美³, 王金凤^1,2, 袁万哲³

1. 河北出入境检验检疫局检验检疫技术中心, 石家庄 050051;
2. 河北省检验检疫科学技术研究院, 石家庄 050051;
3. 河北农业大学动物医学院, 保定 071001

收稿日期:2017-03-28 出版日期:2017-11-20 发布日期:2017-11-21
通讯作者: 袁万哲 E-mail:yuanwanzhe@126.com
作者简介:刘立兵(1986-),男,吉林榆树人,硕士,中级兽医师,研究方向:动物疫病分子生物学,E-mail:bing521564@163.com
基金资助:
河北省自然科学基金青年项目（C2017325001）；猪主要繁殖障碍性疫病防控关键技术研究（16226604D）

Development of the Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Parvovirus

LIU Li-bing^1,2, WANG Jian-chang^1,2, JING Mei³, WANG Jin-feng^1,2, YUAN Wan-zhe³

1. Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang 050051, China;
2. Hebei Academy of Science and Technology for Inspection and Quarantine, Shijiazhuang 050051, China;
3. College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China

Received:2017-03-28 Online:2017-11-20 Published:2017-11-21

摘要/Abstract

摘要：

试验旨在建立一种简单、快速检测猪细小病毒（porcine parvovirus，PPV）的重组酶聚合酶（recombinase polymerase amplification，RPA）检测方法，为中国PPV的防控和诊断提供一种新的、可靠的技术支持。本研究基于PPV VP2基因的保守序列，设计合成1对RPA引物探针，通过对反应时间的优化，建立38℃恒温水浴锅检测PPV的RPA方法。结果表明，所建立的RPA方法在38℃水浴锅中恒温反应30 min，能够特异性的检测PPV；以重组质粒pPPV-VP2作为模板，RPA的检测限为10²拷贝，同本研究中应用的实时荧光定量PCR方法检测限一致，比普通PCR方法高100倍；RPA方法对疑似PPV感染临床样品的阳性检出率为82.6%，略低于实时荧光定量PCR的检出率（86.9%），明显高于普通PCR的阳性检出率（66.7%）。本研究建立的RPA方法操作简单、反应快速，结果确实可靠，适用于PPV的快速检测。

关键词: 猪细小病毒(PPV); 等温扩增; 聚合酶重组酶扩增; 快速检测

Abstract:

This study was aimed to develop a simple and rapid method for the porcine parvovirus (PPV) with recombinase polymerase amplification (RPA), which could be a novel and reliable tool for the control and detect of PPV. The RPA was developed using specific primers for the conserved region of PPV VP2 gene. The reaction time was optimized,the RPA reaction could amplify the PPV, and was performed successfully at 38℃ for 30 min in a water bath. The results showed that the detection limit of RPA was 102 copies of plasmid DNA, which was the same as the Real-time quantitative PCR applied in this study, and 100 times more sensitive than conventional PCR. For the clinical samples from the suspected PPV-infected pigs, the positive detection ratio was 82.6% for RPA, which was lower than that of Real-time quantitative PCR (86.9%), but was much higher than conventional PCR (66.7%). The PPV RPA assay developed in the study was simple, rapid and reliable, and was suitable for rapid detection of PPV.

Key words: porcine parvovirus (PPV); isothermal amplification; recombinase polymerase amplification (RPA); rapid detection

中图分类号:

S852.65⁺9.2

刘立兵, 王建昌, 经美, 王金凤, 袁万哲. 猪细小病毒重组酶聚合酶扩增快速检测方法的建立[J]. 《中国畜牧兽医》, 2017, 44(11): 3320-3326.

LIU Li-bing, WANG Jian-chang, JING Mei, WANG Jin-feng, YUAN Wan-zhe. Development of the Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Parvovirus[J]. , 2017, 44(11): 3320-3326.

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猪细小病毒重组酶聚合酶扩增快速检测方法的建立

Development of the Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Parvovirus

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