猪流行性腹泻病毒实时荧光RPA等温检测方法的建立

doi:10.16431/j.cnki.1671-7236.2017.12.006

摘要/Abstract

摘要：

为建立一种简单、快速的猪流行性腹泻病毒（PEDV）分子检测方法，本研究基于PEDV病毒M基因保守序列，设计一系列扩增引物及其荧光探针，以包含PEDV病毒M基因片段的pUC57质粒为模板，同时以包含猪传染性胃肠炎病毒（TGEV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）及古典猪瘟病毒（CSFV）等病毒膜蛋白或核衣壳蛋白基因序列的质粒作为对照，建立了一种PEDV实时荧光重组酶聚合酶扩增（RPA）等温检测方法，测定了方法的特异性与敏感性。结果表明，该实时荧光RPA方法可在39℃恒温反应20 min特异性扩增PEDV病毒M基因，与TGEV、PRRSV等对照病毒基因无交叉反应，其检测限为10拷贝/μL。应用该实时荧光RPA方法可有效检测出血浆及血浆蛋白粉中的PEDV核酸。本研究建立的实时荧光RPA检测方法简单、快速、灵敏度高，可为PEDV的检测防控提供一种新的、可靠的技术支持。

关键词: 猪流行性腹泻病毒; 分子检测; 重组酶聚合酶扩增(RPA); 等温扩增

Abstract:

To develop a precise and rapid diagnosis method for detecting porcine epidemic diarrhoea virus (PEDV), a series of recombinase polymerase amplification (RPA) primers and exo-probes were established based on the highly conserved M gene of PEDV. Then a Real-time RPA assay was developed to detect PEDV using pUC57 plasmid carrying M gene fragment of PEDV as template, and the membrane or nucleotide capsid proteins from TGEV, PRRSV, PCV2 and CSFV were utilized as control. Then the sensitivity and specificity of this Real-time RPA assay was evaluated. The results showed that the Real-time reaction could detect PEDV specifically at 39℃ within 20 min with the detection limit of 10 copies/μL of plasmid DNA, and there was no cross-reaction with other control viral pathogens. Besides, the established Real-time PRA method could successfully detecte the PEDV M gene in the plasma and plasma protein power. The Real-time established in this study was simple, rapid and sensitive, which could be a novel and reliable method for diagnosing and control of PED.

Key words: porcine epidemic diarrhoea virus (PEDV); molecular diagnosis; recombinase polymerase amplification (RPA); isothermal amplification

中图分类号:

S858.28

吕继洲, 范燕茹, 冯春燕, 袁向芬, 吴绍强. 猪流行性腹泻病毒实时荧光RPA等温检测方法的建立[J]. 《中国畜牧兽医》, 2017, 44(12): 3434-3439.

LV Ji-zhou, FAN Yan-ru, FENG Chun-yan, YUAN Xiang-fen, WU Shao-qiang. Establishment of Real-time Recombinase Polymerase Amplification for Detection of Porcine Epidemic Diarrhoea Virus[J]. , 2017, 44(12): 3434-3439.

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