同时检测PEDV和TGEV及PDCoV的多重RT-PCR方法的建立及初步应用

发布时间：2018-04-01 12:15

  本文选题：多重RT-PCR　切入点：猪流行性腹泻病毒　出处：《中国兽医科学》2016年06期

【摘要】：为建立一种同时检测猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪Delta冠状病毒(PDCo V)的多重RT-PCR方法,根据Gen Bank中PEDV和TGEV基因序列保守区设计2对引物,参照文献合成1对PDCo V引物,优化三对引物在同一RT-PCR扩增体系下的浓度、退火温度等反应条件,同时优化方法的灵敏度、特异性。将初步建立的多重RT-PCR方法用于检测采自四川省及重庆市14个猪场的222份样品。结果表明,本试验建立的多重RT-PCR在引物量分别为PEDV 0.2 L,PDCo V 0.2 L和TGEV 0.4L,退火温度为53℃时的扩增效果最佳;最低检测量分别为PEDV:60.96 pg、PDCo V:58.85 pg、TGEV:102.69 pg;用该法对多种猪传染病病原DNA或c DNA进行扩增时均无非特异性扩增条带出现。对222份腹泻样品的检测结果表明,PEDV阳性检出率为52.2%,PDCo V为2.7%,TGEV为2.3%。同时多重RT-PCR检测方法和单项RT-PCR检测方法符合率分别为PEDV 92.9%、PDCo V 100%、TGEV 100%。表明该法具较高可信度。本试验建立了一种高效、特异地同时检测PEDV、TGEV和PDCo V的多重RT-PCR方法,为病原的实验室诊断和分子流行病学调查提供了技术支持。对四川省及重庆市部分猪场三种病原流行情况进行调查和统计分析,为地区猪腹泻病的防控提供了依据。
[Abstract]:In order to establish a multiplex RT-PCR method for simultaneous detection of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV) and porcine Delta coronavirus (PDCo V), two pairs of primers were designed according to the conserved region of PEDV and TGEV gene in Gen Bank. A pair of PDCo V primers were synthesized with reference to the literature. The reaction conditions such as the concentration of three pairs of primers under the same RT-PCR amplification system and annealing temperature were optimized, and the sensitivity of the method was optimized. The multiplex RT-PCR method was used to detect 222 samples from 14 pig farms in Sichuan province and Chongqing city. In this experiment, the amplification effect of multiple RT-PCR was the best when the number of primers was 0.2L PEDV 0.2L PDCo V 0.2L and TGEV 0.4L, and the annealing temperature was 53 鈩，

本文编号：1695531