猪流行性腹泻病毒RT-LAMP检测方法的建立与应用

Establishment and Application of RT-LAMP for Detection of Porcine Epidemic Diarrhea Virus

关键词:  LAMP  猪流行性腹泻病毒  快速检测  检测方法

Key words:  LAMP  PEDV  rapid detection  test method

摘要:

为建立一种适用于现场快速检测猪流行性腹泻病毒(PEDV)的环介导等温扩增(LAMP)检测方法，针对猪流行性腹泻病毒(PEDV)的M基因编码区序列，分别设计合成3组引物，通过对引物的筛选和反应条件优化，建立了PEDV RT-LAMP可视化快速检测方法。灵敏度和特异性试验结果显示，本方法在65 ℃ 45 min对PEDV的检测灵敏度为2 copies/μL，与猪传染性胃肠炎病毒、猪轮状病毒、猪瘟病毒、猪伪狂犬病毒、猪繁殖与呼吸综合征病毒均无交叉反应。利用该方法对30份送检的临床样品进行检测，检出阳性样品8份，与荧光定量RT-PCR检测结果一致。试验表明，所建立的RT-LAMP检测方法具有特异性好、灵敏度高、快速、结果可视、设备适用范围广等优点，适用于PEDV现场快速检测。

Abstract:

In order to establish a loop-mediated isothermal amplification (LAMP) method for rapid detection of porcine epidemic diarrhea virus (PEDV), three sets of primers for the M gene coding region sequence of PEDV were designed and synthesized, and a visualized rapid detection method of PEDV RT-LAMP was established by screening primers and optimizing reaction conditions. The results of specificity and sensitivity test showed that the sensitivity of this method to detect PEDV was 2 copies/μL, within 45 minute at 65 ℃, and there was no cross reaction with transmissible gastroenteritis virus, swine rotavirus, swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus. The method was used to detect 30 clinical samples, 8 positive samples were detected, which was consistent with the results of fluorescence quantitative RT-PCR. The experimental results show that the RT-LAMP detection method has the advantages of good characteristics, high sensitivity, high speed, visible results, wide application range of equipment and so on. It is suitable for the field rapid detection of PEDV.