猪瘟病毒实时荧光LAMP检测方法的建立

Establishment of Real-time Fluorescence Loop-mediated Isothermal Assay for Detection of Classical Swine Fever Virus

摘要:

为做好猪瘟的早期诊断和及时预防，建立一种快速检测猪瘟病毒(Classical swine fever virus，CSFV)的实时荧光环介导等温扩增方法。试验利用Primer Explorer version 4针对CSFV的5`UTR非编码区设计4组引物，筛选出最佳引物后建立CSFV的实时荧光环介导等温扩增检测方法，并对该方法的特异性、灵敏度以及适用性进行验证。结果显示，本试验建立的实时荧光环介导等温扩增方法对128份临床样本和22份猪瘟活疫苗人工干扰样本的检测结果与实时荧光聚合酶链式反应检测方法一致。该方法仅对猪瘟病毒有扩增反应，且最低检测浓度为10 fg/μL，对猪伪狂犬病毒、高致病性高致病型猪繁殖与呼吸综合征病毒、猪圆环病毒2型等8种病原无扩增反应。本试验建立的实时荧光环介导等温扩增方法特异性强、灵敏度高，检测效果好，操作简单，适用于CSFV临床样本的快速检测。

Abstract:

In order to make early diagnosis and prompt prevention of swine fever, a real-time fluorescent loop-mediated isothermal amplification method for rapid detection of classical swine fever virus (CSFV) was established. Using Primer Explorer version 4 to design 4 sets of primers for the 5'UTR non-coding region of CSFV, the best primers were selected to establish a real-time fluorescent loop-mediated isothermal amplification assay for CSFV, and the specificity, sensitivity and applicability were verified. The results showed that the real-time fluorescent loop-mediated isothermal amplification method established in this experiment was consistent with the real-time fluorescent polymerase chain reaction detection method for 128 clinical samples and 22 artificial interference sample by hog cholera live vaccine. This method only has amplified reaction for classical swine fever virus and the minimum detectable concentration was 10 fg/μL. There were no amplified reactions for eight pathogens such as virulence, highly pathogenic highly pathogenic porcine reproductive and respiratory syndrome virus and porcine circovirus type 2. The real-time fluorescent loop-mediated isothermal amplification method established in this experiment has strong specificity, high sensitivity, good detection effect and simple operation, so is suitable for rapid detection of clinical samples of CSFV.