非洲猪瘟病毒微芯片荧光PCR快速检测技术的建立及初步应用

非洲猪瘟缺乏安全有效的疫苗,其防控有赖于及时、准确的诊断和消毒灭源。本研究在对非洲猪瘟病毒(African swine fever virus,ASFV)B646L基因序列进行分析的基础上设计引物、探针,建立了基于微芯片的ASFV免提取荧光PCR检测技术。特异性试验结果显示,微芯片PCR可用于我国不同地区ASFV毒株的通用检测,对其他主要猪源病毒均为阴性;敏感性试验结果显示其检测下限为10拷贝/μL;对117份临床样品的检测结果显示,该方法可用于猪全血、淋巴结、脾脏、肌肉样本中ASFV核酸的检测,与世界动物卫生组织推荐的荧光PCR检测方法相符率达100%,且显著简化了检测操作步骤,检测用时缩短在30 min之内,获得农业农村部第一批非洲猪瘟现场快速检测试剂的推荐,应用于养殖场、屠宰场和流通环节中ASFV核酸的检测。

Establishment and application of microchip real-time PCR assay for rapid identification of African swine fever virus

No vaccine is available to prevent African swine fever(ASF)infection.The prevention and eradication measures are based on classical disease control methods,including timely and accurate surveillance and stamping out in infected holdings.To detect African swine fever virus(ASFV),primers and probes were designed based on the analysis of the B646L gene of ASFV,and a microchip-based ASFV extraction-free fluorescent PCR detection technology was established.The results of specificity test showed that the assay could be used for the universal detection of ASFV strains in different regions of China,and was negative for other major swine-origin viruses.Sensitivity test results showed the lower limit of detection was 10 copies/μL.The test results of 117 clinical samples detected by the established microchip real-time PCR and the quantitative PCR method recommended by the World Organization for Animal Health(OIE)showed that this method could be used for the detection of ASFV nucleic acid from pig whole blood,lymph nodes,spleen,and muscle samples,the coincidence rate of the two methods was 100%.This method significantly simplifies the detection operation steps,the detection can be completed within 30 min,indicating that it is rapid and suitable for on-site detection.Therefore,it has been recommended as the first batch of ASF rapid detection of reagents by the Ministry of Agriculture and Rural Affairs,and is widely used by farms,slaughter houses and markets.