7种猪繁殖障碍性疫病液相芯片快速检测方法的建立与应用  

Establishment and Application of Liquid Chip Method for Rapid Detection of Seven Porcine(Sus scrofa)Reproductive Diseases

作　　者：邬旭龙 肖璐 杨苗 谢礼 安微 郭杨 张琴 王印[4] 林华 WU Xu-Long;XIAO Lu;YANG Miao;XIE Li;AN Wei;GUO Yang;ZHANG Qin;WANG Yin;LIN Hua(Animal Epidemic Disease Detection Center,Chengdu Agricultural College,Chengdu 611130,China;Key Laboratory of Animal Genetics and Breeding of Sichuan Province,Sichuan Animal Science Academy,Chengdu 610066,China;Key Laboratory of Food Safety Testing of Sichuan Province,Technology Center of Chengdu Customs,Chengdu 610041,China;College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]成都农业科技职业学院动物疫病检测中心,成都611130 [2]四川省畜牧科学研究院动物遗传育种四川省重点实验室,成都610066 [3]成都海关技术中心食品安全检测四川省重点实验室,成都610041 [4]四川农业大学动物医学院,成都611130

出　　处：《农业生物技术学报》2023年第1期203-212,共10页Journal of Agricultural Biotechnology

基　　金：成都农业科技职业学院科研基金项目(22ZR203);海关总署科研项目(2020HK141);四川省重点研发项目(2021YFS0002)。

摘　　要：随着我国养猪业的发展和进步,对于疾病的鉴别诊断和大数据分析提出了更高的要求和目标。为建立一种同时检测7种猪(Sus scrofa)繁殖障碍性疫病的方法,促进高通量检测技术在动物病原检测中的发展和应用,本研究针对猪伪狂犬病毒(Pseudorabies virus,PRV)、猪日本乙型脑炎病毒(Japanese encephalitis virus,JEV)、猪瘟病毒(Classical swine fever virus,CSFV)、猪圆环病毒2型(Porcine circovirus type 2,PCV-2)、猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪细小病毒(Porcine parvovirus,PPV)、非洲猪瘟病毒(African classical swine fever virus,ASFV)7种猪繁殖障碍性疫病病原,设计特异性引物、探针,经反应条件优化、探针与微球偶联和杂交捕获反应,成功建立1种同时检测7种猪疫病的液相芯片检测方法,并将该方法进行临床应用。结果显示,微球与探针偶联效率较高,在液相环境中能实现对特异性目的基因捕获检测,该方法检测灵敏性较高,可同时检测到7种特异性病原的最低检测限度为103 copies/μL;该方法检测特异性好,与猪其他常见病原无交叉反应,重复性试验中组内和组间检测数据一致性较强。利用建立的方法对临床采集四川规模化猪场疑似发病的65份样品和ASFV阳性对照核酸进行检测和验证,共检出49份阳性样品,阳性率达75.38%(49/65),混合感染检出阳性率为23.08%(15/65),经单重PCR复检后结果一致,证实建立的液相芯片检测方法可应用于临床检测。本研究为猪病多种病原快速鉴别诊断提供有力的技术储备,同时为高通量检测技术的进一步发展提供参考。With the development and progress of the pig industry in China,higher expection are required for the differential diagnosis and big data analysis of diseases.Here,a novel method was established,which promoted the development and application of high-throughput detection technology in animal pathogen detection.In detail,specific primers and probes were designed for the Pseudorabies virus(PRV)、Japanese encephalitis virus(JEV)、Classical swine fever virus(CSFV)、Porcine circovirus type 2(PCV-2)、Porcine reproductive and respiratory syndrome virus(PRRSV)、Porcine parvovirus(PPV)、African classical swine fever virus(ASFV)7 pathogens of swine(Sus scrofa)reproductive diseases.After optimizing the reaction conditions,coupling the probe with microspheres,and hybridization capture reaction,a liquid chip detection method to detect 7 pig reproductive diseases at the same time was successfully established and applied to clinical practice.The results showed that the specific target gene could be captured and detected in the liquid environment,the detection limit of 7 specific pathogens was 103 copies/μL.The method had good specificity and no cross reaction with other pathogens.The established method was used to detect the 65 samples collected from large-scale pig farms in Sichuan province.A total of 49 positive samples were detected,with a positive rate of 75.38%(49/65).The positive rate of mixed infection of 2 or more pathogens was 23.08%(15/65).The results of single PCR detection were consistent with the established method,which confirmed that the established liquid chip detection method can be applied to clinical detection.This study provides a powerful technical reserve for rapid differential diagnosis of multiple pathogens of swine disease,and provides a reference for the further development of high-throughput detection technology.

关 键 词：液相芯片 高通量 猪繁殖障碍疫病 临床检测 

分 类 号：S855.3[农业科学—临床兽医学]