猪库布病毒纳米PCR快速检测方法的建立  

Establishment and preliminary application of nano-PCR method for detecting porcine Kubuvirus

作　　者：李睿 凤焱 孙亮[3] 姜秋杰[3] 呼延含荣 张秀森 程敏 郭利 赵立峰[1,2] LI Rui;FENG Yan;SUN Liang;JIANG Qiujie;HUYAN Hanrong;ZHANG Xiusen;CHENG Min;UO Li;ZHAO Lifeng(Jilin Agricultural University,Changchun 130122,China;Jilin Agricultural Science and Technology College,Jilin 132101,China;Jilin Animal Disease Prevention and Control Center,Changchun 133000,China;Liaoyuan Animal Disease Prevention and Control Center,Liaoyuan 136300,China)

机构地区：[1]吉林农业大学,吉林长春130122 [2]吉林农业科技学院,吉林吉林132101 [3]吉林省动物疫病预防控制中心,吉林长春133000 [4]辽源动物疫病预防控制中心,吉林辽源136300

出　　处：《中国兽医科学》2024年第3期325-329,共5页Chinese Veterinary Science

基　　金：吉林省科技厅科技发展计划项目(YDZJ202203CGZH050,20210202101NC);吉林省农业关键核心技术示范推广项目(202300702)。

摘　　要：为了给临床检测猪库布病毒(PKV)提供更加准确便捷的检测方法,本试验设计了1对特异性引物,扩增PKV基因组的VP0区域。在传统PCR反应体系中添加纳米金颗粒材料,优化后建立了一种方便快捷且特异检测PKV的nano-PCR方法。结果显示,该方法能特异性扩增猪库布病毒,与猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪丁型冠状病毒、猪轮状病毒、猪札幌病毒均无交叉反应。nano-PCR相对于普通PCR表现出更高的敏感性,普通PCR的最低核酸检测浓度为5.61×10^(-7)ng/μL,而nano-PCR的最低核酸检测浓度为5.61×10^(-10)ng/μL,敏感性提高了1000倍。对来自不同发病猪场30份腹泻猪的粪便和血液临床样本的检测结果显示,nano-PCR阳性检出率为23.33%(7/30),普通PCR阳性检出率为16.66%(5/30)。结果表明,本试验建立的nano-PCR方法在猪库布病毒的检测中表现出更高的特异性和敏感性,对猪库布病毒的快速诊断和检测具有重要的临床意义。In order to establish a method for rapid diagnosis of porcine kobuvirus and provide a more accurate and convenient method for clinical detection of the virus,this experiment designed a pair of specific primers to amplify the VP0 region of the PKV genome.Nano-gold particle materials were added to the traditional PCR reaction system,and after optimization,a convenient,fast and specific nano-PCR method for detecting PKV was established.The test results show that this method can specifically amplify porcine Kubuvirus and has no cross-reactivity with TGEV,PEDV,PDCo V,PRV and PSa V.The nano-PCR showed a higher sensitivity compared to the normal PCR,with a 1 000-fold increased sensitivity.The lowest concentration of nucleic acid detection for ordinary PCR was 5.61×10^(-7)ng/μL,while the lowest nucleic acid detection concentration for nano-PCR was only 5.61×10^(-10)ng/μL.Thirty feces and blood clinical samples from pigs with diarrhea from different pig farms were tested.The results showed that the positive detection rate of nano-PCR was 23.33%(7/30).The positive detection rate of ordinary PCR is 16.66%(5/30).Therefore,the nano-PCR method established in this trial showed higher specificity and sensitivity in the detection of porcine Kubuvirus,which has important clinical significance for the rapid diagnosis and detection of porcine Kubuvirus.

关 键 词：猪库布病毒 nano-PCR 纳米金颗粒 

分 类 号：S852.659.6[农业科学—基础兽医学]