猪圆环病毒3型微滴数字PCR定量检测方法的建立
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摘要
猪圆环病毒3型是一种新型猪圆环病毒,可引起猪感染相关疾病。本实验根据猪圆环病毒3型的OPF2基因序列,设计引物探针,建立了可对其准确定量检测的微滴数字PCR(droplet digital PCR,dd PCR)方法。通过对dd PCR反应体系中的引物探针浓度和退火温度进行优化,得到dd PCR反应的最佳引物探针浓度比为800 nM:800 nM:400 nM;最佳退火温度为60℃。该dd PCR方法的标准曲线相关系数(R2)为0.999,呈良好的线性关系;灵敏度高,可达4.4 copies/μL;特异性良好,与常见猪病原无交叉反应。通过对临床样品的检测结果证明,本研究建立的猪圆环病毒3型dd PCR检测方法比实时荧光PCR检测方法的灵敏度高1个数量级,检测结果与荧光PCR定性结果一致,并且可对可疑样本进行定量分析。结果表明:新建立的dd PCR方法灵敏度高、特异性强,可用于猪圆环病毒3型的定量检测。
Porcine circovirus type 3 is a novel porcine circovirus that can cause diseases associated with porcine infection. In this experiment, primer probes were designed based on the OPF2 gene sequence of porcine circovirus type 3, and a droplet digital PCR(ddPCR)method for accurate and quantitative detection was established. After optimization of the primer probe concentration and annealing temperature for the dd PCR reaction system, the optimal primer probe concentration ratio was 800 n M: 800 n M: 400 n M; and the optimal annealing temperature was 60 ℃. The correlation coefficient R2 of the dd PCR standard curve was 0.999, showing a good linear relationship. The sensitivity was high with the limit of detection being 4.4 copies/μL and good specificity(no cross-reaction with the common pig pathogens).The detection results of the clinical samples revealed that the ddPCR method established in this study was more sensitive than the real-time fluorescent PCR method by one order of magnitude, and could generate results consistent with those obtained by the fluorescent PCR and analyze quantitatively suspicious samples. The results showed that the newly established ddPCR method in this study had high sensitivity and specificity, thus could be used for quantitative detection of porcine circovirus type 3.
Porcine circovirus type 3 is a novel porcine circovirus that can cause diseases associated with porcine infection. In this experiment, primer probes were designed based on the OPF2 gene sequence of porcine circovirus type 3, and a droplet digital PCR(ddPCR)method for accurate and quantitative detection was established. After optimization of the primer probe concentration and annealing temperature for the dd PCR reaction system, the optimal primer probe concentration ratio was 800 n M: 800 n M: 400 n M; and the optimal annealing temperature was 60 ℃. The correlation coefficient R2 of the dd PCR standard curve was 0.999, showing a good linear relationship. The sensitivity was high with the limit of detection being 4.4 copies/μL and good specificity(no cross-reaction with the common pig pathogens).The detection results of the clinical samples revealed that the ddPCR method established in this study was more sensitive than the real-time fluorescent PCR method by one order of magnitude, and could generate results consistent with those obtained by the fluorescent PCR and analyze quantitatively suspicious samples. The results showed that the newly established ddPCR method in this study had high sensitivity and specificity, thus could be used for quantitative detection of porcine circovirus type 3.
引文
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[16]姜辰龙,严秀文,张日腾,等.猪圆环病毒3型LAMP检测方法的建立与应用[J].畜牧兽医学报,2018,49(6):1314-1319JIANG Chen-long,YAN Xiu-wen,ZHANG Ri-teng,et al.Establishment and application of LAMP method for detection of porcine circovirus type 3[J].Acta veterinaria et Zootechnica Sinica,2018,49(6):1314-1319
[17]徐朋丽,赵宇,张宇,等.猪圆环病毒3型SYBRGreenⅠ荧光定量PCR检测方法的建立[C]//中国畜牧兽医学会动物传染病学分会第九次全国会员代表大会暨第十七次全国学术研讨会论文集.2017XU Peng-li,ZHAO Yu,ZHANG Yu,et al.Establishment of CYBR Green I fluorescence quantitative PCR assay for porcine circovirus type 3[C]//Ninth National Congress of the Chinese Society of Animal Husbandry and Veterinary Medicine and the Tenth National Congress Proceedings of seven national academic seminars.2017
[18]李畅,库旭钢,王俊伟,等.猪圆环病毒3型Taq Man荧光定量PCR方法的建立和初步应用[J].畜牧兽医学报,2018,49(6):1320-1326LI Chang,KU Xu-gang,WANG Jun-wei,et al.Establishment and preliminary application of porcine circovirus type 3 Taq Man fluorescent quantitative PCRmethod[J].Journal of Animal Husbandry and Veterinary Medicine,2018,49(6):1320-1326
[2]Shen H,Liu X,Zhang P,et al.Genome characterization of a porcine circovirus type 3 in South China[J].Transboundary&Emerging Diseases,2018,65(1)
[3]Lu B,Qin Y,Ying H,et al.Complete genome sequence of a novel porcine circovirus type 3 strain,CH/GX/1776D/2017,isolated from guangxi,China[J].Genome Announcements,2018,6(17)
[4]李军,贺会利,潘艳,等.广西猪圆环病毒3型感染的初步调查[C]//中国畜牧兽医学会动物传染病学分会第九次全国会员代表大会暨第十七次全国学术研讨会论文集.2017LI jun,HE Hui-li,PAN Yan,et al.Preliminary investigation of porcine circovirus type 3 infection in guangxi[C]//proceedings of the 9th national congress of the branch of animal infectious diseases of China animal husbandry and veterinary association and the 17th national academic symposium.2017
[5]冯兆民,舒跃龙.数字PCR技术及其应用进展[J].病毒学报,2017,1:103-107FENG Zhao-min,SHU Yue-long.Digital PCR technology and its application progress[J].Journal of viruses,2017,1:103-107
[6]汪惠泽.盐酸克伦特罗单克隆抗体的制备及其Ci ELISA检测方法的建立[D].扬州大学,2012WANG Hui-ze.Preparation of clenbuterol monoclonal antibody and establishment of Ci ELISA detection method[D].Yangzhou university,2012
[7]李静芳,汤水平,张素文,等.实时荧光定量PCR技术在食品检测中的应用[J].实用预防医学,2008,15(6):1997-1999LI Jing-fang,TANG Shui-ping,ZHANG Su-wen et al.Application of real-time quantitative PCR in food detection[J].Practical preventive medicine,2008,15(6):1997-1999
[8]成媛媛.梅毒螺旋体核酸检测及基于微滴式数字PCR技术的梅毒螺旋体核酸绝对定量方法学研究[D].合肥:安徽医科大学,2017CHENG Yuan-yuan.Detection of Treponema pallidum nucleic acid and absolute quantitative methodological study of Treponema pallidum based on microdroplet digital PCRtechnology[D].Hefei:Anhui Medical University,2017
[9]刘静.RPA技术与微滴数字PCR在转基因玉米与大豆检测中的应用[D].上海海洋大学,2017LIU Jing.Application of RPA technology and microdrop digital PCR in detection of transgenic corn and soybean[D].Shanghai Ocean University,2017
[10]陈晨,张岩,李永波,等.微滴式数字PCR对肉制品中羊肉和猪肉定量分析[J].现代食品科技,2018,34(1):221-226,194CHEN Chen,ZHANG Yan,LI Yong-bo,et al.Quantitative analysis of mutton and pork in meat products by micro-droplet digital PCR[J].Modern food technology,2018,(1):221-226,194
[11]陈亚娜,王静,訾占超,等.伪狂犬病病毒微滴数字PCR定量检测方法的建立[J].畜牧兽医学报,2017,48(9):1705-1710CHEN Ya-na,WANG Jing,ZHAI Zhan-chao,et al.Establishment of quantitative PCR detection method for pseudorabies virus microdroplets[J].Journal of Animal Husbandry and Veterinary Medicine,2017,48(9):1705-1710
[12]原霖,訾占超,亢文华,等.猪圆环病毒1型微滴数字PCR定量检测方法的建立[J].中国兽医学报,2017,37(11):2043-2047YUAN Lin,ZHAI Zhan-chao,KANG Wen-hua,et al.Establishment of quantitative detection method for porcine circovirus type 1 microdroplet digital PCR[J].Chinese Journal of Veterinary Medicine,2017,37(11):2043-2047
[13]赵珊.猪圆环病毒2型微滴式数字PCR检测方法的建立与初步应用[D].四川农业大学,2016ZHAO Shan.Establishment and preliminary application of porcine circovirus type 2 microdroplet digital PCR detection method[D].Sichuan Agricultural University,2016
[14]邬旭龙,肖璐,宋勇,等.非洲猪瘟病毒微滴数字PCR(dd PCR)方法的建立及应用[J].微生物学通报,2017,44(12):2839-2846WU Xu-long,XIAO Lu,SONG Yong,et al.Establishment and application of African swine fever virus microdroplet digital PCR(dd PCR)method[J].Bulletin of Microbiology,2017,44(12):2839-2846
[15]万绍贵,鲍登克,杨琪.一种用于绝对定量检测猪繁殖与呼吸综合征病毒的dd PCR方法,引物及其应用和试剂盒,CN107267664A[P].2017WAN Shao-gui,BAO Deng-ke,YANG Qi.A ddPCR method,primer,application and kit for absolute quantitative detection of porcine reproductive and respiratory syndrome virus,CN107267664 A[P].2017
[16]姜辰龙,严秀文,张日腾,等.猪圆环病毒3型LAMP检测方法的建立与应用[J].畜牧兽医学报,2018,49(6):1314-1319JIANG Chen-long,YAN Xiu-wen,ZHANG Ri-teng,et al.Establishment and application of LAMP method for detection of porcine circovirus type 3[J].Acta veterinaria et Zootechnica Sinica,2018,49(6):1314-1319
[17]徐朋丽,赵宇,张宇,等.猪圆环病毒3型SYBRGreenⅠ荧光定量PCR检测方法的建立[C]//中国畜牧兽医学会动物传染病学分会第九次全国会员代表大会暨第十七次全国学术研讨会论文集.2017XU Peng-li,ZHAO Yu,ZHANG Yu,et al.Establishment of CYBR Green I fluorescence quantitative PCR assay for porcine circovirus type 3[C]//Ninth National Congress of the Chinese Society of Animal Husbandry and Veterinary Medicine and the Tenth National Congress Proceedings of seven national academic seminars.2017
[18]李畅,库旭钢,王俊伟,等.猪圆环病毒3型Taq Man荧光定量PCR方法的建立和初步应用[J].畜牧兽医学报,2018,49(6):1320-1326LI Chang,KU Xu-gang,WANG Jun-wei,et al.Establishment and preliminary application of porcine circovirus type 3 Taq Man fluorescent quantitative PCRmethod[J].Journal of Animal Husbandry and Veterinary Medicine,2018,49(6):1320-1326
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