D型流感病毒实时荧光定量PCR方法的建立与应用  

Establishment and application of real-time fluorescence quantitative PCR method for Influenza D virus

作　　者：王旭阳 卢刚 程娇娇 蔡思棋 钟林涛 赖志颖 贾斌 徐亮 欧嘉俊 肖雨晴 胡学瑞 王芳 赖悯婷 邵冉 郑飞燕 远立国 WANG Xuyang;LU Gang;CHENG Jiaojiao;CAI Siqi;ZHONG Lintao;LAI Zhiying;JIA Bin;XU Liang;OU Jiajun;XIAO Yuqing;HU Xuerui;WANG Fang;LAI Minting;SHAO Ran;ZHENG Feiyan;YUAN Liguo(College of Veterinary Medicine/Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases,South China Agricultural University,Guangzhou 510642,China;Branch of Animal Husbandry and Veterinary,Heilongjiang Academy of Agricultural Sciences,Qiqihar 161006,China)

机构地区：[1]华南农业大学兽医学院/广东省兽医临床重大疾病综合防控重点实验室,广州510642 [2]黑龙江省农业科学院畜牧兽医分院,黑龙江齐齐哈尔161006

出　　处：《黑龙江畜牧兽医》2023年第18期83-89,137,共8页Heilongjiang Animal Science And veterinary Medicine

摘　　要：为了建立一种可以快捷、灵敏、安全地检测D型流感病毒(Influenza D virus,IDV)的实时荧光定量PCR(quantitative real time PCR,RT-qPCR)方法,试验首先将IDV NP基因作为检测目标,根据其序列的保守区设计了一对RT-qPCR引物,然后采用单一变量法对RT-qPCR反应条件中的引物浓度和退火温度进行优化,建立了一种可检测IDV的RT-qPCR方法,并对该方法的灵敏度、重复性和特异性进行检测,最后应用该方法进行临床样本检测。结果表明:所建立的RT-qPCR方法的最佳引物浓度和退火温度分别为400 nmol/L和61℃,标准曲线为y=-3.572x+42.02(R^(2)=0.9993);该方法能检测到的最低拷贝数为8.30×10^(1)copies,灵敏度是常规PCR的100倍左右;组内重复和组间重复变异系数(CV)值均小于3%;同时检测牛呼吸道合胞体病毒(BRSV)、牛传染性鼻气管炎病毒(IBRV)、牛病毒性腹泻病毒(BVDV)、牛冠状病毒(BCoV)、牛副流感病毒3型(BPIV-3)、牛疱疹病毒4型(BHV-4)和牛流行热病毒(BEFV)的结果均为阴性;应用该方法对收集到的244份有呼吸道症状的牛鼻拭子样品进行检测,阳性率为1.23%。说明成功建立了IDV RT-qPCR检测方法,该方法具有灵敏度高、重复性和特异性好的特点,为IDV的防控提供了可靠的技术支撑。In order to establish a quick,sensitive and safe real-time quantitative PCR(RT-qPCR)method for the detection of Influenza D virus(IDV),in the experiment,firstly,the IDV NP gene was used as the detection target,and a pair of RT-qPCR primers was designed based on the conserved region of its sequence.Then,the single variable method was used to optimize the primer concentration and annealing temperature in the RT-qPCR reaction conditions,and a RT-qPCR method that could detect IDV was established.The sensitivity,repeatability and specificity of the method were detected;finally,this method was applied for clinical sample detection.The results showed that the optimal primer concentration and annealing temperature of the established RT-qPCR method were 400 nmol/L and 61 C,respectively.The standard curve was y=-3.572x+42.02(R^(2)=0.9993).The minimum number of copies detected by this method was 8.30x10^(1)copies,which was about 100 times the sensitivity of conventional PCR.The values of the coefficient of variation(CV)of repeats within and between groups were less than 3%.The results of Bovine respiratory syncytial virus(BRSV),Bovine alphaherpesvirus 1(IBRV),Bovine viral diarrhea virus(BVDV),Bovine coronavirus(BCoV),Bovine parainfluenza virus type 3(BPIV-3),Bovine herpesvirus type 4(BHV-4)and Bovine ephemeral fever virus(BEFV)were all negative.This method was used to test 244 bovine nasal swab samples with respiratory symptoms collected,and the positive rate was 1.23%.The results suggested that the IDV RT-qPCR detection method was successfully established;this method had the characteristics of high sensitivity,repeatability and specificity,which provided reliable technical support for the prevention and control of IDV.

关 键 词：D型流感病毒(IDV) 实时荧光定量PCR(RT-qPCR) SYBR GreenⅠ染料 方法 建立 临床检测 

分 类 号：S855.3[农业科学—临床兽医学]