狂犬病病毒和犬2型腺病毒双重荧光定量PCR检测方法的建立及应用

Establishment and preliminary application of dual fluorescent quantitative PCR for detection of RV and CAV2

目的为了解CAV2-ΔE3-CGS口服疫苗免疫后排毒情况和掌握犬腺病毒(CAV)感染本底情况,研究建立狂犬病病毒和犬2型腺病毒双重荧光定量PCR检测方法。方法针对CAV2-ΔE3-CGS中犬2型腺病毒E1基因和狂犬病病毒G基因设计特异性引物和探针,建立检测CAV2-ΔE3-CGS的双重荧光定量PCR检测方法,并对实验动物场和犬类留验场流浪犬口鼻拭子和环境样本进行了检测。结果该方法生成的标准曲线分别为Y=-3.351×logX+44.895,R^(2)为0.999和Y=-3.413×logX+45.192,R^(2)为0.996,线性关系良好,最低检测线都为102拷贝/μL。在实验动物场流浪犬和环境中未检测到CAV2-ΔE3-CGS;在犬类留验场流浪犬中检测到CAV感染,其中口鼻拭子阳性率为5.88%(5/85),肛拭子阳性率为8.24%(7/85),环境样本阳性率为4.00%(1/25)。结论研究建立的双重荧光定量PCR检测方法适用于CAV2-ΔE3-CGS排毒情况排查和CAV感染调查。研究表明CAV2-ΔE3-CGS免疫后未检测到排毒,上海市流浪犬CAV感染率较低,因此符合CAV2-ΔE3-CGS的口服免疫要求。

Objective To investigate the shedding of CAV2-ΔE3-CGS after immunization and the background of canine adenovirus(CAV)infection,and to establish a dual fluorescent quantitative PCR detection method for rabies virus(RV)and canine adenovirus type 2(CAV2).Methods A dual fluorescent quantitative PCR detection method was established by designing specific primers and probes for E1 gene of CAV and G gene of RV for the detection of CAV2-ΔE3-CGS.Oral swabs,anal swabs and environmental samples of stray dogs from experimental animal farm and dog detention center were tested.Results The standard curves generated by this method were Y=-3.351×logX+44.895,R^(2)=0.999 and Y=-3.413×logX+45.192,R^(2)=0.996,respectively.The linear relationships were good,and the minimum detection limits were both 102 copies/μL.CAV2-ΔE3-CGS was not detected in experimental animal farm.CAV was detected in dog detention center,and the positive rates were 5.88%(5/85)in oral swabs,8.24%(7/85)in anal swabs,and 4%(1/25)in environmental samples.Conclusion The dual fluorescent quantitative PCR method can be used for the detection of CAV2-ΔE3-CGS after immunization and the investigation of CAV infection.The present study has shown that no CAV2-ΔE3-CGS has been detected after immunization and CAV infection rate of stay dogs is low in Shanghai.CAV2-ΔE3-CGS oral immunization meets requirement and is applicable.