猪圆环病毒3型套式PCR检测方法的建立及应用
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摘要
为了建立一种准确、快速检测猪圆环病毒3型(PCV-3)的方法,试验根据GenBank中PCV-3基因序列设计合成了内、外两对引物,建立了套式PCR方法,并检验了该方法的特异性、敏感性、重复性,同时用该方法对124份临床疑似病料进行检测。结果表明:用建立的套式PCR方法检测PCV-2、PRV、PPV、PRRSV、CSFV、TGEV、PEDV,均为阴性,外引物PCR扩增的检测灵敏度为17 ng DNA,内引物PCR扩增的检测灵敏度为0. 17 ng DNA。用该方法检测124份临床病料样品,外引物共检测出阳性样品22份,内引物共检测出阳性样品28份,青海省部分地区PCV-3的感染率达22. 58%(28/124)。说明该套式PCR具有良好的特异性、敏感性、重复性和临床适用性,可以用于临床病料中低含量PCV-3的快速检测。
In order to establish an accurate and rapid detection method for porcine circovirus type 3( PCV-3),based on the sequence of PCV-3 gene in GenBank,two pairs of primers were designed and synthesized,and the nested PCR method was established. The specificity,sensitivity and repeatability of the method were tested,and 124 clinical suspected samples were detected by this method.The results showed that PCV-2,PRV,PPV,PRRSV,CSFV,TGEV and PEDV were all negative by nested PCR. The sensitivity of external primer and internal primer was 17 ng DNA and 0. 17 ng DNA respectively. 124 clinical samples were detected by this method,22 positive samples were detected by external primers and 28 positive samples were detected by internal primers. The infection rate of PCV-3 in some areas of Qinghai province was22. 58%( 28/124). It indicats that the nested PCR has good specificity,sensitivity,repeatability,accuracy and clinical applicability,and it can be used for rapid and low-content detection of PCV-3 in clinical materials.
In order to establish an accurate and rapid detection method for porcine circovirus type 3( PCV-3),based on the sequence of PCV-3 gene in GenBank,two pairs of primers were designed and synthesized,and the nested PCR method was established. The specificity,sensitivity and repeatability of the method were tested,and 124 clinical suspected samples were detected by this method.The results showed that PCV-2,PRV,PPV,PRRSV,CSFV,TGEV and PEDV were all negative by nested PCR. The sensitivity of external primer and internal primer was 17 ng DNA and 0. 17 ng DNA respectively. 124 clinical samples were detected by this method,22 positive samples were detected by external primers and 28 positive samples were detected by internal primers. The infection rate of PCV-3 in some areas of Qinghai province was22. 58%( 28/124). It indicats that the nested PCR has good specificity,sensitivity,repeatability,accuracy and clinical applicability,and it can be used for rapid and low-content detection of PCV-3 in clinical materials.
引文
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[9]朱树全,邬孝红.鸭疱疹病毒1型套式PCR检测方法的建立及应用[J].中国畜牧兽医,2013,40(8):210-213.
[10]索南卓玛.犬细小病毒套式PCR检测方法的建立及应用[J].畜牧与兽医,2013,45(6):71-74.
[2] FAN S,KU X,CHEN F,et al. Complete genome sequence of a novel porcine circovirus type 3 strain,PCV3/CN/Hubei-618/2016,isolated from China[J]. Genome Announc,2017,5(15):e00100-17.
[3] ZHENG S,WU X,ZHANG L,et al.The occurrence of porcine circovirus 3 without clinical infection signs in Shandong Province[J].Transbound Emerg Dis,2017,64(5):1337-1341.
[4] KU X,CHEN F,LI P,et al. Identification and genetic characterization of porcine circovirus type 3 in China[J].Transbound Emerg Dis,2017,64(3):703-708.
[5]贺会利,李军,潘艳,等.广西首例猪圆环病毒3型的发现及其衣壳蛋白序列分析[J].南方农业学报,2017,48(8):1499-1503.
[6]代振江,王伟丞,曾智勇,等.猪圆环病毒基因组结构与功能研究进展[J].畜牧与兽医,2016,48(3):151-154.
[7]杜兴华.圆环病毒病在猪场中的危害及防治[J].中国畜牧兽医文摘,2017,33(7):139.
[8]徐朋丽,张鸿鑫,张宇,等.猪圆环病毒3型PCR检测方法的建立及应用[J].中国预防兽医学报,2017,39(9):727-730.
[9]朱树全,邬孝红.鸭疱疹病毒1型套式PCR检测方法的建立及应用[J].中国畜牧兽医,2013,40(8):210-213.
[10]索南卓玛.犬细小病毒套式PCR检测方法的建立及应用[J].畜牧与兽医,2013,45(6):71-74.