中国兽药杂志
中國獸藥雜誌
중국수약잡지
CHINESE JOURNAL OF VETERINARY DRUG
2014年
12期
17-21
,共5页
孙庆歌%李晶梅%朱薇%肖爱芳%赖志%薛霜%高俊锋%马慧慧%祝春花%谢红玲
孫慶歌%李晶梅%硃薇%肖愛芳%賴誌%薛霜%高俊鋒%馬慧慧%祝春花%謝紅玲
손경가%리정매%주미%초애방%뢰지%설상%고준봉%마혜혜%축춘화%사홍령
血清1型鸭甲型肝炎病毒%实时定量PCR%TaqMan探针
血清1型鴨甲型肝炎病毒%實時定量PCR%TaqMan探針
혈청1형압갑형간염병독%실시정량PCR%TaqMan탐침
duck hepatitis A virus serotype 1%real-time quantitative PCR%TaqMan probe
为建立一种可检测血清1型鸭甲型肝炎病毒（ DHAV－1）的实时定量PCR方法，根据GenBank中DHAV－15’非编码区的保守区，设计合成1对引物和1条TaqMan探针，以构建的重组质粒作为标准品，绘制标准曲线，并对所建立方法进行了特异性、敏感性和可重复性试验以及临床病料检测初步应用。结果，该方法与血清3型鸭甲型肝炎病毒、鸭瘟病毒、新城疫病毒、禽流感病毒、呼肠孤病毒、传染性支气管炎病毒等无交叉反应性；最低可以检测到10 copies／μL；组内和组间变异系数均小于3％；临床病料的检测结果与测序检测结果一致。结果表明，所建立的实时定量检测方法具有特异、敏感、稳定等优点，可用于DHAV－1的快速检测与定量分析。
為建立一種可檢測血清1型鴨甲型肝炎病毒（ DHAV－1）的實時定量PCR方法，根據GenBank中DHAV－15’非編碼區的保守區，設計閤成1對引物和1條TaqMan探針，以構建的重組質粒作為標準品，繪製標準麯線，併對所建立方法進行瞭特異性、敏感性和可重複性試驗以及臨床病料檢測初步應用。結果，該方法與血清3型鴨甲型肝炎病毒、鴨瘟病毒、新城疫病毒、禽流感病毒、呼腸孤病毒、傳染性支氣管炎病毒等無交扠反應性；最低可以檢測到10 copies／μL；組內和組間變異繫數均小于3％；臨床病料的檢測結果與測序檢測結果一緻。結果錶明，所建立的實時定量檢測方法具有特異、敏感、穩定等優點，可用于DHAV－1的快速檢測與定量分析。
위건립일충가검측혈청1형압갑형간염병독（ DHAV－1）적실시정량PCR방법，근거GenBank중DHAV－15’비편마구적보수구，설계합성1대인물화1조TaqMan탐침，이구건적중조질립작위표준품，회제표준곡선，병대소건립방법진행료특이성、민감성화가중복성시험이급림상병료검측초보응용。결과，해방법여혈청3형압갑형간염병독、압온병독、신성역병독、금류감병독、호장고병독、전염성지기관염병독등무교차반응성；최저가이검측도10 copies／μL；조내화조간변이계수균소우3％；림상병료적검측결과여측서검측결과일치。결과표명，소건립적실시정량검측방법구유특이、민감、은정등우점，가용우DHAV－1적쾌속검측여정량분석。
In order to develop a real-time quantitative PCR to detect duck hepatitis A virus serotype 1 ( DHAV-1) , a pair of primers and one TaqMan probe were designed and synthesized, according to the 5 ’ untranslated sequences of DHAV-1 published on GenBank, The recombinant plasmid was built as a standard control for the method, the specificity, sensitivity, and repeatability of the method were determined, and also preliminarily applicated in the detection of clinical samples. The results showed that the detection assay was specific for DHAV-1, and there was no cross reaction between duck hepatitis A virus serotype 3( DHAV-3) and the other viruses including duck plague virus, Newcastle disease virus, avian influenza virus, duck reovirus, avian infectious bronchitis virus, etc. The method also showed high sensitivity, as low as 10 copies virus could be detected for each reaction, and good reproducibility, there was a coefficient of variations less than 3% for both inter-assay and intra-assay. The detection results of clinical samples were consistent with the sequencing. These results indicated that the developed TaqMan fluorescence quantitative PCR assay had the advantages of good specificity, sensitivity and repeatability;it was useful for the rapid diagnosis and quantification analysis of DHAV-1.
