国际流行病学传染病学杂志
國際流行病學傳染病學雜誌
국제류행병학전염병학잡지
INTERNATIONAL JOURNAL OF EPIDEMIOLOGY AND INFECTIOUS DISEASE
2014年
6期
382-386
,共5页
张舟听%夏文英%王忠发%刘妙%翁瑛瑛
張舟聽%夏文英%王忠髮%劉妙%翁瑛瑛
장주은%하문영%왕충발%류묘%옹영영
禽流感%RT-PCR%H7N9%早期诊断%监测
禽流感%RT-PCR%H7N9%早期診斷%鑑測
금류감%RT-PCR%H7N9%조기진단%감측
Avian influenza%RT-PCR%H7N9%Early diagnosis%Surveillance
目的 构建一种灵敏、特异、快速的实时荧光定量RT-PCR反应体系用于临床H7N9禽流感疑似病例早期诊断及外环境H7N9病毒的监测.方法 以H7N9禽流感病毒H7基因和N9基因为靶基因设计引物以及TaqMan探针,并对引物、探针及反应条件进行优化,验证该反应体系检测的特异性、灵敏度、重复性和检测速度,然后与两种商品化试剂盒(试剂盒I和试剂盒Ⅱ)进行各项指标及184份现场样本作平行比对.结果 新建立的H7N9禽流感病毒实时荧光定量RT-PCR反应体系呈典型的扩增曲线、扩增时间约50 min,H7基因与N9基因的最低检出限分别为28拷贝/μL与41拷贝/μL.重复性测定显示,H7和N9检测Ct值的变异系数(CV)分别为0.17％～0.89％和0.33％～0.59％,扩增效率分别为0.9491和0.9713;与其他常见禽流感病毒或肠道病毒无明显交叉反应.184份现场可疑样本阳性检出率为5.43％(10/184),与试剂盒Ⅱ结果一致.结论 本研究建立的实时荧光定量RT-PCR反应体系具有检测速度快、灵敏度高、特异性强、重复性好、结果可靠等特点,可用于临床疑似病例的应急检测、早期诊断及养鸡场、活禽交易市场等外环境H7N9禽流感病毒监测.
目的 構建一種靈敏、特異、快速的實時熒光定量RT-PCR反應體繫用于臨床H7N9禽流感疑似病例早期診斷及外環境H7N9病毒的鑑測.方法 以H7N9禽流感病毒H7基因和N9基因為靶基因設計引物以及TaqMan探針,併對引物、探針及反應條件進行優化,驗證該反應體繫檢測的特異性、靈敏度、重複性和檢測速度,然後與兩種商品化試劑盒(試劑盒I和試劑盒Ⅱ)進行各項指標及184份現場樣本作平行比對.結果 新建立的H7N9禽流感病毒實時熒光定量RT-PCR反應體繫呈典型的擴增麯線、擴增時間約50 min,H7基因與N9基因的最低檢齣限分彆為28拷貝/μL與41拷貝/μL.重複性測定顯示,H7和N9檢測Ct值的變異繫數(CV)分彆為0.17％～0.89％和0.33％～0.59％,擴增效率分彆為0.9491和0.9713;與其他常見禽流感病毒或腸道病毒無明顯交扠反應.184份現場可疑樣本暘性檢齣率為5.43％(10/184),與試劑盒Ⅱ結果一緻.結論 本研究建立的實時熒光定量RT-PCR反應體繫具有檢測速度快、靈敏度高、特異性彊、重複性好、結果可靠等特點,可用于臨床疑似病例的應急檢測、早期診斷及養鷄場、活禽交易市場等外環境H7N9禽流感病毒鑑測.
목적 구건일충령민、특이、쾌속적실시형광정량RT-PCR반응체계용우림상H7N9금류감의사병례조기진단급외배경H7N9병독적감측.방법 이H7N9금류감병독H7기인화N9기인위파기인설계인물이급TaqMan탐침,병대인물、탐침급반응조건진행우화,험증해반응체계검측적특이성、령민도、중복성화검측속도,연후여량충상품화시제합(시제합I화시제합Ⅱ)진행각항지표급184빈현장양본작평행비대.결과 신건립적H7N9금류감병독실시형광정량RT-PCR반응체계정전형적확증곡선、확증시간약50 min,H7기인여N9기인적최저검출한분별위28고패/μL여41고패/μL.중복성측정현시,H7화N9검측Ct치적변이계수(CV)분별위0.17％～0.89％화0.33％～0.59％,확증효솔분별위0.9491화0.9713;여기타상견금류감병독혹장도병독무명현교차반응.184빈현장가의양본양성검출솔위5.43％(10/184),여시제합Ⅱ결과일치.결론 본연구건립적실시형광정량RT-PCR반응체계구유검측속도쾌、령민도고、특이성강、중복성호、결과가고등특점,가용우림상의사병례적응급검측、조기진단급양계장、활금교역시장등외배경H7N9금류감병독감측.
Objective To develop a sensitive,specific and rapid reaction system of real-time fluorescence quantitative RT-PCR for early diagnosing clinical H7N9 avian influenza suspected patients and monitoring environmental pollutions.Methods Primers and TaqMan probes were designed according to the H7 and N9 genes of H7N9 avian influenza virus,then the primers,probes and reaction conditions were optimized,the specificity,sensitivity,repeatability and detection speed were verified.The RT-PCR method developed was compared with two commercial kits (Kit I and Kit Ⅱ)in every parameter and 184 field samples.Results The real-time fluorescence quantitative RT-PCR developed for detecting H7N9 avian influenza virus had typical amplification curves,the detection time was 50 minutes,the detection limit of H7 gene and N9 gene was 28 copies/μL and 41 copies/μL,respectively.Repeatability assay showed that coefficients of variation of Ct value were 0.17％-0.89％and 0.33％-0.59％,respectively,amplification efficiencies were 0.9491 and 0.9713.No significant cross-reaction was found between H7N9 avian influenza virus and other common avian influenza viruses or enteroviruses.The positive rate was 5.43％(10/184) in 184 field suspicious samples,which was consistent with Kit Ⅱ.Conclusions The real-time fluorescence quantitative RT-PCR reaction system develops for rapid assay of H7N9 avian influnenza virus gene,good sensitivity,specificity,repeatability and reliable results,and can be used for emergency detection and early diagnosis of clinical suspected patients with H7N9 avian influenza virus infection,and for surveillance of the virus in external environments like chicken farms and live poultry markets.
