农业科学与技术（英文版）
農業科學與技術（英文版）
농업과학여기술（영문판）
AGRICULTURAL SCIENCE & TECHNOLOGY
2014年
9期
1487-1490
,共4页
俞正玉%徐向伟%孙冰%何孔旺%郭容利%杜露平%温立斌%张雪寒%茅爱华%倪艳秀%李彬
俞正玉%徐嚮偉%孫冰%何孔旺%郭容利%杜露平%溫立斌%張雪寒%茅愛華%倪豔秀%李彬
유정옥%서향위%손빙%하공왕%곽용리%두로평%온립빈%장설한%모애화%예염수%리빈
猪传染性胃肠炎病毒%TaqMan荧光定量PCR%检测
豬傳染性胃腸炎病毒%TaqMan熒光定量PCR%檢測
저전염성위장염병독%TaqMan형광정량PCR%검측
Transmissible gastroenteritis virus (TGEV)%TaqMan-based real-time PCR%Detection
[目的]建立检测猪传染性胃肠炎病毒（transmissible gastroenteritis virus, TGEV）的 TaqMan荧光定量 PCR方法。[方法]根据TGEV基因组中保守的 N基因序列设计引物和探针，以梯度稀释的含有 N基因的重组质粒作为标准品，进行定量 PCR反应。[结果]该试验建立的荧光定量 PCR方法在102~1010拷贝/μl之间具有良好的线性关系，相关系数达到0.99以上，扩增效率在90%~110%之间。该方法的检测灵敏度为10拷贝，敏感性较高，而且特异性较好，与猪的其他病毒核酸均无交叉反应；批内和批间的变异系数低于3%，表明该方法的重复性较好。应用该方法可以对疫苗的免疫效力进行定量检测和评价，也可以对临床病料进行检测。[结论]该研究建立的荧光定量 PCR方法灵敏度高、特异性好，可为TGEV的流行病学调查、疫苗开发和发病机制等研究提供可靠的工具。
[目的]建立檢測豬傳染性胃腸炎病毒（transmissible gastroenteritis virus, TGEV）的 TaqMan熒光定量 PCR方法。[方法]根據TGEV基因組中保守的 N基因序列設計引物和探針，以梯度稀釋的含有 N基因的重組質粒作為標準品，進行定量 PCR反應。[結果]該試驗建立的熒光定量 PCR方法在102~1010拷貝/μl之間具有良好的線性關繫，相關繫數達到0.99以上，擴增效率在90%~110%之間。該方法的檢測靈敏度為10拷貝，敏感性較高，而且特異性較好，與豬的其他病毒覈痠均無交扠反應；批內和批間的變異繫數低于3%，錶明該方法的重複性較好。應用該方法可以對疫苗的免疫效力進行定量檢測和評價，也可以對臨床病料進行檢測。[結論]該研究建立的熒光定量 PCR方法靈敏度高、特異性好，可為TGEV的流行病學調查、疫苗開髮和髮病機製等研究提供可靠的工具。
[목적]건립검측저전염성위장염병독（transmissible gastroenteritis virus, TGEV）적 TaqMan형광정량 PCR방법。[방법]근거TGEV기인조중보수적 N기인서렬설계인물화탐침，이제도희석적함유 N기인적중조질립작위표준품，진행정량 PCR반응。[결과]해시험건립적형광정량 PCR방법재102~1010고패/μl지간구유량호적선성관계，상관계수체도0.99이상，확증효솔재90%~110%지간。해방법적검측령민도위10고패，민감성교고，이차특이성교호，여저적기타병독핵산균무교차반응；비내화비간적변이계수저우3%，표명해방법적중복성교호。응용해방법가이대역묘적면역효력진행정량검측화평개，야가이대림상병료진행검측。[결론]해연구건립적형광정량 PCR방법령민도고、특이성호，가위TGEV적류행병학조사、역묘개발화발병궤제등연구제공가고적공구。
This study aimed to establish a TaqMan-based real-time PCR assay for detecting transmissible gastroenteritis virus (TGEV). [Method] Primers and a probe were designed according to the conserved sequence of N gene in TGEV genome. After gradient dilution, the recombinant plasmid harboring the N gene was used as a standard for real-time PCR assay to establish the standard curve. [Re-sult] The results showed that the established real-time PCR assay exhibited a good linear relationship within the range of 102-1010 copies/ul; the correlation coefficient was above 0.99 and the amplification efficiency ranged from 90% to 110%. The de-tection limit of real-time PCR assay for TGEV was 10 copies/μl, suggesting a high sensitivity; there was no cross reaction with other porcine viruses, indicating a good specificity; coefficients of variation within and among batches were lower than 3%, suggesting a good repeatability. The established real-time PCR method could be ap-plied in quantitative analysis and evaluation of the immune efficacy of TGEV vac-cines and detection of TGEV in clinical samples. [Conclusion] The TaqMan-based real-time PCR assay established in this study is highly sensitive and specific, which can provide technical means for the epidemiological survey of TGEV, development of TGEV vaccines and investigation of the pathogenesis of TGE.
