Research of fluorescent reagent in detection of IBV by loop mediated isothermal amplification (LAMP) | 基于荧光显色的IBV LAMP检测方法研究

Chino. 建立基于荧光显色的可视化禽传染性支气管炎病毒（IBV）环介导等温扩增（LAMP）检测体系，为IBV的快速临床检测提供有力的技术支持。根据NCBI中收录的IBV 5a+5b基因的8个区域设计了3对LAMP引物，通过对反应体系各组分和条件的优化，建立IBV LAMP检测方法，并对该方法的特异性和敏感性进行验证。分别用SYBR Green I和一定浓度比的钙黄绿素/锰离子混合物作为显色剂，对IBV LAMP检测结果进行可视化判定。应用该检测体系和PCR方法对临床送检的60份样品同时进行检测，比较2种方法的阳性检出率。成功建立了IBV LAMP检测方法，该方法能够在等温（63 ℃）条件下1 h内完成反应，具有良好的敏感性和特异性，最低可以检出1拷贝/μL的阳性重组质粒，比普通PCR方法敏感100倍，而对其他病毒检测结果均为阴性。应用SYBR Green I和钙黄绿素/锰离子显色时，阴阳性样品均有强烈的颜色对比，其中0.05 mmol/L钙黄绿素与0.6 mmol/L锰离子混合液可以用于LAMP扩增产物的判断，其结果与琼脂糖凝胶电泳结果一致。在对60份临床样本的检测中，LAMP和PCR方法检测出的阳性样本数量分别为49份和42份。建立了基于荧光显色的IBV LAMP检测方法，该方法具有在科研机构、基层实验室特别是检测现场推广和应用的潜力。

Inglés. In order to provide support for infectious bronchitis virus detection, a novel method namely loop-mediated isothermal amplification (LAMP) was reported using fluorescent reagent as the product judgment. With one set of primers containing six special primers targeting eight part of 5a+5b gene obtained from NCBI, the LAMP system was optimized by sifting the time, temperature and concentration of all components. Then specificity and sensitivity test was conducted. SYBR Green I and calcein/Mn2+ reagent were respectively used in LAMP product judgment with naked eyes. In order to compare the LAMP and PCR method, sixty clinical samples were detected. The LAMP method was successfully established, and the reaction could complete within 1 hour at 63 ℃ with favorable sensitivity and specificity. The detection limit of the assay was 1 copy virus, which was 100 times more sensitive than traditional PCR. In the fluorescent reagent test, SYBR Green I and calcein reagent which contained 0.05 mmol/L calcein and 0.6 mmol/L Mn2+ were respectively used in LAMP product judgment, and the result showed strong colorimetric comparison between positive and negative and was consistent with agarose gel electrophoresis. In the detection of 60 clinical samples, 49 and 42 samples were respectively positive by LAMP and PCR assays. LAMP assay with fluorescent reagent provided a useful tool for infectious bronchitis virus rapidly detection.