基于颜色判定的环介导等温扩增方法检测EV71  

Colorimetric detection of EV71 with reverse transcription loop-mediate isothermal amplification

作　　者：戴颖[1] 刘峰涛[2] 何书[1] 汪奇伟[1] 

机构地区：[1]河南大学淮河医院儿科,开封475003 [2]河南大学医学院免疫学研究所,开封475004

出　　处：《现代免疫学》2014年第2期151-154,共4页Current Immunology

摘　　要：建立了一种基于颜色判定的灵敏、快速和经济的逆转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)方法应用于肠道病毒71(enterovirus 71,EV71)基因检测。针对EV71病毒VP1基因特异性序列的设计出6条特异引物,在等温条件下(63℃)进行扩增反应60min。在扩增前加入染料羟基萘酚蓝(HNB)作为反应指示剂,以其颜色变化作为结果判断标准。进行特异性/灵敏度分析,同时与RT-PCR方法进行比较,并对93份疑似患者中咽拭子的病毒核酸进行了检测。通过颜色变化能观察到LAMP扩增产物的存在,且与柯萨奇病毒A16型(CA16)、轮状病毒、诺如病毒无交叉反应发生。所建立的RT-LAMP检测方法灵敏,灵敏度为500copy/tube,与定量PCR灵敏度相同。93份咽拭子标本中,RT-LAMP检测为46份阳性,荧光定量PCR检测有为44份阳性,二者统计学无显著差异。RTLAMP是一种快速、敏感、特异、经济的方法,适合用于基层医疗机构临床检测。To establish a simple, rapid and sensitive colorimetric reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay to detect enterovirus 71（EV71） , six primers which recognized 8 distinct regions on the VP1 gene of EV71 were designed for RT-LAMP assay, and the sensitivity and specificity were also evaluated. RT-LAMP assay was optimized to amplify VP1 gene in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63 ~C for 1 h. The amplified products were observed with HNB staining. There was no cross reaction with other viruses. The detection limit of RT-LAMP assay was 500 copy/tube, quivalent to that of RT-PCR. Of 93 swab specimens, 46 were EV71 positive by RT-LAMP assay, while 44 were EVT1 positive by fluorescence quantitative fluorescent RT-PCR , indicating that the two methods have no significant difference. Altogether, RT-LAMP is a rapid, sensitive, specific and economic method for detection of EV71 in clinical specimens, and thus especially suitable for primary medical care agencies.

关 键 词：逆转录-环介导等温扩增技术 肠道病毒71型 手足口病 

分 类 号：R511[医药卫生—内科学]