反转录环介导等温扩增技术对丙型肝炎病毒可视化分型检测

Visual genotyping detection of HCV by reverse transcription loop-mediated isothermal amplification technique

目的应用反转录环介导等温扩增技术(RT-LAMP)可视化检测丙型肝炎病毒(HCV)1b和2a基因型,为RTLAMP技术应用于现场或基层医院提供初步探索经验。方法首先,收集临床阳性样本并用荧光定量PCR分型检测,把样本基本分为1型和非1型。然后分别根据HCV 1b和2a基因型设计RT-LAMP引物并优化反应条件,通过互换模板实验验证这2套引物的特异性,并通过检测稀释的模板以验证引物的灵敏度。同时用钙黄绿素进行产物显色,使HCV分型检测可视化。最后用RT-LAMP方法检测临床样本,并计算阳性率,应用配对卡方检验比较RT-LAMP和荧光定量PCR的统计学差异。结果优化好的RT-LAMP体系的特异性好,对HCV 1b型的检测灵敏度为103 IU/mL,2a型的检测灵敏度为104 IU/mL。另外,利用钙黄绿素进行产物检测的效果和电泳结果相当。通过对临床样本分型检测,HCV 1b型样本的RTLAMP检测结果和荧光定量PCR差异无统计学意义(P>0.05),HCV 2a型样本的RT-LAMP检测结果和荧光定量PCR差异有统计学意义(P<0.05),但是未检出的样本中有2例为HCV 2b型。结论 RT-LAMP对HCV的可视化分型检测具有较好的特异性和灵敏度,操作简便,具有在现场或基层医院的应用前景。

Reverse transcription loop-mediated isothermal amplification（RT-LAMP）method was applied to detect hepatitis C virus（HCV）genotypes 1band 2avisually,and provided prelimary exploration experience for its promotion at field or in primary care hospitals.First,clinical samples were collected and genotyped by fluorescent quantitative PCR,which could genotype these samples into HCV 1genotype and non-1genotype in general.After that,RT-LAMP primers were designed according to HCV genotypes 1band 2a,the reaction condition was tested for optimization.The specificity of the 2sets of primers was validated by interchange of templates.The sensitivity of the 2sets of primers was validated through detection of dilute templates.In the meantime,products strain was realized by calcein for visual HCV genotyping detection.At last,clinical samples were detected by RT-LAMP,and positive rate of RT-LAMP was calculated,the chi-square test was applied to compare the RT-LAMP with fluorescent quantitative PCR.Results showed that optimized RT-LAMP had good specificity,which the sensitivity for detection of HCV genotype 1bwas 103 IU/mL and for HCV genotype 2awas 104 IU/mL,respectively.In addition,calcein showed the same effect with electrophoresis in products detection.After detection of clinical samples,RT-LAMP had no statistics difference with fluorescent quantitative PCR（P〉0.05）for detection of HCV genotype 1b.RT-LAMP had statistics difference with fluorescent quantitative PCR（P〈0.05）for detection of HCV genotype 2a,but we found that there were two HCV 2bsamples in the undetected samples.In conclusion,the RT-LAMP method performs well for visualized genotyping detection of HCV.It is easy to operate,with good specificity and sensitivity,and has the application prospect at field or in primary care hospitals.