Detection of HCV genotypes 1b and 2a by a reverse transcription loop-mediated isothermal amplification assay
Detection of HCV genotypes 1b and 2a by a reverse transcription loop-mediated isothermal amplification assay
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DOI：
10.1002/jmv.24747
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发表时间：
2017-06-01
影响因子：
12.7
通讯作者：
Sun，Dianxing
中科院分区：
文献类型：
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作者：
Zhao，Na;Liu，Jinxia;Sun，Dianxing
丙型肝炎病毒（HCV）基因型1b和2a是中国北方肝病的主要原因;然而，传统的检测工具耗费人力，技术要求高，成本高。在这里，我们评估了逆转录环介导等温扩增（RT-LAMP）检测HCV基因型1b和2a的特异性，灵敏度和临床实用性。首先，从HCV基因型1b和2a感染的患者中收集临床样品，并提取RNA。其次，检测HCV基因型1b和2a的RT-LAMP检测法的特异性与其他肝炎病毒的病毒基因组进行了测试。使用标准HCV基因型1b和2a的系列稀释液确定RT-LAMP测定的灵敏度。扩增产物通过电泳和钙黄绿素/Mn 2+依赖性视觉方法检测。最后，我们比较了RT-LAMP和真实的-time PCR的临床检出率。RT-LAMP检测HCV基因型1b和2b的特异性较高，与其他肝炎病毒无交叉反应。通过电泳或钙黄绿素/Mn 2+依赖性目视方法检测的两种基因型的RT-LAMP灵敏度均为100 IU/mL。RT-LAMP检测在临床样本中的检出率也与真实的-time PCR相当，两种方法之间无显著差异。本研究提出了一种新开发的用于检测HCV基因型1b和2a的RT-LAMP检测方法。RT-LAMP是一种高度特异性、敏感性和简单的诊断工具，特别是在资源有限的环境中，可用于HCV的筛查和早期诊断。
Hepatitis C virus (HCV) genotypes 1b and 2a are the major cause of liver disease in northern China; however, conventional detection tools are labor‐consuming, technically demanding, and costly. Here, we assessed the specificity, sensitivity, and clinical utility of reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assay for detection of HCV genotypes 1b and 2a. Firstly, clinical samples were collected from HCV genotype 1b and 2a infected patients and the RNA were extracted. Secondly, specificity of RT‐LAMP assay for detection HCV genotypes 1b and 2a were tested against viral genomes of other hepatitis viruses. Sensitivity of RT‐LAMP assay was determined using serial dilutions of standard HCV genotypes 1b and 2a. The amplified products were detected by both electrophoresis and calcein/Mn2+‐dependent visual methods. Finally, we compared the clinical detection rate of RT‐LAMP to that of real‐time PCR. RT‐LAMP assay showed high specificity to detect HCV genotypes 1b and 2b since there was no cross‐reactivity with other hepatitis viruses. Sensitivity of RT‐LAMP was 100 IU/mL for both genotypes detected by either electrophoresis or calcein/Mn2+‐dependent visual methods. The detection rate of RT‐LAMP assay in clinical samples was also comparable to that of real‐time PCR without significant difference between the both assays. This study proposes a newly developed RT‐LAMP assay for detection of HCV genotypes 1b and 2a. RT‐LAMP is highly specific, sensitive, and simple diagnostic tool which would be useful for screening and early diagnosis of HCV especially in resource‐limited environments.