Wang Jing, Zhang Hua, Yan Shengyuan, Wang Yichao, Lu Xin, Fu Hongwei
Clin Lab. 2017 May 1;63(5):901-905. doi: 10.7754/Clin.Lab.2017.160919.
Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in areas with poor sanitation. Rabbit is one of important animal reservoirs of the virus. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system is a novel nucleic acid amplification assay, for which the specificity and sensitivity are much higher than that of conventional PCR. However, previously reported RT-LAMP system cannot detect rabbit HEV, which was identified recently and seems to be another potential source of human HEV infection.
In this study, genotype 4 HEV strains and rabbit HEV strains were used to verify the applicability of the previously reported HEV RT-LAMP. A new specific one step RT-LAMP system was developed and evaluated to amplify rabbit HEV RNA. In order to test the sensitivity of the newly-developed assay, serial dilutions (from 5 x 103 to 5 x 10-1/µL) of the rabbit HEV RNA were used as template to be detected by RT-LAMP, real-time RTPCR, and nested RT-PCR. Specificity of the new assay was further evaluated using HAV, HBV, and HCV. With this new assay, 46 rabbit fecal samples were retrospectively investigated with real-time RT-PCR and nested RTPCR in parallel.
The detection limit of this newly-developed RT-LAMP assay reached as low as 10 copies/reaction and no cross-reactivity was observed with other hepatitis viruses including hepatitis A, B, and C virus, which indicated that this assay has much higher sensitivity and specificity than that of nested RT-PCR and real time RT-PCR. Furthermore, among 46 rabbit fecal samples, there were four positive samples determined by those three assays and one positive sample only detected by HEV RT-LAMP and real-time RT-PCR.
Using a combination of sensitivity, specificity, and evaluation of clinical samples, this study provides the first data on the usefulness of RT-LAMP in the diagnosis of rabbit HEV RNA.
戊型肝炎病毒（HEV）是卫生条件差的地区急性病毒性肝炎的主要病因。兔是该病毒重要的动物宿主之一。逆转录环介导等温扩增（RT-LAMP）系统是一种新型核酸扩增检测方法，其特异性和灵敏度远高于传统PCR。然而，先前报道的RT-LAMP系统无法检测到最近鉴定出的兔HEV，而兔HEV似乎是人类HEV感染的另一个潜在来源。
在本研究中，使用4型HEV毒株和兔HEV毒株验证先前报道的HEV RT-LAMP的适用性。开发并评估了一种新的特异性一步RT-LAMP系统，用于扩增兔HEV RNA。为了测试新开发检测方法的灵敏度，将兔HEV RNA的系列稀释液（从5×10³到5×10⁻¹/μL）用作模板，通过RT-LAMP、实时RT-PCR和巢式RT-PCR进行检测。使用甲型肝炎病毒、乙型肝炎病毒和丙型肝炎病毒进一步评估新检测方法的特异性。使用这种新检测方法，对46份兔粪便样本同时进行实时RT-PCR和巢式RT-PCR回顾性研究。
这种新开发的RT-LAMP检测方法的检测限低至10拷贝/反应，并且未观察到与其他肝炎病毒（包括甲型、乙型和丙型肝炎病毒）的交叉反应，这表明该检测方法的灵敏度和特异性远高于巢式RT-PCR和实时RT-PCR。此外，在46份兔粪便样本中，这三种检测方法确定有4份阳性样本，1份阳性样本仅通过HEV RT-LAMP和实时RT-PCR检测到。
本研究结合灵敏度、特异性及临床样本评估，首次提供了RT-LAMP在诊断兔HEV RNA方面实用性的数据。
