环介导逆转录等温扩增技术(RT-LAMP)在丙型肝炎病毒基因检测中的应用

该文介绍了一种便捷、灵敏而又特异的环介导逆转录等温扩增基因检测技术,该技术分别使用特异对应于靶序列中8个基因区段的3对特殊引物,并在反转录酶和Bst-DNA聚合酶的作用下对靶序列进行等温核酸扩增反应,整个检测反应只需1~2h。利用这种技术成功检测了丙型肝炎病毒基因,对60份经Real-time PCR或RT-PCR验证阳性的血清样品检测,阳性符合率为98%。同时,对扩增终产物进一步进行酶切分析,并与HIV、HBV和不同亚型流感病毒RNA进行交叉反应和特异性测试,均与预期结果吻合。将Real-time PCR定量后的RNA系列稀释后对检测方法的灵敏度进行了测试。结果显示,该技术的检测灵敏度在理论上可达到10个拷贝的RNA分子。以上结果证明,RT-LAMP扩增技术是一种检测程序简单、灵敏度和特异性较高的基因检测手段,在丙型肝炎病毒的快速检测方面具有一定的开发潜力。     

逆转录环介导等温核酸扩增技术(RT-LAMP)在H5N1禽流感病毒基因检测中的应用

建立一种便捷、灵敏的检测方法,即逆转录环介导等温核酸扩增技术(RT-LAMP)用于H5N1亚型禽流感病毒基因检测.该技术使用特异对应于靶序列中8个基因区段的6条特异引物,在等温条件下进行核酸扩增反应.对51份实验感染动物及病毒培养标本的H5N1亚型禽流感病毒的HA、NA基因区进行了RT-LAMP检测,并以SYBR Green Ⅰ为反应指示剂进行了逆转录环介导等温核酸扩增技术,对该反应进行实时监控,经对扩增产物做内切酶验证和测序分析,证明RT-LAMP技术的特异性;同时,用10倍系列稀释的RNA样品对该检测方法的灵敏度进行了测试.结果显示:利用RT-LAMP技术成功检测到H5N1禽流感病毒的HA、NA基因区,且RT-LAMP与Real-time PCR结果呈现很好的一致性.此方法的灵敏度可达到能检测10个拷贝RNA分子水平.因此,RT-LAMP技术应用于H5N1亚型禽流感病毒的快速检测是一种可行的方法.     

河北省丙型肝炎病毒基因分型研究

Using nested RT-PCR and type special primers of HCV, we performed genotype analysis of HCV RNA from five representative cities in Hebei Province. Among 168 samples with HCV RNA, 104 sera was typelb(61.9%), type 2a account for 22.6% (38/168) , and lb/2a mixed-type was 15.5% (26/168) . The results indicate that typelb is the dominance strain in Hebei Province. Compared with type 2a, typelb has important relationship with chronic hepatitis C. This study build up some foundations for diagnosis, treatment and vaccine study of hepatitis C.     

Ultracentrifugation of serum samples allows detection of hepatitis C virus RNA in patients with occult hepatitis C

Occult hepatitis C virus (HCV) infection of patients with abnormal liver function tests of unknown origin who are anti-HCV and serum HCV RNA negative but who have HCV RNA in the liver has been described. As HCV replicates in the liver cells of these patients, it could be that the amount of circulating viral particles is under the detection limit of the most sensitive techniques. To prove this hypothesis, serum samples from 106 patients with occult HCV infection were analyzed. Two milliliters of serum was ultracentrifuged over a 10% sucrose cushion for 17 h at 100,000 x g(av), where av means average, and HCV RNA detection was performed by strand-specific real-time PCR. Out of the 106 patients, 62 (58.5%) had detectable serum HCV RNA levels after ultracentrifugation, with a median load of 70.5 copies/ml (range, 18 to 192). Iodixanol density gradient studies revealed that HCV RNA was positive at densities of 1.03 to 1.04 and from 1.08 to 1.19 g/ml, which were very similar to those found in the sera of patients with classical chronic HCV infection. Antigenomic HCV RNA was found in the livers of 56 of 62 (90.3%) patients with detectable serum HCV RNA levels after ultracentrifugation, compared to 27 of 44 (61.4%) negative patients (P < 0.001). No differences in the median loads of antigenomic HCV RNA between patients with an those without serum HCV RNA (4.5 x 10(4) [range, 7.9 x 10(2) to 1.0 x 10(6)] versus 2.3 x 10(4) [range, 4.0 x 10(2) to 2.2 x 10(5)]) were found. Alanine aminotransferase and gamma-glutamyl transpeptidase levels, liver necroinflammatory activity, and fibrosis did not differ between both groups. In conclusion, HCV RNA can be detected in the sera of patients with occult HCV infection after circulating viral particles are concentrated by ultracentrifugation.     

戊型肝炎病毒荧光定量RT-PCR快速检测技术的建立和初步应用

利用DNAMAN软件对GenBank登录的戊型肝炎病毒四个主要基因型代表株的序列进行分析, 选择其高度保守的ORF2区域设计合成引物和探针, 并用包含有扩增区域的核苷酸片段进行体外转录制备标准品cRNA。在对荧光定量RT-PCR的反应条件优化的基础上, 建立了适用于戊型肝炎病毒主要基因型检测的荧光定量RT-PCR检测技术。该检测技术可以有效检测I型和IV型戊型肝炎阳性病料, 而对猪的其它几种疫病阳性病料则为阴性结果, 证实本技术的特异性强、可靠性好。对阳性标准品的检测结果表明, 所建立的TaqMan荧光定量RT-PCR灵敏度可达2.0×101拷贝/反应, 相比于巢式RT-PCR方法, 其灵敏度高10~100倍以上。在对54份临床样品的检测中, 进一步证实了该方法快速、灵敏且重复性好, 可满足戊型肝炎病毒早期快速诊断的需要。     

戊型肝炎病毒荧光定量RT-PCR快速检测技术的建立和初步应用

利用DNAMAN软件对GenBank登录的戊型肝炎病毒四个主要基因型代表株的序列进行分析, 选择其高度保守的ORF2区域设计合成引物和探针, 并用包含有扩增区域的核苷酸片段进行体外转录制备标准品cRNA。在对荧光定量RT-PCR的反应条件优化的基础上, 建立了适用于戊型肝炎病毒主要基因型检测的荧光定量RT-PCR检测技术。该检测技术可以有效检测I型和IV型戊型肝炎阳性病料, 而对猪的其它几种疫病阳性病料则为阴性结果, 证实本技术的特异性强、可靠性好。对阳性标准品的检测结果表明, 所建立的TaqMan荧光定量RT-PCR灵敏度可达2.0×101拷贝/反应, 相比于巢式RT-PCR方法, 其灵敏度高10~100倍以上。在对54份临床样品的检测中, 进一步证实了该方法快速、灵敏且重复性好, 可满足戊型肝炎病毒早期快速诊断的需要。     

Chip-based detection of hepatitis C virus using RNA aptamers that specifically bind to HCV core antigen

The development of reagents with high affinity and specificity to the antigens of hepatitis C virus (HCV) is important for the early stage diagnosis of its infection. Aptamers are short, single-stranded oligonucleotides with the ability to specifically recognize target molecules with high affinity. Herein, we report the selection of RNA aptamers that bind to the core antigen of HCV. High affinity aptamers were isolated from a 10(15) random library of 60 mer RNAs using the SELEX procedure. Importantly, the selected aptamers specifically bound to the core antigen, but not to another HCV antigen, NS5, in a protein chip-based assay. Using these aptamers, we developed an aptamer-based biosensor for HCV diagnosis and detected the core antigen from HCV infected patients' sera with good specificity. This novel aptamer-based antigen detection sensor could be applied to the early diagnosis of HCV infection.