School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
J Med Microbiol. 2011 Apr;60(Pt 4):481-485. doi: 10.1099/jmm.0.027433-0. Epub 2010 Dec 23.
Vibrio cholerae has caused severe outbreaks of cholera worldwide with thousands of recorded deaths annually. Molecular diagnosis for cholera has become increasingly important for rapid detection of cholera as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for cholera which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of V. cholerae based on the cholera toxin A (ctxA) and outer-membrane lipoprotein (lolB) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of trehalose, which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100 % sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 7 months at 24 °C. The limit of detection of the thermostabilized triplex PCR assay was 2×10(4) c.f.u. at the bacterial cell level and 100 pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic V. cholerae which requires minimal pipetting steps and is cold chain-free.
霍乱弧菌在全球范围内引发了严重的霍乱疫情，每年都有数千人因此死亡。分子诊断对于霍乱的快速检测变得越来越重要，因为传统方法耗时耗力。然而，传统的 PCR 测试仍然需要冷链运输和储存，以及经过培训的人员来进行操作，这使得它们不便于用户使用。本研究旨在开发一种热稳定的三重 PCR 检测方法，用于霍乱的检测，这种检测方法为即用型，无需冷链。该 PCR 检测方法专门基于霍乱毒素 A (ctxA) 和外膜脂蛋白 (lolB) 基因来检测产毒性和非产毒性的霍乱弧菌。热稳定的三重 PCR 还结合了内部扩增对照，有助于检查样品中的 PCR 抑制剂。PCR 试剂和特定的引物在海藻糖存在的情况下被冻干成颗粒形式，海藻糖可以作为酶稳定剂。该三重 PCR 已用 174 份细菌污染的粪便标本进行了验证，结果显示其灵敏度和特异性均为 100%。使用 Q10 法评估了热稳定 PCR 的稳定性，结果发现其在 24°C 下稳定约 7 个月。热稳定三重 PCR 检测方法的检测限为 2×10(4) c.f.u. 细菌细胞水平和 100 pg 基因组 DNA 水平，与传统 PCR 方法相当。总之，开发了一种快速热稳定的三重 PCR 检测方法，用于检测产毒性和非产毒性的霍乱弧菌，该方法需要的移液步骤最少，且无需冷链。
