Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia.
Biomed Res Int. 2013;2013:412370. doi: 10.1155/2013/412370. Epub 2013 Feb 13.
The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 10(4) CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.
发展中国家的志贺菌病的严重程度在很大程度上是未知的，因为没有负担得起的检测方法。目前对志贺氏菌属的实验室诊断既费力又费时，而且敏感性低。因此，在本研究中，开发了一种基于分子的诊断检测方法，该方法可同时扩增四个特定基因，以鉴定志贺菌属的 invC、S. flexneri 的 rfc、S. sonnei 的 wbgZ 和 S. dysenteriae 的 rfpB，以及一个内部对照（ompA）基因，以在单个反应中检测和区分志贺氏菌属。用 120 株志贺氏菌和 37 株非志贺氏菌菌株进行验证，特异性为 100%。PCR 的灵敏度为 100 pg 基因组 DNA、5.4×10(4) CFU/ml，或约 120 CFU 每反应混合物中的细菌。在粪便样本中预先孵育革兰氏阴性肉汤后，五重 PCR 检测的灵敏度进一步提高。对 30 份腹泻标本的初步研究表明，与其他测试的非志贺氏菌菌株没有交叉反应。我们得出结论，开发的五重 PCR 检测方法稳健可靠，可提供有关鉴定志贺菌属和引起大多数志贺菌病的三种志贺氏菌所必需的四个靶基因的信息。
