Noor-Shazleen-Husnie M M, Emelia O, Ahmad-Firdaus M S, Zainol-Ariffin P, Aishah-Hani A
Department of Parasitology and Medical Entomology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre (UKMMC), 56000 Kuala Lumpur, Malaysia.
Department of Health and Environment, Kuala Lumpur City Hall (DBKL), 56100 Kuala Lumpur, Malaysia.
Trop Biomed. 2018 Jun 1;35(2):330-346.
Wolbachia, a naturally endosymbiotic bacteria, has shown its potential as one of biological control agents for vector borne diseases. Due to large number of mosquitoes involved in Wolbachia screening, Loop-mediated isothermal amplification (LAMP) is recommended as a convenient and time-saving technique. This study aimed to evaluate a newly developed LAMP assay for detection of Wolbachia by targeting 16S rDNA gene in samples of wild mosquito populations. The LAMP products were confirmed by colorimetric detection using hydroxy naphthol blue (HNB), digestion with RsaI restriction enzyme and gel electrophoresis. The restriction enzyme digestion of PCR products was performed to differentiate between Wolbachia supergroups A and B. Out of 765 mosquito samples tested, 349 (45.6%) and 237 (31%) of the samples were positive for LAMP and PCR techniques respectively. The prevalence of Wolbachia detected in mosquitoes was significantly higher using LAMP as compared to PCR. There is significant association between numbers of mosquitoes positive with Wolbachia detected using LAMP and PCR (χ=61.31; df=1; p < 0.05) with a kappa (κ) value of 0.27 and Phi value, 0.283. This study highlighted the potential of LAMP as a sensitive, specific and rapid tool for screening of Wolbachia in mosquitoes, thus it presents as an alternative to PCR-based assays.
沃尔巴克氏体是一种天然的共生细菌，已显示出其作为媒介传播疾病生物防治剂之一的潜力。由于参与沃尔巴克氏体筛选的蚊子数量众多，推荐环介导等温扩增技术（LAMP）作为一种便捷省时的技术。本研究旨在评估一种新开发的LAMP检测方法，该方法通过靶向野生蚊子种群样本中的16S rDNA基因来检测沃尔巴克氏体。LAMP产物通过使用羟基萘酚蓝（HNB）进行比色检测、用RsaI限制性内切酶消化和凝胶电泳来确认。对PCR产物进行限制性内切酶消化以区分沃尔巴克氏体A和B超群。在测试的765个蚊子样本中，分别有349个（45.6%）和237个（31%）样本的LAMP和PCR技术检测呈阳性。与PCR相比，使用LAMP检测到的蚊子中沃尔巴克氏体的流行率显著更高。使用LAMP和PCR检测到的沃尔巴克氏体阳性蚊子数量之间存在显著关联（χ=61.31；自由度=1；p<0.05），kappa（κ）值为0.27，Phi值为0.283。本研究强调了LAMP作为一种灵敏、特异且快速的工具用于筛选蚊子中沃尔巴克氏体的潜力，因此它可作为基于PCR检测方法的替代方法。
