Center of Excellence for Vectors and Vector-Borne Diseases, Faculty of Science, Mahidol University at Salaya, Nakhon Pathom, Thailand.
Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand.
PLoS Negl Trop Dis. 2022 May 13;16(5):e0009600. doi: 10.1371/journal.pntd.0009600. eCollection 2022 May.
Wolbachia is an endosymbiont bacterium generally found in about 40% of insects, including mosquitoes, but it is absent in Aedes aegypti which is an important vector of several arboviral diseases. The evidence that Wolbachia trans-infected Ae. aegypti mosquitoes lost their vectorial competence and became less capable of transmitting arboviruses to human hosts highlights the potential of using Wolbachia-based approaches for prevention and control of arboviral diseases. Recently, release of Wolbachia trans-infected Ae. aegypti has been deployed widely in many countries for the control of mosquito-borne viral diseases. Field surveillance and monitoring of Wolbachia presence in released mosquitoes is important for the success of these control programs. So far, a number of studies have reported the development of loop mediated isothermal amplification (LAMP) assays to detect Wolbachia in mosquitoes, but the methods still have some specificity and cost issues.
METHODOLOGY/PRINCIPAL FINDINGS: We describe here the development of a LAMP assay combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae. aegypti. Our developed LAMP primers used a low-cost dye detecting system and 4 oligo nucleotide primers which can reduce the cost of analysis while the specificity is comparable to the previous methods. The detection capacity of our LAMP technique was 1.4 nM and the detection limit reduced to 2.2 fM when combined with the BIOSENSOR. Our study demonstrates that a BIOSENSOR can also be applied as a stand-alone method for detecting Wolbachia; and it showed high sensitivity when used with the crude DNA extracts of macerated mosquito samples without DNA purification.
CONCLUSIONS/SIGNIFICANCE: Our results suggest that both LAMP and BIOSENSOR, either used in combination or stand-alone, are robust and sensitive. The methods have good potential for routine detection of Wolbachia in mosquitoes during field surveillance and monitoring of Wolbachia-based release programs, especially in countries with limited resources.
沃尔巴克氏体是一种内共生菌，通常存在于约 40%的昆虫中，包括蚊子，但埃及伊蚊中不存在，而埃及伊蚊是几种虫媒病毒病的重要传播媒介。证据表明，沃尔巴克氏体感染的埃及伊蚊失去了媒介能力，传播虫媒病毒给人类宿主的能力降低，这凸显了利用沃尔巴克氏体方法预防和控制虫媒病毒病的潜力。最近，释放感染沃尔巴克氏体的埃及伊蚊已在许多国家广泛用于控制蚊媒病毒病。在释放的蚊子中监测沃尔巴克氏体的存在对于这些控制计划的成功至关重要。到目前为止，已经有许多研究报告了开发环介导等温扩增（LAMP）检测方法来检测蚊子中的沃尔巴克氏体，但这些方法仍然存在一些特异性和成本问题。
方法/主要发现：我们在这里描述了一种 LAMP 检测方法与基于 DNA 链置换的电化学传感器（BIOSENSOR）方法相结合，用于检测感染的埃及伊蚊中的 wAlbB 沃尔巴克氏体。我们开发的 LAMP 引物使用了低成本染料检测系统和 4 个寡核苷酸引物，可以降低分析成本，而特异性与以前的方法相当。当与 BIOSENSOR 结合使用时，我们的 LAMP 技术的检测能力为 1.4 nM，检测限降低至 2.2 fM。我们的研究表明，BIOSENSOR 也可以作为一种独立的方法用于检测沃尔巴克氏体；并且当与未经 DNA 纯化的研磨蚊子样本的粗 DNA 提取物一起使用时，它显示出很高的灵敏度。
结论/意义：我们的结果表明，LAMP 和 BIOSENSOR 无论是单独使用还是联合使用，都具有强大的敏感性。这些方法在现场监测和监测基于沃尔巴克氏体的释放计划中检测蚊子中的沃尔巴克氏体具有良好的潜力，尤其是在资源有限的国家。
