Discipline of Genetics, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal,Westville Campus, Durban, South Africa.
Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Westville Campus, Durban, South Africa.
Lett Appl Microbiol. 2019 Oct;69(4):264-270. doi: 10.1111/lam.13200. Epub 2019 Aug 9.
Contaminated wastewater plays an important role in the transmission of Listeria monocytogenes in the environment. In this study, a loop-mediated isothermal amplification (LAMP) assay for sensitive detection of L. monocytogenes in wastewater from treatment plants was developed, validated and compared to conventional PCR. The lmo0753 gene which codes for a Crp/Fnr family transcription factor, was targeted to design four specific primers to detect L. monocytogenes in 60 min at 63°C in a water bath. Amplification products were visualized by agarose gel electrophoresis. The detection limit of the LAMP assay was 65 fg µl of DNA and 38 CFU per ml. Conventional PCR was 10 times less sensitive than LAMP assay with primers targeting the HlyA gene. A total of 70 crude wastewater samples collected at different treatment stages (aeration tank, pre chlorination and post chlorination), were tested directly by LAMP and PCR. Samples from aeration and pre-chlorination stages tested positive with LAMP and culture method but not with conventional PCR. LAMP assay was tolerant to inhibitors present in wastewater and circumvented the need for isolation of pure DNA for detection. Both LAMP assay and culture method failed to detect L. monocytogenes in post-chlorinated wastewater, confirming the efficiency of the treatment process in the removal of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated wastewater effluent contains Listeria monocytogenes which survives conventional wastewater treatment processes and can re-enter human food chain, thus it is imperative to detect L. monocytogenes using a rapid and an inexpensive method. To the best of our knowledge, this is the first report of a loop-mediated isothermal amplification (LAMP) assay, targeting the lmo0753 gene for detection of L. monocytogenes in wastewater from treatment plants. The LAMP assay detects L. monocytogenes in 60 min at 63°C in a water bath. LAMP does not require isolation of pure genomic DNA hence it is a user friendly method for L. monocytogenes detection.
受污染的废水在环境中李斯特菌的传播中起着重要作用。在这项研究中，开发了一种环介导等温扩增（LAMP）检测方法，用于敏感检测处理厂废水中的李斯特菌。该方法针对编码 Crp/Fnr 家族转录因子的 lmo0753 基因设计了四个特异性引物，在 63°C水浴中 60 分钟内即可检测到李斯特菌。通过琼脂糖凝胶电泳可视化扩增产物。LAMP 检测的检测限为 65 fg µl DNA 和 38 CFU/ml。针对 HlyA 基因的常规 PCR 比 LAMP 检测方法的灵敏度低 10 倍。用 LAMP 和培养方法直接检测了从不同处理阶段（曝气池、预氯化和后氯化）收集的 70 个粗废水样品。LAMP 和培养方法检测到曝气和预氯化阶段的样品呈阳性，但常规 PCR 检测不到。LAMP 检测法对废水中存在的抑制剂具有耐受性，并且避免了为检测而分离纯 DNA 的需要。LAMP 检测法和培养法均未能检测到后氯化废水中的李斯特菌，证实了处理过程去除李斯特菌的效率。研究的意义和影响：处理后的废水含有李斯特菌，它能在常规废水处理过程中存活下来，并重新进入人类食物链，因此必须使用快速且廉价的方法来检测李斯特菌。据我们所知，这是首次报道使用环介导等温扩增（LAMP）检测方法，针对 lmo0753 基因检测处理厂废水中的李斯特菌。LAMP 检测法在 63°C水浴中 60 分钟内即可检测到李斯特菌。LAMP 不需要分离纯基因组 DNA，因此是一种方便用户使用的李斯特菌检测方法。
