Department of Life Sciences, University of Calicut, Malappuram, Kerala-673635, India.
Mar Biotechnol (NY). 2021 Dec;23(6):955-963. doi: 10.1007/s10126-021-10078-9. Epub 2021 Oct 29.
Waterborne microbial diseases are regarded as a major public health concern, particularly in nations with poor sanitation, a lack of social awareness, and problems linked with low socioeconomic status. Waterborne pathogen identification using traditional culture methods is time-consuming and labor-intensive. As a result, there is a growing demand for quick pathogen detection technologies. High sensitivity, specificity, and rapidity are all advantages of using molecular techniques like polymerase chain reaction (PCR) in such instances. In this study, we designed multiplex PCR and quantitative real-time PCR (qPCR) assays for the co-detection and enumeration of waterborne pathogens such as Aeromonas hydrophila, Pseudomonas aeruginosa, Salmonella enterica, Yersinia enterocolitica, Escherichia coli, Vibrio cholerae, and Shigella spp. Specific primers were selected against the virulence and species-specific genes of the seven target pathogens. For all seven target organisms, the detection limits for conventional culture methods were in the range of 10-10 cells/ml. While employing multiplex PCR method in this study, Pseudomonas aeruginosa and Shigella spp. have a detection sensitivity of 10 cells/ml, Vibrio cholerae and Aeromonas hydrophila have a detection sensitivity of 10 cells/ml, whereas Salmonella enterica, E. coli, and Yersinia enterocolitica have a detection sensitivity of only 10 cells/ml. According to our cost-benefit analysis, these molecular technologies are less expensive, with unit analysis costs of ₹52 and ₹173 for qPCR and multiplex PCR, respectively. Furthermore, all of the target genes had a detection limit of 1 cell/ml in qPCR. Because of their speed, sensitivity, specificity, and cost-effectiveness, these multiplex and qPCR assays could be employed for successful co-detection of aquatic pathogens.
水媒微生物疾病被认为是一个主要的公共卫生关注点，特别是在那些卫生条件差、社会意识薄弱以及与低社会经济地位相关的问题较多的国家。使用传统培养方法进行水媒病原体识别既费时又费力。因此，人们越来越需要快速的病原体检测技术。在这种情况下，聚合酶链反应（PCR）等分子技术具有高灵敏度、特异性和快速性的优势。在本研究中，我们设计了多重 PCR 和定量实时 PCR（qPCR）检测方法，用于同时检测和计数水媒病原体，如嗜水气单胞菌、铜绿假单胞菌、肠炎沙门氏菌、小肠结肠炎耶尔森菌、大肠埃希氏菌、霍乱弧菌和志贺氏菌。选择了针对这七种目标病原体的毒力和种特异性基因的特异性引物。对于所有七种目标生物，传统培养方法的检测限在 10-10 细胞/ml 范围内。在本研究中使用多重 PCR 方法时，铜绿假单胞菌和志贺氏菌的检测灵敏度为 10 细胞/ml，霍乱弧菌和嗜水气单胞菌的检测灵敏度为 10 细胞/ml，而肠炎沙门氏菌、大肠埃希氏菌和小肠结肠炎耶尔森菌的检测灵敏度仅为 10 细胞/ml。根据我们的成本效益分析，这些分子技术更便宜，qPCR 和多重 PCR 的单位分析成本分别为₹52 和₹173。此外，qPCR 中所有目标基因的检测限均为 1 细胞/ml。由于这些多重和 qPCR 检测方法具有速度快、灵敏度高、特异性强和具有成本效益的特点，因此可以成功地同时检测水生病原体。
