Development of a SYBR Green-Based Real-Time PCR Assay to Detect Oncomelania hupensis quadrasi DNA in Environmental Water Samples.

IF 2.8 4区医学 Q2 INFECTIOUS DISEASES

Tropical Medicine and Infectious Disease Pub Date : 2025-05-20 DOI:10.3390/tropicalmed10050140

Daria L Manalo, Jude Karlo G Bolivar, Karl Ian T Ermino, Jeromir G Bondoc, Mark Joseph M Espino, Efraim P Panganiban, Kathyleen S Nogrado, Raffy Jay C Fornillos, Mario A Jiz, Lydia R Leonardo, Ian Kendrich C Fontanilla

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Abstract

Oncomelania hupensis quadrasi is the intermediate host of S. japonicum, the causative species of schistosomiasis in the Philippines. Conventionally, risk areas are identified by procedures requiring highly skilled personnel and constant surveillance efforts. Recent developments in disease diagnostics explore the utilization of environmental DNA as targets for polymerase chain reactions in disease surveillance. In this study, a low-cost, specific, and efficient SYBR Green-based real-time PCR assay to detect O. h. quadrasi DNA from water samples was developed, optimized, and validated. Primers were designed based on the A18 microsatellite region of O. h. quadrasi. The assay exhibited a detection limit of one copy number per microliter at 99.4% efficiency and R² = 0.999, which specifically amplified O. h. quadrasi DNA only. Validation of this assay in environmental water samples demonstrated 100% PPV and NPV values, suggesting its potential as a tool for identifying risk areas, pathogen-directed surveillance, policy making, and disease control.

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基于SYBR绿色的实时荧光定量PCR检测环境水样中湖北钉螺DNA的建立。

方形猪钉螺是菲律宾血吸虫病的致病种日本血吸虫的中间宿主。通常，风险区域是通过需要高技能人员和持续监测努力的程序确定的。疾病诊断的最新发展探索了利用环境DNA作为疾病监测中聚合酶链反应的靶标。本研究建立了一种低成本、特异、高效的基于SYBR green的实时PCR方法，用于检测水样中的方形腹虫DNA，并进行了优化和验证。引物的设计是基于圆叶螟A18微卫星区。该方法的检测限为每微升1个拷贝数，检测效率为99.4%,R2 = 0.999，特异扩增了方形腹虫的DNA。该方法在环境水样中的验证显示出100%的PPV和NPV值，表明其有潜力作为确定风险区域、病原体导向监测、政策制定和疾病控制的工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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