An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples.
An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples.
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DOI：
10.1371/journal.pone.0160523
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Stoddard RA
中科院分区：
文献类型：
--
作者：
Riediger IN;Hoffmaster AR;Casanovas-Massana A;Biondo AW;Ko AI;Stoddard RA
钩端螺旋体病是一种人畜共患疾病，通常通过接触受感染动物尿液污染的水而获得。然而，很少有分子方法用于监测或量化环境水样中的致病性钩端螺旋体。在这里，我们使用先前描述的基于 Taqman 的 qPCR 方法优化了钩端螺旋体定量的 DNA 提取方法，该方法针对 lipL32，这是致病性钩端螺旋体独有且高度保守的基因。 QIAamp DNA mini、MO BIO PowerWater DNA 和 PowerSoil DNA Isolation 试剂盒经过评估，可从掺有钩端螺旋体的污水、池塘、河流和超纯水样本中提取 DNA。每个试剂盒的性能因样品类型而异。由于 PowerSoil DNA 试剂盒在浑浊水样上的性能和重现性，我们进一步评估和优化了样品处理方法。比较了离心速度、水量和使用大肠杆菌作为载体以提高 DNA 回收率。所有基质在 106 至 10° 钩端螺旋体/mL 的浓度范围内均表现出很强的线性，检测下限范围为从<1 个细胞/mL（河水）到 36 个细胞/mL（以大肠杆菌为载体的超纯水）。总之，我们优化了一种定量环境水域（河流、池塘和污水）中致病性钩端螺旋体的方法，该方法包括通过在 4°C 下以 15,000×g 离心 20 分钟来浓缩 40 mL 样品，然后使用 PowerSoil DNA 分离试剂盒提取 DNA。尽管本文描述的方法需要在环境研究中得到验证，但它可能为有效、及时和敏感地评估环境钩端螺旋体负担提供机会。
Leptospirosis is a zoonotic disease usually acquired by contact with water contaminated with urine of infected animals. However, few molecular methods have been used to monitor or quantify pathogenic Leptospira in environmental water samples. Here we optimized a DNA extraction method for the quantification of leptospires using a previously described Taqman-based qPCR method targeting lipL32, a gene unique to and highly conserved in pathogenic Leptospira. QIAamp DNA mini, MO BIO PowerWater DNA and PowerSoil DNA Isolation kits were evaluated to extract DNA from sewage, pond, river and ultrapure water samples spiked with leptospires. Performance of each kit varied with sample type. Sample processing methods were further evaluated and optimized using the PowerSoil DNA kit due to its performance on turbid water samples and reproducibility. Centrifugation speeds, water volumes and use of Escherichia coli as a carrier were compared to improve DNA recovery. All matrices showed a strong linearity in a range of concentrations from 106 to 10° leptospires/mL and lower limits of detection ranging from <1 cell /ml for river water to 36 cells/mL for ultrapure water with E. coli as a carrier. In conclusion, we optimized a method to quantify pathogenic Leptospira in environmental waters (river, pond and sewage) which consists of the concentration of 40 mL samples by centrifugation at 15,000×g for 20 minutes at 4°C, followed by DNA extraction with the PowerSoil DNA Isolation kit. Although the method described herein needs to be validated in environmental studies, it potentially provides the opportunity for effective, timely and sensitive assessment of environmental leptospiral burden.