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首页> 外文期刊>Applied Microbiology >Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9
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Loop-Mediated Isothermal Amplification (LAMP) for Rapid Detection and Quantification of Dehalococcoides Biomarker Genes in Commercial Reductive Dechlorinating Cultures KB-1 and SDC-9

机译：环路介导的等温扩增（LAMP），用于快速检测和定量商业还原性脱氯培养物KB-1和SDC-9中的脱卤球菌生物标志物基因

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Real-time quantitative PCR (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA , bvcA , and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent-contaminated sites. In this study, loop-mediated isothermal amplification (LAMP) was developed as an alternative approach for the quantification of these genes. LAMP does not require a real-time thermal cycler (i.e., amplification is isothermal), allowing the method to be performed using less-expensive and potentially field-deployable detection devices. Six LAMP primers were designed for each of three RDase genes ( vcrA , bvcA , and tceA ) using Primer Explorer V4. The LAMP assays were compared to conventional qPCR approaches using plasmid standards, two commercially available bioaugmentation cultures, KB-1 and SDC-9 (both contain Dehalococcoides species). DNA was extracted over a growth cycle from KB-1 and SDC-9 cultures amended with trichloroethene and vinyl chloride, respectively. All three genes were quantified for KB-1, whereas only vcrA was quantified for SDC-9. A comparison of LAMP and qPCR using standard plasmids indicated that quantification results were similar over a large range of gene concentrations. In addition, the quantitative increase in gene concentrations over one growth cycle of KB-1 and SDC-9 using LAMP was comparable to that of qPCR. The developed LAMP assays for vcrA and tceA genes were validated by comparing quantification on the Gene-Z handheld platform and a real-time thermal cycler using DNA isolated from eight groundwater samples obtained from an SDC-9-bioaugmented site (Tulsa, OK). These assays will be particularly useful at sites subject to bioaugmentation with these two commonly used Dehalococcoides species-containing cultures.
机译：特定于还原性脱卤素酶（RDase）基因vcrA，bvcA和tceA的实时定量PCR（qPCR）协议通常用于定量Dehalococcoides spp。来自氯化溶剂污染场所的地下水中。在这项研究中，开发了环介​​导的等温扩增（LAMP）作为量化这些基因的替代方法。 LAMP不需要实时热循环仪（即，扩增是等温的），从而允许使用价格较低且可能在现场部署的检测设备执行该方法。使用Primer Explorer V4为三个RDase基因（vcrA，bvcA和tceA）的每一个设计了六个LAMP引物。使用质粒标准品，两种市售生物增强培养物KB-1和SDC-9（均包含Dehaloccocoides菌种），将LAMP分析与常规qPCR方法进行了比较。在整个生长周期中，从分别用三氯乙烯和氯乙烯修正的KB-1和SDC-9培养物中提取DNA。所有三个基因的KB-1定量，而仅vcrA的SDC-9定量。使用标准质粒对LAMP和qPCR的比较表明，在较大的基因浓度范围内，定量结果相似。此外，使用LAMP在KB-1和SDC-9的一个生长周期中基因浓度的定量增加与qPCR相当。通过比较从Gene-Z手持平台和实时热循环仪上的定量数据，使用从SDC-9生物富集位点（塔尔萨，俄克拉何马州，塔尔萨）获得的八个地下水样品中分离得到的DNA，对vcrA和tceA基因开发的LAMP分析进行了验证。这些测定在使用这两种常用的含有脱卤球菌物种的培养物进行生物增强的部位上特别有用。

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