Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater.

摘要: Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use filtration, elution, direct isothermal (i.e. no DNA extraction purification) as a field-able means quantify Dehalococcoides spp. groundwater. This study expands previous work with loop mediated (LAMP) for detection quantification Dehalobacter Experiments tested without crude lysis varying concentrations humic acid. Three separate methods biomass concentration eight aquifer samples were also tested, comparing LAMP traditional quantitative PCR (qPCR). A new technique was developed where filters amplified directly within disposable Gene-Z chips. The filter (DFA) method eliminated an elution step provided limit 102Dehalobacter cells per 100mL. crudely lysed had negligible effect on threshold time sensitivity compared samples. assay more resilient than qPCR sample, amplifying up 100mg L reaction 1mg qPCR. Of methods, DFA lowest coefficient variation among spiked groundwater indicating high capture efficiency low inhibition. While demonstrated Dehalobacter, can potentially be number applications requiring field-able, rapid (<60min) highly sensitive microorganisms environmental water