You are here:
Progress report on development of modifications to EPA Method C to mitigate sample matrix interferences
Citation:
Haugland, Rich AND S. Hertel. Progress report on development of modifications to EPA Method C to mitigate sample matrix interferences. MiNET Fall 2021 Meeting, Muskegon, MI, October 14 - 15, 2021.
Impact/Purpose:
Inform public health officials and scientists on the current status of EPA efforts to support implementation of rapid recreational beach monitoring by quantitative PCR methods in Michigan and Great Lakes region (EPA Region 5).
Description:
In 2020, a Regional Applied Research Effort (RARE) grant, sponsored by EPA Region 5, was awarded for EPA/ORD researchers to support efforts by MiNet labs to interpret and improve analysis results from recreational beaches by EPA Draft Method C for E. coli. A major component of the proposed effort was to develop more robust sample preparation procedures that would address relatively rare but nevertheless significant instances of sample matrix interference by some water samples to qPCR analyses. Work has proceeded on this component of the project and current results indicate that the addition of a proposed stabilizing material to the extraction buffer used in the method is highly effective in mitigating losses of DNA recovery and subsequent interferences to qPCR analysis results caused by a surrogate, naturally occuring interfering substance in the environment (kaolinite clay). Results have further indicated that this stabilizing material has no significant effects itself on qPCR measurements by the sample processing control (SPC) assay or by the qPCR assay used for measuring E. coli target sequences in EPA Draft Method C when used at defined concentrations that were effective in mitigating kaolinite clay interferences. Optimal concentrations of the stabilizing material have been identified and a modified method, incorporating this material, is now ready for paired sample testing with the current method on freezer-archived recreational beach water filter samples, collected with support from the RARE grant by selected MiNet and other laboratories. Results of this testing are anticipated to show similar results between the two methods in normal sample analyses and to demonstrate mitigation of sample matrix interference by the modified method in samples where interference is observed from SPC analysis results in the current method.