Performance Testing for Polymerase Chain Reaction Assays
Project ID: 7954
Principal Investigator: Kevin Kelly
Research Topic: Invasive Species
Priority Area Assignments: 2015 (Zebra and Quagga Mussels)
Funded Fiscal Years: 2014 and 2015
Keywords: polymerase chain reaction, dreissena spp., plasmid, dna
Can we directly measure and compare the performance of polymerase chain reaction (PCR) assays and individual laboratories using a positive control plasmid?
Can live, dead, and cross-contamination sources of target DNA be distinguished during PCR assays that will help water managers determine if a reservoir is truly infested with live mussels?
We have recently completed a national three phase round robin study involving 19 laboratories utilizing polymerase chain reaction and microscopy-based laboratory assays for the early detection of Dreissena spp. (zebra and quagga mussels). The second phase of our study was published (Frischer ME, Kelly KL, and Nierzwicki-Bauer SA. 2012. Accuracy and Reliability of Dreissena spp. Larvae Detection by Cross-Polarized Light Microscopy, Imaging Flow Cytometry, and Polymerase Chain Reaction Assays. Lake Reservoir Management, 28:265-276). Overall, results indicated that PCR was the least reliable assay (75.8% accuracy). Lessons learned from this study has led to valuable insights on how we can improve the quality and defend the integrity of our PCR results, upon which water managers are often reliant for making management decisions.
Need and Benefit
Contaminating DNA in the laboratory can originate from three sources: 1. From other samples ("sample cross-contamination"), 2. From introduced experimental materials such as recombinant clones, and 3. From DNA generated by previous PCR amplification of the same target sequence ("carry-over contamination"). The first and third sources are the most likely sources of contamination occurring in our labs. We need to make sure these false positives are not happening for strong data defensibility. Failure to ensure that these false positives do not occur jeapordizes the integrity and reputation of our zebra and quagga mussel monitoring programs. This research project proposed to use methods published and endorsed by Metropolitan Water District to prevent carry-over contamination. In addition, this project proposes to utilized artificially designed DNA (so not to be confused with actual target DNA) to test for the source of contamination and current procedures for preventing contamination, test for the performance of extant PCR methods, and inter-laboratory consensus for method robustness and reproducibility that will allow us to obtain second-lab confirmation (i.e., Metropolitan Water District). All extant PCR sequences were compiled together by the PI (Kelly) and created by a private contractor (i.e., SeqWright). However, testing must be performed internally by Reclamation and other laboratories as described above to determine effectiveness prior to routine use.
Currently, all analytical methods being used by Reclamation to monitor for zebra and quagga mussels do not determine whether an positive ID resulted from a live or dead source. The inability to distinguish dead source from a live source in a reservoir complicate decisions by water managers. Simply detecting the presence of a shell (i.e., flowcam, cross-polarized microscopy, electron microscopy) or target DNA (PCR) is not proof the reservoirs is infested. However, DNA from a dead source has a limited life and rapidly decay. This project proposes to determine the feasibility of utilizing established bacteriology procedures to distiguish live versus dead sources of zm/qm DNA. A few other federal agencies have begun to use this technique with success, but it's uncertain how robust it is for a larger geographical area.
Report of laboratory testing results from: a. Plasmid dilution series containing all of the extant PCR and qPCR reaction assays. b. Uracil-DNA-glycoslase reaction. If it works well, laboratory standard operating procedure for incorporating Uracil-DNA-glycosylase in any PCR assays. And c. Performance of PMA on spiked dead ("naked") DNA versus decaying veligers versus freshly preserved veligers. We will also provide a summary report on the investigation of the feasibility of utilizing qPCR by adapting or upgrading existing equipment in the laboratory. The report will include the background, description of methods performed, results, and interpretation of the results. Novel procedures or applications (especially with plasmids, which has never been done before) may be published in peer reviewed publications.