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Validation of Master Mix Storage and RNA Extraction Methods for a Real-Time RT-PCT (RRT-PCR) Assay for Avian Influenza


The objective of the study is to evaluate the stability of bulk lots of master mix solution for use with real-time RT-PCR for avian influenza virus. The master mix will be evaluated for stability under common laboratory conditions as compared to fresh master mix solution. The second objective will be the comparison of different RNA extraction procedures for use with wild bird and poultry fecal samples. An internal control will be included to evaluate the ability of the various RNA extraction procedures to remove PCR inhibitors that may be present in the fecal samples and could interfere with the PCR test.

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APPROACH: The preparation of the master mix for RRT-PCR includes primers, probes, buffer, dNTPs, internal control RNA, RNase inhibitors, and polymerase enzymes. Currently there is a requirement to do this preparation on a daily basis; however, the laboratories that perform the tests have expressed interest in preparing larger bulk mixes that can be stored and used as needed to improve efficiency of the testing process. A comparison of the stability of the bulk master mix will be evaluated for reproducibility of test results over storage time of the master mix. The master mix will be kept either frozen at -20 to -80 C or at 4 C. At select time points after preparation, aliquots of the master mix will be thawed and used to detect avian influenza virus. Comparison with freshly prepared master mix will provide a standard. Additionally, viral RNA extraction procedures will be compared. Various readily available extraction platforms will be compared, as will high throughput procedures that rely on vacuum or magnetic bead extraction techniques. Some of those procedures will include use of a commercially available extraction robot. Wild aquatic bird fecal samples and domestic poultry fecal samples will serve as the sample matrix. The fecal samples will be spiked with known concentrations of virus. An internal control will be used to detect the presence of PCR inhibitors. The study also will attempt to provide an estimate of economy for the different procedures, taking into account materials cost, labor, time, and assay performance.

Michigan State University
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