Reference Materials For Specific PIK3CA Gene Mutation Testing :establishment And Application

Objective:To develop can simulate real clinical samples containing specific PIK3 CA gene mutation detection quality, clinical laboratory gene mutations and use it to measure the quality evaluation between pre-research, discusses practicability of the quality control and clinical laboratory PIK3 CA gene mutation detection ability. Methods: Artificial preparation contains five different type PIK3 CA gene in human breast cancer cell line and human colorectal cancer cell line of formaldehyde fixed, paraffin embedding samples by 4% neutral formalin fixed dehydration, transparent and xylene, ethanol gradient again after paraffin embedding embedding slice; Slice evaluation directly by HE staining cells content; Every 100 preliminary extract six samples of the quality control for DNA extraction and concentration, and the preparation of good quality material placed at 37 ° C, respectively at room temperature(20 ~ 25 ° C) and 2 ~ 8 ° C stability test; PCR- Sanger sequencing and amplification hinder mutation system method to validate different genotype, composed of PIK3 CA gene mutations room quality evaluation between pre-study sample plate issued to each clinical laboratory, at the same time, the sample plate to three domestic authoritative laboratory for testing; Statistical analysis of returns from the laboratory results. Results:Chip cell content enough, uniform distribution, each sample DNA concentrations were greater than 500 ng, at 37 ° C, room temperature(20 ~ 25 ° C) and 2 ~ 8 ° C conditions can keep stable for at least 6 months; PCR- Sanger sequencing and amplification hinder the mutation system method of different genotypes with expectations for the results of the validation; To return from the laboratory results were statistically analyzed, according to a 2015 PIK3 CA chamber interstitial advance research received 75 valid returns the result of evaluation. Test results completely correct laboratory proportion was 25.33%(19/75), all eligible laboratory tests of the false negative rate was 18.67%(112/600), false positive rate was 17.33%(26/150). All eligible laboratory tests positive samples coincidence rate was 62.83%(377/600), the negative samples coincidence rate was 79.33%(119/150). The most widely used method is amplified block mutation system method(ARMS). Conclusion: This study successfully established the artificial preparation containing specific PIK3 CA gene mutation detection method of quality control to the quality evaluation between pre-research and applied to the chamber; Develop quality content than clinical pathological sample source wide, more stable, heterogeneity is small, easy to a lot of preparation, can be effectively used to measure the PIK3 CA gene mutations between quality evaluation; Room 2015, according to the results of quality evaluation pre-research in each lab samples on the overall coincidence rate is not high, false positive and false negative results has always been the major problems affecting mutation detection accuracy.