ABSTRACT
Objective:Use the droplet digital PCR (ddRCR) technology to establish, optimize and evaluate the method of EGFR-T790M mutation detection.Methods:The relevant probes and primers were designed for EGFR-T790M mutations. The ddPCR reaction system was established, the optimal annealing temperature was set and the basic performance of the method was tested. On this basis, from January 2019 to October 2019, 72 cell-free DNA (cfDNA) samples from NSCLC patients were collected from Shandong Cancer Hospital Affiliated to Shandong First Medical University, and clinically verified. The consistency of the gene mutation detections with Bole ddPCR products was analyzed using Kappa test.Results:The ddPCR reaction system was established and optimized. Linear evaluation showed the R2 value was greater than 0.99. Using ddPCR, the blank detection limit was determined to be the numbers of mutant droplets≥2, with excellent specificity. For the sensitivity analysis, the lower limit of mutation detection was determined to be at least 0.05%. In the repeatability and inter-assay precision tests, the results had a coefficient of variation( CV)<20%. The relative deviation of the results was within the range of±10% for the accuracy analysis. Using the established T790M mutation detection method, 72 samples from the NSCLC patients were tested for genetic mutation in cfDNA, and the overall agreement with the Bole ddPCR products was 91.67% (66/72, Kappa=0.749; P<0.001). Conclusion:Using ddPCR, the method of EGFR-T790M mutation detection for NSCLC was successfully established.
ABSTRACT
<p><b>OBJECTIVE</b>To study contractility of guinea pig ileum in vitro,and analyze the mechanism of anti-emetic effects of Lianqiao.</p><p><b>METHOD</b>Using emesis-relating agonists as drugs, the inhibitory effects of Lianqiao on guinea pig ileum contractility in vitro were observed in organ bath.</p><p><b>RESULT</b>Lianqiao could inhibit guinea pig ileum spontaneous contractions, reducing the tone of contractions dose-dependently. Acetylcholine (Ach), histamine (His), 5-hydroxytryptamine (5-HT) stimulated contractions of the guinea pig ileum, enhanced the tone and amplitude. All the three doses (10, 5, 2 g x L(-1)) of Lianqiao could suppress the contractility, significantly reduced the tone and amplitude of ileum contractions stimulated by drugs but not the frequency. Dopamine could inhibit the spontaneous contraction tone and amplitude of ileum; Both the large doses (10, 5 g x L(-1)) of Lianqiao could antagonise the inhibitory effect of DA, enhance the tone and amplitude. Small dose(2 g x L(-1)) had additive effects on tone of ileum contractions with DA,but enhanced the amplitude not the frequency.</p><p><b>CONCLUSION</b>Lianqiao have an inhibitory effect on guinea pig ileum contractions,the mechanism might be blocking M receptor, H1 receptor, 5-HT receptor and D2 receptor or directly suppressed ileum smooth muscle. The mechanisms of anti-emetic effect of Lianqiao needs further study.</p>