Non-small Cell Lung Cancer Egfr Gene Mutation Detection Kit Developed

Part I Construction of29Recombinants containing mutant EGFR genesObjective:To construct the twenty-nine recombinants containing EGFR mutant genes by using overlap extension PCR, plasmid ligation and transformation.Method:Firstly, the twenty-nine most common mutant EGFR genes [including three types of point mutation in exon18(G719A, G719S and G719C); nineteen types of deletion mutation in exon19; five types of mutation in exon20(T790M, S768I and three types of insertion mutation); two types of point mutation in exon21(L859R and L861Q)] were established and amplified by overlap extension PCR. Secondly, the mutant EGFR genes were ligated to pMD19plasimid and then the products of liagation were transformed to DH5a competent cells. Subsequently, the transformed DH5a competent cells were inoculated on the solid culture medium containing ampicillin. White bacterial colonies indicated the success of gene insertion and blue bacterial colonies indicated the failure of gene insertion. Finally, white bacterial colonies were selected and added to the LB liquid medium to culture. The sequence identification by PCR and sequencing was used to judge whether the recombinants were established successfully.Results:The results of overlap extension PCR showed corresponding outer and mutant primers did successfully yield half fragments with complimentary ends, each half fragment containing mutant bases. And Self-priming of the complementary half fragments and subsequent amplification created final products with mutant bases.The products of mutant bases could be successfully cloned into pMD19plasmid. The sequences of each recombinant plasmid were confirmed by sequencing and were the same as the expected.Conclusions:The twenty-nine recombinants containing EGFR mutant genes could be constructed successfully by using overlap extension PCR, plasmid ligation and transformation. Part II Establishment of ARMS and Taqman fluorescence quantitative real-time PCR systemObjective:To establish a real-time PCR kit for the detection of29mutations in EGFR gene using ARMS specific primers and Taqman fluorescence probes.Method:The ARMS specific primers for the29EGFR gene mutations and Taqman fluorescence probes for the detection of target sequence were carefully designed by the Primer Express3.0software. The optimal ARMS primers and Taqman probes were determined by repeating screening experiments and during this process the reaction conditions were optimized and the kit was established. Then using the recombinants containing mutant EGFR genes constructed in the part I experiment as the study objects, we further anlysed the sensitivity and the lower limit of this kit and determined the cutoff value ofâ–³Ct to judge specific or non-specific amplification. Finally, we compared the sensitivity of direct sequencing and our Taqman ARMS method for the EGGR gene mutation detection in the mimic plasm samples.Result:The optimal ARMS primers and Taqman probes were listed in Tabble3. The lower limit of this kit for the detection of EGFR gene mutation was10copies if no disturbance of wild-type EGFR gene or background DNA. As for the specificity of this kit, non-specific amplifications were presented when it was used for detecting T790M and21L858R mutation in leucocytes DNA samples from healthy volunteers (DNA concentration ranged from1to50ng/ul). But the minimalâ–³CT values were11.36and14.48respectively. Non-specific amplifications were not presented for the detection of G719X,19Del,20Tns, L861Q and S768T mutation. To further test the specificity of this kit, ninty plasma DNA samples from healthy volunteers (DNA concentration ranged from1to15ng/ul) were used, but non-specific amplications for all mutant types of EGFR gene were not found. As for the sensitivity of this kit, the detection resolution was as high as1%and0.1-0.5%in the background of500copies and5000copies wild-type EGFR gene respectively. In the mimic plasm samples, the sensitivity of direct sequencing was10%, but the sensitivity of our kit was0.5%and much better than direct sequencing.Conclusions:Our kit for the EGFR gene mutations detection had high sensitivity and specificity, lower price and shorter operation time which made it can be used in clinical setting. Part III:EGFR gene mutation detection in tissues of NSCLC pateitns by ARMS Taqman real-time PCRObjective:To evaluate the characteristics of the EGFR gene mutation detection kit which was established with ARMS and Taqman probe in Part II experiment.Method:One hundred clinical samples were collected and analysed for EGFR gene mutation by Taqman ARMS. Among these samples, twenty-nine clinical samples were analysed for EGFR gene mutation by Scorpions ARMS and Taqman ARMS respectively. By comparing the results of these two methods, we evaluated the concordant rate of these two methods. DNA sequencing validation was performed in those mutant samples indicated by our Taqman ARMS method. Among these samples, another twenty-nine clinical samples were detected EGFR gene mutation by ME-PCR and our Taqman ARMS method, respectively. By comparing the results of these two methods, we evaluated the concordant rate of these two methods.Results:In one hundred clinical samples, forty-one mutations were detected (19Del,21L858R,20Ins and G719X were21,18,1and1respectively) by Taqman ARMS and the total mutation rate was41%. The distribution of EGFR gene mutation was displaied as the following:19Del51.2%;21L858R43.9%;20Ins2.4%; G719X2.4%; no T790M mutation detected.In twenty-nine clinical samples, nine mutations were detected (19Del and21L858R were3and6respectively) by Scorpions ARMS and the total mutation rate was31.0%. However, eight mutations were detected (19Del and21L858R were3and5respectively) by Taqman-ARMS and the total rate were27.6%. The results of DNA sequencing confirmation were the same as the Taqman-ARMS method except one clinical sample in which DNA sequencing was failed. The agreements of these two tests were93.1%and96.6%. with a Kappa value of0.627and0.890for19Del and21L858R, respectively.In another twenty-nine clinical samples, sixteen mutations (19Del and21L858R were8and8respectively) were detected by ME-PCR-squencing and the total mutation rate was55.2%. Eighteen mutations were detected (19Del,21L858R,20Ins and G719X were9.7.1and1respectively) by Taqman-ARMS and the total rate were62.1%. The agreements of these two methods were96.6%and89.7%with a Kappa value of0.918and0.732for19Del and21L858R, respectively.Conclusions:The Taqman ARMS real-time PCR kit for the deteciont of29mutations in the epidermal growth factor receptor (EGFR) gene designed by us was a rapid, simple, economic, sensitive and specific method and worth of using in the clinical setting.