ABSTRACT
Objective To establish a simple,rapid and sensitive nucleotide polymorphisms genotyping method in order to conduct the routine clinical detections under the simple laboratory condition by this method.Methods Based on the ligase-agarose gel electrophoresis,the oligonucleotide detection probes of mutational sites was designed.The detection underwent the detection probe connecting,purification and universal amplification,finally the mutation genotypes of detection sites were judged by the ap-peared bands in the agarose gel electrophoresis(AGE).With the 3 SNP sites EGFR,c.2573T>G(L858R),EGFR,c.2582T> A (L861Q)and EGFR,c.2155 G>T(G719C)in epidermal growth factor receptor(EGFR)gene as the detection objects,the plasmid template and plasma circulating DNA sample in lung cancer were performed the detection.Results The established method was easy to operate with higher specificity and sensitivity.After 20-30 cycles of PCR amplification,the genotype of detection sites was clearly estimated according to the amplification band.When detecting the mixed alleles in the heterogeneous sample,minimal 2.5%mutation alleles could be detected out.This method and the direct sequencing method could respectively detect 6 cases and 2 cases of heterozygotes mutation in the SNP site of L858R among 62 samples of lung cancer.Conclusion The established detection method for SNP genotyping is suitable to the routine mutation detection on the heterogeneous samples under the simple laboratory condi-tion.
ABSTRACT
Objective To evaluate the clinical applications and surgical methods of combined laparoscopic common bile duct (CBD) exploration with choledochoscopy. Methods From 2006 to 2009,clinical data of 42 patients with choledocholithiasis undergoing laparoscopic common bile duct exploration were retrospectively analyzed. We applied a step-by-step electric coagulating incision technique on the CBD,the step-by-step suturing technique, and the step-by-step clamping technique with alligator forceps, and soft tube irrigating technique with suctioning by selecting the proper exploration route, improving the common bile duct incision technique and calculus removing techniques. Results Procedures were successful in all the cases. There was no conversions to open surgery, no postoperative bleeding and no operative mortality. The mean operating time was 120 minutes (ranging, 90 to 150 minutes) with minimal intraoperative blood loss ( ranging, 20 to 40 ml). Ductal stone clearance was successful in 41 out of 42 patients ( 93% ). The largest number of the common bile duct stones was 16. With the diameter of stones larger than 15 mm in 18 cases in which the biggest was 30 mm. Bile leak developed in 1 patient, retained stones found in 3 patients,including intrahepatic cholelithiasis in one case. As a result, 38 out of 42 patients underwent common bile duct exploration. 35 patients were placed on T-tubes. Four patients underwent cystic duct exploration in which 3 had primary suture of the cystic duct and 1 had drainage. There was no infection and stenosis of biliary tract in the 42 followed-up cases. Conclusions Laparoscopic common bile duct exploration with stone extraction can be performed with high efficiency, minimal morbidity and without mortality. Improving the way of operation and selecting suitable exploration can result in better clinical outcomes.