Establish Of The Method Of Quantitative Detection Of K-ras Gene Mutation At Cod On12and13Through Single Tube Pcr And Valuing Of Diagnosis Of Pancreatic Cancer

The incidence of pancreatic cancer (PC) increases year by year, it has been seen asthe king of malignant tumor, and because of its concealment, PC comes to be theadvanced stage when found, the median survival is about1year, and prognosis of PC isbad. Occasionally early stage of PC is diagnosed, after surgery, patient can live longerobviously. Nowadays, clinically there is no way to diagnose PC in its early stage, onlyserum CA19-9testing is positive correlation with tumor clinical staging, but we can getpositive testing consequence only when PC is in advanced stage, which is not able toscreen PC in early stage. Besides, chronic panceatitis has the similar clinicalcharacteristics as PC, the differential diagnosis is also the difficulty in clinic. How toraise the early diagnosis rate is an important way for most patients to get the chance toexcise PC. It is common of K-ras gene mutation in PC tissue, the mutation rate is75%～95%. The hot spot of mutation usually at codon12、13and61which can activate K-rasgene permanently and enhance the downward genes and proteins.It has many methods of testing K-ras gene mutation, such as: PCR-DSM(Directsequencing method), PCR-RFLP(restriction fragment length), PCR-SSCP (single strandconformation Plymorphism), PCR-MASA (mutant allele-specific amplification), ARMS(amplification refractory mutation system) and so on, and the methods as Enriched-PCR&Enzyme Linked Mini-sequence Assay (ELMA), LigAmp assay, High resolution meltinganalysis which can rough quantitation test K-ras gene mutation has been found recently,however, these methods all have disadvantage of long time testing and difficult operation.Besides, they are limited for widely use as the result of their low sensitivity and specificity.Our laboratory have established the method of quantitative detection of K-ras genemutation at codon12using PNA-mediated PCR clamping with different fluorescencelabeled probes.The conclusion supported that the method can detect all of the six types ofK-ras gene mutation at codon12effectively with high sensitivity（1/107-1/105）.And thismethod is reliable and efficient through the test result of6pancreatic cancer cell.Ourresearch is aim to set up an effective method of quantitative detect of K-ras genemutation at codon12and13, basing on the original method of detection increase thesensitivity and specificity. Then detecte K-ras gene mutation in the blood of three typesof people: pancreatic cancer, chronic pancreatitis and normal disease-free people usingthe method, explore the differential diagnostic value directing at pancreatic cancer. A：Establish the method of quantitative detection of K-rasgene mutation at codon12and13through single tube PCRAim: To set up the method of quantitative detection of K-ras gene mutation at codon12and13through single tube PCR and verify its sensitivity and specificity.Materials and Methods: Design K-ras-FAM Taqman MGB probe and K-ras-VICTaqman MGB probe which was labeled with FAM and VIC individually. K-ras-FAMTaqman MGB probe was a kind of probe directing at the six types of K-ras genemutation at codon12,and K-ras-VIC Taqman MGB probe was directing at the six typesof K-ras gene mutation at codon13.Design the PNA which focus at the codon12and13of K-ras gene at exon2. Compose the standard K-ras wild type and K-ras mutant typeDNA and diluted with10times to do the absolute quantitative detection.Then optimizethe probe、primer and reaction system basing on the original method of detection.Results: The method of quantitative detection of K-ras gene mutation at codon12and13can through single tube PCR can detect all of the six types of K-ras gene mutationat codon12and all of the six types of K-ras gene mutation at codon13. The method hasthe high sensitivity. The test result of7pancreatic cancer cell supports that this method isreliable and efficient.Conclusion：The method of quantitative detection of K-ras gene mutation at codon12and13can through single tube PCR can detect K-ras gene mutation efficiently withhigh sensitivity and specificity.B：The application of the method of quantitative detectionof K-ras gene mutation at codon12and13through single tubePCR in the peripheral bloodAim: To analyze the mutation of K-ras gene mutation in the peripheral blood andexploration of the differential diagnostic value of K-ras gene mutation with clinicalparameters.Material and method: The condition of K-ras gene mutation was quantitativelydetected using the method of single tube PCR in the blood of three types of people:pancreatic cancer, chronic pancreatitis and normal disease-free people from nine clinicalmedical centers. Result: The percent of K-ras gene mutation in pancreatic cancer group, chronicpancreatitis and normal disease-free people had statistical significance, all P<0.05.Conclusion: It is an effective way to differential diagnosis pancreatic cancer usingthe method of quantitative detection of K-ras gene mutation at codon12and13throughsingle tube PCR.