| Tumor is a new organization caused by excessive proliferation of somatic cells deficient of normal regulation,and can invade the surrounding tissue or develop metastasis.The tumor brings threats to the public health and heavy burden to the suffered family.According to the report,the number of cancer deaths in China was 2.81 million in 2015,that means the average daily death tolls about 7500 people.The prevention and control situation in China is still grim.To control the occurrence and development of cancer as early as possibleand to reduce its morbidity and mortality has become an urgent problem to be solved.With the development of molecular biology,it has been revealed that the changes of tumor associated gene loci are closely related to the occurrence,development,diagnosis,treatment and prognosis of the tumor.Among which,the change of the tumor related gene site,which is the gene mutation,refers to the change of the composition or sequence of the base pair.Of the genes which could cause tumor malignantproliferation,the genes mutations of KRAS and its downstream core molecular-BRAF are the research focus in recent years.The gene mutation of KRAS or BRAF leads to continuous activation of mitogen activated protein kinase pathway,result in malignant hyperplasia development.More clinical studies have demonstrated that mutations in the KRAS gene often leads to failure of commonly used monoclonal antibody drugs in tumor therapy,so the FDA has recommended it as one of the pre drug test for the monoclonal antibody.With the diversification of clinical samples and the in-depth study of circulating tumor DNA,except for blood and tissues,other clinical samples such as urine,feces,saliva,and oral mucosal shedding cells,could be used in noninvasive detection of tumor derived mutant DNA.Screening of early gene mutations can provide an important basis for early diagnosis and prognosis of tumors.Because the tumor associated mutant genes are often hidden in a large number of wild type genes,it is very important to effectively detect the rare tumor derived gene mutations in clinical samples.Detection of rare gene mutations can effectively enrich and find the occurrence of trace mutations of targeted genesin the background of a large number of wild type genes,combined with laboratory data of sample,the mutation rate could be precisely calculated.Together,these provide a strong basis for individualized treatment of cancer.In a wide variety of detection techniques,as one of the most effective methods for detection of rare gene mutations,the main principle of clamp-based PCR is to use the lock nucleic acid probe to improve the ability of base mismatch recognition,so that the detection sensitivity is higher,in accordance with the requirements of the detection of rare gene mutations.However,there are still many problems in this method at present,such asthe need for specificity of DNA polymerase,the high requirements for the amplification of the DNA,the target gene non-specific amplificationcaused bythe amplification process with base artificial mismatch,etc.In view of this,we intends to use the internal competitive PCR to reduce the artificial mismatch in the detection process,so as to avoid non-specific amplification of target genes,and to improve the screening of a large number of wild type genes to highlight the mutation sites of rare gene in the purpose of constructingthe PCR(wire PCR)based on the internal competitive amplification to enhance the wild type inhibitory.With wirePCR,the mutation specific amplification of the rare gene mutation could be detected by real-time fluorescence quantitative PCR.Objectives:1.To construct a high sensitivity detection method of KRAS and BRAF gene mutation in colorectal neoplasms,and to optimize the reaction conditions of this detection method based on the common gene mutation sites in the tumor,combining with the method of restraining the wild type background gene by locked nucleid acid through the internal competitive amplification fragment.2.To evaluate this wire PCR detection method systematically,and to apply detection and verification analysis of therare KRAS and BRAF genemutation in colonoscopy biopsy specimens with the optimized reaction conditions.3.To further apply the internal competitive amplified fragment to the digital PCR,so as to detect the rare mutation in circulating tumor DNA.4.To construct aninternal competitive amplified fragment engaged,wild type gene restrained multiple fluorescence PCR reaction system,and finally realizes the simultaneous detection of the rare mutation in multiple genes.Methods:1.The primers were designed and synthesized,and the fluorescence probe was used to optimize the concentration of the primer pair and the fluorescent probe.The temperature gradient PCR was used to optimize the reaction conditions of the annealing temperature.Constructed the rare gene mutation quantitative fluorescent detection system by design and synthesizethe LNA/DNA chimeric probes withthe known common mutation site in KRAS and BRAF gene and use Leptin gene as the internal competitive amplification fragments involved in the reaction.And further optimized the reaction conditions such as primer,fluorescent probe,and Locked nucleic acid probe.2.The mutant HT-29 human colon cancer cell line and wild type DNA,which were mixed in proportion,was used as amplification template.With the constructed method above and the optimized reaction system,the templates with mutation rate of 50%,25%,10%,1%,0.1%,and 0.01% were amplified by wirePCR reaction,and the amplified products were sequenced to evaluate the sensitivity,specificity,and accuracy of the internal competitive amplified fragment engaged,wild type gene restrained rare gene mutation detection method.3.50 cases of suspected rectal cancer were collected,the concentration of DNA was detected and the concentration was adjusted to 100ng/ul.According to the optimized wirePCR reaction conditions,the KRAS and BRAF gene mutations were detected,and the results were verified by the results of the pathological analysis and direct sequencing of HE stained sections.4.In order to adapt to the reaction conditions and operation process ofdroplet digital PCR,the upstream and downstream primers of KRAS and BRAF gene were upgraded,and the primer pair with higher TM value was used.The corresponding fluorescent probes were also made corresponding improvements.The MGB probes with VIC and FAM fluorescence were selected to participate in the reaction system.The rare mutations of KRAS and BRAF gene in circulating tumor DNA were detected by internal competitive amplified fragment engaged,wild type gene restrained digital PCR.5.In triple fluorescence reaction system of the internal competitive amplified fragment engaged,wild type gene restrained rare gene mutation detection method,LEPTIN reference gene was used as an internal competition amplified fragment,and the primers and probe concentration of which consistent with the previous experiments.The fluorophore of fluorescent probeused in the rare gene mutation detection of KRAS and BRAF gene in the fluorescent probes were VIC,HEX,and FAM,combined with optimized primers and screened wild type LNA/DNA chimeric probe concentration to participate in the reaction.Results:1.The design and synthesis of primers and LNA/DNA probes were completed,the primer pair for the internal competitive amplification fragment of the reaction system was HQ-329/330,and the final concentration of 500 nM fluorescent probe was 100 n M.The optimal reaction system for detecting the mutation of KRAS gene was determined : The final concentration of primer was 500 nM,and the final concentration offluorescent probewas 250 nM,the LNA/DNA chimeric probe(HQ-144)designed forthe wild type of KRAS genecan effectively shield the 50-150ng/?L wild type template at its final concentration of 500 n M;the optimal reaction system detection BRAF gene mutations: the final concentration of primer was 500 n M,and the final concentration of fluorescent probe was 250 nM,the LNA/DNA chimeric probe(HQ-356)designed for the wild type of BRAF gene can effectively shield the 50-200ng/?L wild type template at its final concentration of 500 nM.Because the effect of the internal competitive amplification fragment and the screened wild type LNA/DNA chimeric probe,the phenomenon of nonspecific amplification will not occur in 60 cycles.2.In the evaluation of the sensitivity and specificity of the method for the detection of the wild type blocked gene,which is involved in the amplified fragments,the optimal annealing temperature of the wirePCR gene of BRAF was 60°C,and the template with mutant DNA ratio of only 0.01% could be effectively detected with good reproducibility.The correlation coefficient R2 of the method was 0.996,and the amplification efficiency was higher.3.50 cases of suspected colorectal cancer endoscopy tissues were detected by using wirePCR,among which,18 cases of KRAS gene mutation type(36%)and 8 cases of BRAF mutations(16%)were detected,all the mutant specimens were KRAS or BRAF single mutant,without simultaneous mutation.The results of the detection and HE staining were consistent with the results of pathological analysis and direct sequencing.4.In the internal competitive amplified fragment engaged,wild type gene restrained rare gene mutation detection droplet digital PCR,the primer concentration in the system is 900 nM,the final concentration of the probe is 200 nM,and template DNA concentration is 50 ng/?L.In the detection of the KRAS gene rare mutation reaction system,the HQ-329/330 were used as amplified primers forinternal competitive amplified fragment,HQ-1433 were selected as FAM fluorescent probes,and HQ-1595/1596 were used as KRAS gene primers,the VIC fluorescent probesof which were HQ-1438.The total number of oil droplets was 1145800,and the detection efficiency of KRAS gene could reach 0.15%.In the detection of the BRAF gene rare mutation reaction system,the HQ-1434 with VIC were used as amplified primers forinternal competitive amplified fragment,HQ-671 with FAM were selected as FAM fluorescent probes,and HQ-1592/1594 were used as BRAF gene primers.The total number of oil droplets was 928355,and the detection efficiency of BRAF gene was 0.11%.5.In triple fluorescence reaction system of the internal competitive amplified fragment engaged,wild type gene restrained rare gene mutation detection method,the primers of reference LEPTIN gene,KRAS and BRAF gene were the same as the previous experiments.In order to acquire relatively equal fluorescence intensity at the same CT value,the optimized probes were respectively: the final concentration of probes for internal competitive amplified fragment LEPTIN HQ-1294 with Texasred fluorophorewas 100 n M,MGB probes with VIC fluorescent signal HQ-1438 were used in KRAS gene mutation detection,and MGB probeswith FAM fluorescent HQ-671 were used in BRAF gene mutation detection,the final concentration of fluorescent probe was 250 n M,in order to avoid non-specific amplification,the annealing temperature was 60°C,and the number of cycles was 60.Conclusions:1.The method of real-time fluorescence quantitative detection for specific amplification of rare gene mutation based on the improved wild type constraining by internal competitive amplified fragments was successfully constructed.The primers and probes suitable for the detection of KRAS gene mutations in colorectal tumors were selected,and the reaction conditions such as the annealing temperature and cyc le number of the reaction system were also explored,and the optimal concentration of KRAS gene was detected by adding the internal competitive PCR fragments.2.The method of real-time fluorescence quantitative detection for specific amplification of rare gene mutation in BRAF based on the improved wild type constraining by internal competitive amplified fragments was successfully constructed.The primer,fluorescent probe and locked nucleic acid probe were optimized,so the reaction system can effectively shield the specimens in the wild type DNA template,with strong ability of selective amplification.BRAF gene mutation identification sensitivity can reach 0.01%,andthe detection condition for the rare gene mutation is satisfied.3.The method of real-time fluorescence quantitative detection for specific amplification of rare gene mutation based on the improved wild type constraining by internal competitive amplified fragments was applied to the detection of rare gene mutation in KRAS and BRAF genes in 50 cases of colorectal cancer tissues.The samples of mutation rates were 36% and 16% respectively.With high specificity and sensitivity,and the operating costs advantage compared to the direct sequencing method,this method we constructed can be widely used in clinical detection of gene mutation,providing a reference for tumor monitoring and personalized medicine.4.Combined with the digital droplet PCR,the detectionmethod for specific amplification of rare gene mutation based on the improved wild type constraining by internal competitive amplified fragmentscould be used to detect the rare gene mutation in circulating tumor DNA.The absolute quantification detection of single copy number gene mutation could be realized by restrain the wild type gene amplification,so this method could also be used to evaluate the sensitivity of other rare gene mutation detection method.5.The result of the triple fluorescencedetectionmethod for specific amplification of rare gene mutation based on the improved wild type constraining by internal competitive amplified fragmentsrevealed that the constructed rare gene mutation detection method can be applied to a variety of combined detection of rare gene mutation.The detection was carried out within closed tube,the data and results of the detection is more accuratewhile operated in the same reaction conditions,and the reaction time has been greatly shortened,and the experimental cost and repeated experimental operationhas reduced.As a result,there is a huge clinical use value for the detection method we constructed in the research here. |
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