Establishment Of Mutant-enrich HRM Detection System And The Application Of Detecting Mutations In Circulating Peripheral Blood

Many studies have confirmed that mutations in circulating peripheral blood are consistent with the tumor issues. As the blood samples are easy to get and can be detected dynamically, cell-free circulating DNA (cfDNA) in plasma or serum is becoming a promising biomarker for detection of cancer alterations. To achieve the purpose of detecting tumor-associated mutations in peripheral blood, many assays focused on this field were developed by technical researchers. However, the actual results are unsatisfactory. This is due to the low concentration of cfDNA and the dilution effect of DNA fragments and wild-type DNA in circulation. Because of the difference of individuals and tumor development stages, the total amount of circulating DNA is instable, which requires detection technologies do not rely on DNA concentration. Existing assays have not yet been perfectly developed with the sensitivity being reported quite different. And commercialization of technological development is not mature. New approaches are needed.High resolution melting analysis (HRM) is a post-PCR technique for detecting mutations, which is based on the different melt curves of DNA sequences. As a closed-tube, simple, fast and high sensitivity, good repeatability, high accuracy, high throughput genotyping detection screening method, it has incomparable superiority in clinical promotion. This study was designed to develop a more accurate HRM technology, by increasing the sensitivity and reducing the judgement difficulty. This experiment aims to broaden the detection range and finally to get it better into use in clinic.We invented a new technology for enrichment amplification--Snapback primer ARMS (SP-ARMS), and received nearly 1000-fold enrichment efficiency. Then we combined this new technology with Snapback HRM, which achieved a millionth or even higher detection sensitivity and met the requirements for difficult contamination, high sensitivity and specificity, simple and easy judgment for results. Then we successfully applied this technique in detecting EGFR/KRAS mutations in tumor tissue and plasma samples of patients with non-small cell lung (NSCLC). At the same time, compared with DNA sequencing and ADx-EGRR/KRAS mutation detection kit, we got a higher sensitivity and accuracy.Part Ⅰ:The invention of Snapback primer ARMS (SP-ARMS) and the establishment of Snapback primer ARMS-HRM (SPA-HRM) detection systemObjectives:Invent SP-ARMS technology to realize efficient enrichment of mutant template and meet the requirements for detection of low-frequent mutations. And then set up an easy popularized and one-step detection system after combining SP-ARMS with Snapback primer HRM technology. Methods:Firstly, we set up Snapback primer HRM in detecting EGFR, KRAS gene mutations and verified it in the test of 47 samples in non-small cell lung cancer (NSCLC) patients. Then based on it, we developed a new method for enrichment PCR--SP-ARMS. Cooperated with snapback primer HRM, the new SPA-HRM mutation detection system was established. Use different mutant ratio of templates to detect the enrichment efficiency of the technology, when comparing the results of normal snapback primer HRM. Results:It could be seen from the sequencing results that 1/104-EGFR exon 19 deletion and 1/100-point mutations of L858R and KRAS got to be tested after SP-ARMS enrichment, while only 1/10-mutant could be detected after normal PCR. Then 10-fold the sensitivity of the sequencing after SP-ARMS was obtained by SPA-HRM mutation detection system. Conclusions:This study invented an enrichment amplification technology--Snapback primer ARMS, the enrichment efficiency is nearly 1000-fold, it is an efficient, simple, low-cost enrichment amplification technology. Combined it with Snapback HRM technology, we can achieve 1/1000-1/104 or even higher detection sensitivity.Part Ⅱ:The amplification in tumor issue and peripheral blood samples with the method—SPA-HRMObjectives:Detecting mutations in tumor tissue and peripheral blood of NSCLC patients to examine the accuracy of SPA-HRM system, when compared with detection by DNA sequencing and ADx-EGRR/KRAS mutation detection kit. Methods:After collecting and extracting DNA from 150 cases of tumor tissue and the 85 cases of corresponding plasma of NSCLC patients, we detected them with SPA-HRM, then identified the stability and accuracy of SPA-HRM detection system by comparing with the results of DNA sequencing and ADx-mutation detection kit. Through designing the Snapback primer, we detected the common mutations of EGFR exon19 deletion, L858R, and KRAS mutations on codon 12、13 of exon 2. Secondly, we explored the factors affecting the detection results, including the selection of plasma or serum, appropriate amount of peripheral blood, collection and extraction methods. Results:In the 85 cases of plasma samples, compared with tissues, we found that the positive detection rate is 74% by SPA-HRM, while sequencing is 26% and ADx-mutation detection kit is 46%. Conclusions:The SPA-HRM we developed can be successfully used in cfDNA mutations detection. Compared with the current highest sensitivity method--ADx-EGFR/KRAS detection kit, it is more available for use and worth of clinical application for such a fast, cheap, accurate technology with one-step detection.