External Quality Assessment Of Septin9 Gene Methylation Detection And Establishment Of Chromosome 13 Labeling Method Based On CRISPR/dCas9

Colorectal cancer(CRC)is a highly lethal malignant tumor,with the incidence and mortality ranking the second and third in the world.The postoperative 5-year survival rate of CRC patients decreases significantly with the stage of cancer(stage I to IV),about 90%in the early stage and about 10%in the late stage.In current research and clinical practice,colonoscopic observation and biopsy of suspicious tumor lesions are still the gold standard for the diagnosis of CRC.However,multiple invasive examinations lead to a decline in general acceptance of patients,limited sensitivity,high cost,and inability to monitor tumor progression and heterogeneity in a single biopsy are unavoidable problems of colonoscopy.Other methods widely used in CRC screening include fecal immunochemical test(FIT)and fecal occult blood testing(FOBT).However,these stool-based methods are limited by the sampling method or specificity.As CRC is highly dependent on early diagnosis and early treatment,there is an urgent need for a method that can be diagnosed early and easily accepted.In addition,chromosomal abnormalities are a common cause of birth defects,with an overall incidence of approximately 1 in 150 neonates.The most common chromosomal abnormality was chromosome aneuploidy,among which the main autosomal aneuploidy was trisomy 21,18 and 13,with the incidence of 1/800,1/7500 and 1/15000,respectively.Therefore,it is very important to prevent the birth of children with chromosomal defects,and prenatal examination and examination should be carried out actively.At present,the gold standard for prenatal diagnosis is chorionic villus sampling or amniocentesis,but it is invasive and has the risk of miscarriage.Other commonly used methods include ultrasound combined with serum biochemical markers and non-invasive cell-free DNA detection,but there are problems of insufficient specificity and sensitivity.At present,fluorescence in situ hybridization(FISH)is recognized as the "gold standard"for chromosome ploidy and structural changes,but its limitations include long time,high cost and many steps.Recently,a new diagnostic concept called "liquid biopsy" has entered the vision of researchers,in which the body fluid of patients is analyzed to find specific information of malignant tumors.Liquid biopsy has shown advantages over traditional biopsy in that it is simple to perform,noninvasive or minimally invasive,easy to sample multiple times,free from heterogeneity,and can be monitored in real time.Among them,the early detection of circulating tumor cells(CTCs)and cell-free DNA(cfDNA)is the direction of clinical attention.In the first part of this thesis,we focused on the laboratory detection and levels of blood CRC detection markers.The 2022 NCCN guidelines clearly indicate that methylated Septin9 gene(mSEPT9)in the blood circulation can be used as a screening marker for specific populations,such as adults over 50 years of age with average risk of colorectal cancer who have been offered but have not completed routine fecal occult blood screening plus colonoscopy.In view of the differences in methylation detection methods and quality management measures adopted by laboratories,the accuracy of test results may be affected,which may interfere with the diagnosis and treatment of diseases.Therefore,to understand the current general situation and clinical performance of mSEPT9 detection in various laboratories in China,this study carried out a national survey on the application of mSEPT9.The general situation of mSEPT9 detection in laboratories was investigated through electronic questionnaires,including methods and ranges,reagents and samples,and analytical performance.In addition,using micrococcal nuclease(MNase)technology,the reference material for mSEPT9 detection with biological characteristics most similar to that of clinical plasma samples was obtained.The external quality assessment(EQA)study further evaluated the standardization and accuracy of mSEPT9 for CRC diagnosis in national laboratories.The questionnaire showed that the detection method of mSEPT9 in most laboratories in China was qualitative methylation method based on quantitative real-time PCR(realtime PCR),and a few laboratories used quantitative methylation method based on NGS sequencing or digital PCR.The detected region was CGI3 of Septin9 gene V2 transcript and covered more than three CpG sites.The detection reagents included 3 NMPA certified commercial reagents and laboratory-developed tests(LDTs).The samples were mainly plasma samples,and the DNA extraction methods were magnetic bead method and centrifugal column method.All samples were converted to bisulfite and purified.Most laboratories have performed performance verification or validation of mSEPT9 detection reagents.In this EQA,a total of 60 laboratories submitted results,of which 96.67%were qualified and 3.33%were unqualified.real-time PCR was used in 59 laboratories and targeted bisulfite sequencing was used in 1 laboratory.Different laboratories choose different reagents for cfDNA extraction,sulfite conversion and Septin9 gene methylation detection.The results showed that the overall positive coincidence rate of the reagents was 97.22%(525/540)and the overall negative coincidence rate was 100%(60/60).The results of this EQA showed that most laboratories in China could perform mSEPT9 detection.In the second part of the paper,we use CRISPR technology to develop a simple,rapid,and cost-effective novel labeling method that can be used to image chromosomes.Each dCas9/sgRNA complex carries a fluorescent group that binds to a target sequence to produce a fluorescent signal.When the binding target is highly repetitive sequence,the fluorescence signal can be concentrated and amplified to reach the visible signal under the microscope.Using this method,we can achieve the labeling of chromosome 13,which presents three signal points in chromosome 13 triploid cells and two signal points in human normal diploid cells.Moreover,the detection time is greatly shortened,the detection process is simplified,and the detection cost is reduced.Therefore,this method can realize the specific and visual detection of chromosome 13,and has the advantages of rapid,simple and economical.To sum up,there are differences in the detection methods and scopes,detection reagents and samples of mSEPT9 in Chinese laboratories.In order to ensure the accuracy and reliability of the test results,all laboratories should standardize the performance verification and performance confirmation work.In this study,reference substances for methylated cfDNA in plasma were prepared by MNase digestion for the first time.By carrying out EQA research,the mSEPT9 detection ability of participating laboratories was evaluated,which is helpful to improve the laboratory’s diagnostic ability and solve the problems existing in daily work.In addition,a new rapid detection method based on CRISPR/dCas9 in situ labeling technique is proposed,which realizes the visual labeling of chromosome 13 and promotes the application and improvement in clinical detection.