| People with CD36 deficiency can develop anti-CD36 antibodies after transfusion or during pregnancy,leading to the human alloimmune thrombocytopenia.Currently,Most commonly used detection methods for CD36 gene mutations are PCR-RFLP,PCR-SSP and direct sequencing.However,these methods have the disadvantages of complicated operation,time-consuming,and easy pollution,and are not suitable for large-scale clinical use.High resolution melting curve analysis(High resolution melting,HRM)is a method for detecting genes that has emerged in recent years.Because of its high sensitivity,low cost,no influence by mutant base sites,and no sequence-specific probes,it has been widely used in zoology,life sciences,clinical medicine and other fields.Based on the characteristics of the HRM method,this study intends to establish a method for screening CD3 6 gene polymorphisms by HRM technology,and to initially explore its clinical application value.Chapter 1 Construction of Three Common CD36 Gene Mutant Standard PlasmidsObjective:To construct three common CD36 gene mutation plasmids and provide mutation standards for establishing HRM methods.Methods:The CD36 gene mutation sites A1237C,C268T,and 329-330delAC,which are common in Guangzhou population,were selected as the research sites.Overlap extension PCR and TA cloning technology were used to construct a standard plasmid containing the above mutation sites,and sequenced them.Results:DNA sequencing results confirmed that the three recombinant plasmids did contain CD36 gene A1237C,C268T,and 329-330delAC mutations,respectively.Conclusion:Three common CD36 gene mutant standard plasmids were successfully constructed,which laid a material foundation for the subsequent establishment of HRM method for screening CD36 gene polymorphisms.Chapter 2 Establishment of HRM Screening Method for CD36 Gene PolymorphismObjective:To establish a low-cost and rapid screening HRM technology for CD36 gene polymorphism,and provide technical support for large-scale clinical screening of CD36 gene polymorphism.Methods:Using the plasmid standard constructed in our laboratory as a genotyping control,a method for screening CD36 gene polymorphisms by HRM technology was established,and it was tested from four aspects:specificity,repeatability,sensitivity,and heterozygous mutation ability Methodological evaluation.Results:The established HRM method can only amplify human blood genomic DNA without the generation of non-specific amplified fragments and primer dimers;the coefficients of variation of Tm and Ct values between various genotype plasmids are less than 5%;This method can detect genomic DNA as low as 1pg and detect heterozygous mutations as low as 15%.Conclusion:A low-cost and rapid HRM method for detecting CD36 gene polymorphism has been successfully established,which provides a laboratory diagnostic basis for further screening for CD36 gene polymorphism.Chapter 3 Screening CD36 Gene Polymorphisms with HRM TechnologyObjective:To apply HRM technology to screen CD36 gene polymorphisms and explore the accuracy of HRM technology to screen single nucleotide polymorphisms.Methods:HRM method was used to screen 758 human blood genomic DNA samples for CD36 gene polymorphism,and the results were compared with DNA sequencing.Results:A total of 21 mutations were detected in 758 clinical specimens,of which 10 were 329-330delAC mutations,2 were C268T mutations,and 9 were new mutations.No A123 7C mutation was detected.Using the sequencing results as a reference,the sensitivity and specificity of A1237C detected by HRM method were 100%;the sensitivity and specificity of C268T were 100%;the sensitivity and specificity of 329-330delAC were 100%and 98.58%,respectively.Conclusion:HRM method not only has good sensitivity and specificity,but also can detect unknown mutations,which provides a more reliable method for clinical screening of CD36 gene polymorphism in the future. |
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