Multi-site Rapid Detection Of Deletion Genetic Diseases

Copy number variation is a common cause of genetic diseases,and the gene large fragments deletion is the main cause of many genetic diseases.At present,the commonly used large deletion detection method has the disadvantages of cumbersome operation,high cost,and need for post-PCR processing.Multicolor Melting Curve Analysis(MMCA)is a simple,efficient,economical,and of no post-PCR processing method for gene detection.Therefore,we used 21-hydroxylase deficiency(21-hydroxylase deficiency,21-OHD)and Duchenne Muscular Dystrophy(DMD)as research objects to investigate the feasibility of MMCA in the rapid detection of large deletion of genetic diseases.In the first chapter,the concept and types of genetic diseases and copy number variation were briefly introduced.The large deletion is an important type of copy number variation and is the main cause of many genetic diseases.Current detection methods for large deletions suffered from many disadvantages.In thesis,through the establishment of 21-hydroxylase deficiency and Duchenne muscular dystrophy gene large deletion detection assay,the feasibility of MMCA applied to the detection of large fragments of genetic diseases was investigated.In the second chapter,the 21-hydroxylase deficiency gene CYP21A2 complete deletion was used as the detection target.Combined with the first round of pre-amplification and the second round of MMCA detection,the complete deletion detection assay of CYP21A2 gene was established.Detection of the four types of CYP21A2 gene complete deletions was achieved.The sensitivity of the assay was examined using human genomic DNA and the results showed a sensitivity of up to 500 pg/reaction.Using 40 wild-type genomic specimens for repeatability,study the melting temperature of the wild-type was 70.34±0.23(?,mean±3SD),and the coefficient of variation(CV)was 0.11%.The assay was validated by clinical specimens,and the results were consistent with the results of the control method multiplex ligation-dependent probe amplification,indicating that the assay could be used for the detection of clinical specimens,and had the advantages of simple operation,low cost,and no post-PCR treatment.In the third chapter,we chose 17 high frequency deletion exons of Duchenne muscular dystrophy gene(DMD)based on the literature,and established a single tube 18-plex DMD deletion detection assay by MeltArray,which can detect more than 98%of the DMD deletion types.The sensitivity of the assay was examined using a wild-type human genomic DNA,and the results showed that the sensitivity of the assay was 2.5 ng/reaction.The repeatability of the assay was investigated by single and three repeated tests of 40 random samples.The Tm value fluctuation range of the melting peak was the within 1?,and the CV value was smaller than.We detected 26 samples with different type deletion in 41 clinical specimens,and the frequency of gene deletion was 63.4%(26/41),which was consistent with the reported frequency.The specimens were verified by MLPA,and the agreement between our detection method and the MLPA was 98%(40/41).The established assay could detect patients with large DMD gene deletion,and had the advantages of no post-PCR treatment,short detection time,simple operation,good repeatability and low cost.It would be suitable for large-scale screening of DMD deletion in China.