The Rapid Detection Of Mitochondrial 12S RRNA 1494 C>T And 1555 A>G Mutations By PCR-HRM

Objective:The mitochondrial 12S rRNA gene is one of the mutant hotspots of maternal hereditary deafness.Our study aim to establish a rapid method for the detection of mitochondrial 12S rRNA gene 1494 C>T and 1555 A>G mutations related to drug-induced deafness based on polymerase chain reaction with high-resolution melting(PCR-HRM)analysis.Methods:We designed the specific primers for the target site and analyzed the melting curve by saturation fluorescent dyes.Mutational plasmid DNA standard materials were constructed by the site-directed mutagenesis cloning strategy.The PCR-HRM system to analysis the target mutation was established and the system was optimized.The PCR-HRM system was developed to detect the mitochondrial 12S rRNA mutations of 106 non-syndromic deafness patients,and the results were further verified with DNA sequencing.Results:A stability and high sensitivity method for rapid and accurate detection of mtDNA1494 C>T and 1555 A>G mutations based on PCR with HRM was successfully established and the melting curve of each genotype were obvious and easy to be analyzed.In the 106 cases with non-syndrnmic deafness,6 cases of 1555 A>G mutation were detected and the results were consistent with those of DNA sequencing.Conclusion:A new method based on PCR-HRM for rapid detection of mitochondrial 12S rRNA gene 1494 C>T and 1555 A>G mutations related to drug-induced deafness is established,which is simple,rapid and accurate,and may be applied to the mutation screening and clinical molecular diagnosis.Genetic diseases of epathogenic genes have highly homologous sequences with genome-wide range of other gene,which can cause the detection result of false-negative.The most classic example is alpha-thalassemia due to mutations of alpha globin gene.Thalassemia is one of the most common genetic diseases in the world that has the greatest impact on human health,in which a-thalassemia is highly prevalent in South China.The mutation of alpha globin gene mainly occur in the a2 globin gene,because of highly homologous with al globin gene(97%),the results of detection a2 globin gene mutations are likely to cause false negative.It is also one of the main reasons why the detection method of mutation in a2 globin is slow to update.At present,the method of detection a2 globin gene mutation has many disadvantages,such as complex operation,low-flux and high cost and so on,which is not suitable for large-scale population screening and routine molecular diagnosis.Our study established a rapid,accurate detection of the mutation in a2 globin gene,including the sites of WS,QS,CS,CD30,CD31,which based on nested asymmetric PCR combined with melting curve analysis.The new method can solve the problem of false negative caused by high homology effectively.Firstly,the specifically amplified primers of a2 globin gene and the target site of single chain were designed for enrichment of α2-globin gene and exclusion the influence of homologous al globin gene,and then the target site-specific probes were synthesized for detecting mutation.We established a method to solve the problem of high homology and to detect a2 globin gene mutation accurately through adjusting and optimizing condition of primers structure and composition,amplification components and reaction procedures and so on.1250 cases a-thalassemia of non-deletion and normal genotype gDNA were collected to evaluate the new method of stability,specificity,sensitivity and practicality and to evaluate the clinical of application by blind analysis.At last,the new system as a laboratory self-built detection method to detect mutation of a2 globin gene in routine diagnosis and the method for industrialization and application were promoted.The result showed that the primers and probes can effectively detect the corresponding genotypes.Through optimizating and evaluating of the detection system,a method based on fluorescence PCR could detect the non-deletion ofα-thalassemia quickly and accurately.The results of detection 1250 samples showed that accuracy rate was as high as 100%and the system was stable,sensitive,which can detect low concentration of template.Compared with the traditional detection method,the new method has the advantages of simple operation,time saving,economic,applied,high efficiency and it can solve problem of false negative.In the conclusion,the fast,high-throughput and accurate detection of a2 globin gene mutation based on nested asymmetric PCR combined with fluorescence probe hybridization and melting curve analysis is established,which can settle the problem of high homology.The method is suitable for large-scale and routine molecular diagnosis,and may provide new detection methods for other gene mutations.