A New Method For Rapid And Visualized Genotyping Of Genetic Polymorphism And Its Preliminary Clinical Evaluation

With the development of individualized medicine,more and more genetic markers have been found having relationships with drug action.As an important kind of gene marker,single nucleotide polymorphism(SNP)can explain the individual differences in drug efficacy.Patients with different types of SNP may have good therapeutic effect,poor therapeutic effect,inefficacy and even toxic reaction for the same drug.Therefore,SNP is widely used in clinical individualized drug therapy to help determine the kind and dose of drugs,reduce the occurrence of serious adverse reactions,and ensure the effectiveness and safety of drug treatment.At present,the standard method for clinical detection of SNP is sequencing,which is precise,expensive,tedious to operate,can not provide SNP information in time,and is not suitable for large-scale use in limited resources areas.Therefore,in order to promote personalized medicine,a method for rapid,cost-effective and large-scale SNP detection is still needed in clinic.In order to solve the above problems,based on the establishment of visualized detection platform in the laboratory,the visualized detection method of SNP is developed.In this method,the nucleic acid is amplified by PCR,the nucleic acid invasion reaction recognizes the single base difference and amplifies the signal,and the AuNPs is used as the reporter molecule.The whole reaction is carried out in turn with the change of program temperature in a single tube,and the result can be read by the naked eye.The detection can be completed with only one PCR instrument,which can get rid of the dependence on precision instruments.This method can detected 20 copies/reaction,and the accuracy was high.19 samples for CYP2C19*2 and CYP2C19*3 were correctly typed,which agreed with pyrosequencing.In order to simplify the operation steps and realize the rapid detection of SNP,further,we established a rapid visualized detection method of SNP with the rapid lysis of oral swabs.In this method,gDNA can be obtained from oral swabs within 6 min,and the process from sample to result can be completed in 90 min.Different from the detection method of collecting blood and extracting gDNA in clinic,this method simplifies the processing process of the sample,shortens the processing time,and realizes the rapid,simple and non-invasive detection of SNP.This method can detected 20 copies/reaction.20 oral swab samples for CYP2C19*2 and CYP2C19*3 were correctly typed,which agreed with pyrosequencing,Finally,in order to show that the established detection method of rapid fragmentation and visualization of samples is suitable for the detection of different clinical samples,a visual detection method of SNP based on rapid lysis of whole blood is established.In this method,blood is used as the sample,and the gDNA can be obtained in about 6 min after simple treatment of the lysate.Combined with the visual SNP detection method in the previous step,the process from the blood sample to the result can be completed in 90 min.20 blood samples for CYP2C19*2 and CYP2C19*3 were correctly typed,which agreed with pyrosequencing,To sum up,this paper establishes a visualized genotyping method which is fast,simple,cost-effective and does not rely on precision instruments,which can meet the needs of SNP detection in areas with limited resources,and has clinical application value.