The Establishment Of "LAMP-GoldMag Lateral Flow Assay " And Its Applications For MTHFR C677t Genotyping

The development of genetic testing technology show great benefit to the application of precision medicine in large medical institutions,but the problem of difficulty and expensive in medical treatment still exists.The development of genotyping technology which can be easily used in all levels of medical institutions is a problem that needs to be solved.Therefore,the simple,fast,efficient and low-cost suitable gene detection technology plays an important role in graded diagnosis and treatment.At present,genotyping methods such as gene sequencing,restriction fragment length polymorphic polymerase chain reaction(RFLP-PCR),allele-specific polymerase chain reaction(ASPCR)and other technologies are complicated to operate,time-consuming,large instruments and professional analysis system,depended,which limit the promotion and application of these methods in hierarchical diagnosis and treatment.In this study,a " LAMP-Gold Mag Lateral Flow Immunoassay " genotyping technology platform was established with MTHFR c.677 C > T genotyping as the model by combining loop-mediated isothermal amplification(LAMP)with Gold Mag Lateral Flow Immunoassay.Through one-step rapid LAMP amplification,the genotype of the sample can be determined by visual interpretation of the color change of the strip at the detection line or by automatic recognition by magnetic quantifier.This paper firstly constructed a positive quality control system for MTHFR c.677 C > T gene,which is used to verify whether the system is working properly.Secondly,through designing and synthesizing specific primers for loop-mediated isothermal amplification,and optimizing the amplification system and Lateral Flow Immunoassay system,the " LAMP-Gold Mag Lateral Flow Immunoassay " genotyping technology was established.To evaluate the specificity,sensitivity and stability of the established method with quality control products.Finally,200 clinical samples buy using this technology platform.At the same time,using sequencing as a control,the effectiveness and applicability of the clinical application of the methodology were evaluated." LAMP-Gold Mag Lateral Flow Immunoassay " genotyping technology can complete the detection of the sample within 20 minutes with good specificity and stability and high sensitivity(up to 0.5ng genomic DNA).In addition,the results of 200 clinical samples were compared with the sequencing.The statistical agreement between the two detection methods was 100%,which determined that the method had good clinical application effectiveness and applicability.Based on MTHFR c.677 C > T,this paper established the " LAMP-Gold Mag Lateral Flow Immunoassay " gene detection technology.The MTHFR c.677 C ? T gene detection technology provides immediate and effective personalized information of genetic polymorphisms for a reasonable administration of folic acid doses in cardiocerebrovascular diseases and birth defect-related diseases.And it has important application value in guiding the prevention and control of birth defects and primary prevention of stroke.