Establishment Of ARMS-PCR-GoldMag Lateral Flow Assay And Its Application In Gene Polymorphism Detection

The morbidity and mortality of cardiovascular and cerebrovascular diseases in China are increasing year by year.The death accounts for 40%of the residents’disease deaths,and the total hospitalization cost exceeds 100 billion yuan every year.The key to carry out primary prevention of cardiovascular and cerebrovascular diseases is to establish a patient-centered,comprehensive prevention and control management mode with hypertension,dyslipidemia and other risk factors management as a breakthrough.Single nucleotide polymorphism（SNP）has been widely used in disease diagnosis,disease susceptibility prediction,pharmacogenetic analysis and evolution research.The commonly used SNP detection technologies,including DNA sequencing,microarray technology,Real-time PCR,mass spectrometry etc.provide accurate methods for SNP analysis.However,these methods rely heavily on sophisticated instruments,tedious operation steps,professional personnel and a long turn-around time（TAT）before patients getting their test reports,which limited their application in laboratories with limited resources and expanded in clinical practice.It draw great attention to develop a more rapid,accurate,simple and convenient molecular diagnosis technology which can be used in different types of hospitals and foundation clinical laboratories and CDC for rapid screening,and guide and intervene in the individual prevention and treatment of diseases.Since 2007,lateral flow assay（LFA）has been used in genotyping.By analyzing the allele specific products with LFA,the genotyping results can be obtained quickly.Compared with other SNP detection methods,LFA-based detection methods show great advantages such as,low cost,indenpended on expensive instruments and professional training for personnel,which applicable to multiple scenarios.However,for the acquisition of allele-specific products,all of these methods contain an allele discrimination step after PCR amplification,which could be further simplified to fit the demand of genotyping in clinical applications.Thus,the running out of PCR amplification and allele discrimination in single step became a key issue in the development of LFA based SNP detection method for large scale samples screening in clinical practice.Supporting by National Major Scientific and Technological Special Project for“Significant New Drugs Development”,in present study,a new SNP detection method has been established that combines amplification refractory mutation system PCR（ARMS-PCR）and LFA,named as ARMS-PCR-GoldMag-LFA.It has been applied to detect MTHFR C677T,MTHFR A1298C and MTRR A66G polymorphisms.Through ARMS-PCR,the allele discrimination could be carried out along with PCR amplification,and allele specific products can be obtained in one step,that simplifying the procedure and saving time.The detection of products can be completed within 5 minutes by using this GoldMag LFA device.The MTHFR C677T gene detection kit developed by ARMS-PCR-GoldMag-LFA technology was identified as national"Innovative medical device product"by the China State Food and Drug Administration in 2014,and has been widely used in clinical testing and large-scale screening.Meanwhile,colleagues in our group also applied this technology to detect multiple SNPs of genes,such as CYP2C19,ApoE,PLD3,ALDH₂ and so on.In order to carry out the detection in a single tube instead of two PCR reactions and two LFA strips for the detection of single SNP,a further developed method called T-ARMS-PCR-GoldMag-LFA has been established which combining tetra-primers ARMS-PCR（T-ARMS-PCR）and GoldMag LFA system.T-ARMS-PCR-GoldMag-LFA allowing us to do genotyping with one PCR reaction and present the result with one strip.This newly developed method saves at least 30%detection time without compromising on sensitivity,specificity and accuracy.Moreover,with optimization,this method could be used for the direct detection of the whole blood sample,which would further simplify the operating procedure and shorten the whole operation time.T-ARMS-PCR-GoldMag-LFA is suitable for the development of automation platform and has broad clinical application prospects.The contents of this study include:1.Establishment of an ARMS-PCR-GoldMag-LFA technology for genotyping.Based on two allele-specific primers and a common primer,the allele discrimination could be achieved during PCR amplification by two ARMS-PCR reaction.After that,the allele-specific products were detected by two GoldMag-LFA strips.Takeing DNA as template,the genotyping results of MTHFR C677T,MTHFR A1298C and MTRR A66G by ARMS-PCR-GoldMag-LFA can be obtained within 105 minutes.2.Through verification of more than 1000 sample,the concordance rate of the genotyping results detected by ARMS-PCR-GoldMag-LFA was up to 99%when compared with the sequencing results.A case-control trial was carried out to investigate the correlation between MTHFR C677T/A1298C and MTRR A66G polymorphisms and homocysteine（Hcy）level and the risk of ischemic stroke（IS）in Shaanxi Han population.These three gene polymorphisms were detected by ARMS-PCR-GoldMag-LFA.The results showed that MTHFR C677T/A1298C and MTRR A66G polymorphisms are associated with the increased risk of IS.The screening of these three loci play an important role in the prevention of IS in high-risk population such as hypertension and hyperlipidemia.3.An optical reader for ARMS-PCR-GoldMag-LFA results interpretation is designed and produced.The optical reader can photograph the detection area of the LFA device.By analyzing of the color value of this image,the built-in program will give the genotyping results according to the result interpretation process and the key parameters of the interpretation process by testing samples with different genotypes.The analysis accuracy of the optical reader are evaluated with 500 samples,and the results are in accordance with those of sequencing.4.Establishment of a T-ARMS-PCR-GoldMag-LFA technology for genotyping.Instead of ARMS-PCR-GoldMag-LFA,T-ARMS-PCR-GoldMag-LFA enables simultaneous visual detection of both alleles in one PCR reaction by using four primers including wild-type primer pair and mutation primer pair.The allele specific products can be detected by single LFA strip.Using whold blood as template,the genotyping results of MTHFR C677T,MTHFR A1298C and MTRR A66G by ARMS-PCR-GoldMag-LFA can be obtained within75 minutes.Through verification by 100 sample,the concordance rate of the genotyping results obtained by T-ARMS-PCR-GoldMag-LFA was up to 100%when compared with the sequencing results.In summary,this present study report an ARMS-PCR-GoldMag-LFA and a T-ARMS-PCR-GoldMag-LFA methods,collectively known as ARMS-PCR GoldMag-LFA platform.This technology provides a more rapid and accurate SNP detection method.By virtue of its simple and convenient operation,indenpend of expensive instruments and professional personnel,it can be used in the laboratories of all levels of hospitals and medical institutions,especially for laboratories with limited resources.With the help of optical reader,a large number of clinical samples can be detected rapidly in clinical application.The application of this ARMS-PCR-GoldMag-LFA platform in genotyping of MTHFR C677T/A1298C and MTRR A66G gene polymorphisms of hypertension patients show great potential in hypertension chronic disease management and early prevention and intervention of stroke.