| Background:There are many important enzymes in the folate metabolism pathway,such as 5,10-methylenetetrahydrofolate reductase(MTHFR),which is a key enzyme.Mutations in single nucleotide polymorphisms(SNP)in MTHFR can cause abnormal folate metabolism and decreased levels of active folate,thereby increasing the risk of developing various diseases such as Down’s syndrome,cleft lip and palate,and tumours.The Single-nucleotide polymorphism of MTHFR genes can be detected by a variety of methods,such as polymerase chain reaction-restriction fragment length polymorphism analysis,which requires less equipment but the process is cumbersome and requires a longer period of time;sequencing is the gold standard of genetic testing,which requires expensive sequencing instruments,a complex testing process,and requires a high level of technical personnel,and is not suitable for large-scale clinical analysis;The amplication refractory mutation systemPCR is simple,rapid and low cost,but its amplification specificity is affected by a variety of factors and is easy to cause mistyping of genes.Gene chip technology is automated,high throughput and easy to interpret results,but requires high quality DNA,relies on scanners for result interpretation,is expensive and time consuming,etc.Most of the methods are complicated to operate and have a long process,and are not suitable for widespread clinical use.Therefore,it is necessary to establish a simple,non-special equipment,low-cost and short time-consuming technique for SNP testing in order to facilitate testing and Point-of-care testing(POCT)in primary laboratories where equipment is lacking and resources are limited,and to guide individualised clinical prevention and treatment.Objective:To develop a simple,rapid,low-cost and portable visual assay for accurate typing of the C677T polymorphic locus of the MTHFR gene,which affects folate metabolism.Method:(1)A new isothermal amplification technique(Bridging primer assisted slippage isothermal amplification,BASIS)was used to amplify the kit-extracted MTHFR gene and to optimize the reaction conditions.Direct amplification of samples treated with NaOH base solution was attempted to compare its amplification with that of kit-extracted DNA.(2)Based on clustered regularly interspaced short palindromic repeats(CRISPR)and the related endonuclease cas12a,two systems were established:A fluorescence detection system and a visual detection system for coupled Gquadruplex chromogenic reaction,and optimize their reaction conditions.According to the cut-off values A and B,the MTHFR gene was determined to be CC,CT or TT by fluorescence detection system.The visual detection system is coupled to the G-quadruplex/Hemin color system to determine the type of single nucleotide polymorphism at the C677T locus of the MTHFR gene based on the color change of the system.Results:(1)The MTHFR gene can be amplified quickly,sensitively and specifically by using BASIS isothermal amplification technique.The reaction system can detect approximately 1.0×102 copies of DNA molecules without interference from other nucleic acids,and can be completed in about 20 minutes.Under the suitable dilution condition(the ratio of samples treated with NaOH to normal saline is 1:4,1:6,1:8)and the suitable alkaline condition(pH is 8.0-9.0),there was no significant difference in the amplification of DNA extracted by NaOH direct extraction and the kit.(2)Under the optimal conditions of Cas12a:gRNA=1:1,Reporter(10 μM)3 μLand cutting time 30 min,the cut-off value A was calculated to be 2727.4,and the cut-off value B was calculated to be 2923.7.When the fluorescence value of the sample in C-type gRNA system is greater than A,and that in T-type gRNA system is less than B,then the sample is wild-type(CC);When the fluorescence value of the samples added to the C-type gRNA system was greater than that of A,and the fluorescence value of the T-type gRNA system was greater than that of B,the samples were heterozygous mutant(CT);When the fluorescence value of the sample in the system of adding C-type gRNA is less than A,and the fluorescence value in the system of T-type gRNA is more than B,then the sample is homozygous mutant(TT).(3)Under the optimum conditions of 3 μL G-quadruplex(10 μM),3 μL Hemin(50 μM)and cutting time of 20 min,The visual detection system coupled with G-quadruplex/Hemin color development system had the best color development effect.When the reaction system contains targets,the Gquadruplex is trans-cleaved and the system is colorless.In contrast,the targetfree system showed a blue-green color because the g-quadruplex was not cleaved.The two systems were added with C-type and T-type gRNA respectively.When the system added with C-type gRNA finally showed colorless and the system added with T-type gRNA showed blue green,the sample was CC type;When the system added with C-type gRNA finally shows blue green and the system added with T-type gRNA shows colorless,the sample is TT type;When both systems finally show colorless,the sample is CT type.Conclusion:In this study,a BASIS isothermal amplification-based method was developed for the visualization of the folate MTHFR gene.The method is simple,sensitive,specific,short time consuming,low cost,accurate and does not require special equipment and is not restricted by location.Only a water bath or metal bath is required for the detection of SNP loci by POCT. |
