Study And Preliminary Application Of SNP High-throughput Microfluidic Chip Method For Risk Assessment Associated Human Micronutrient Deficiency

BackgroudAflter the restriction enzyme fragment length polymorphism and short tandem repeats,single nucleotide polymorphism loci?single-nucleotide polymorphism.SNP?has become the third-generation polymorphism markers by its high stability,high density model to detect the characteristics and easy to realize automation.The human genomics research shows great application prospect in the genetic diagnosis,genetic risk assessment,linkage disequilibrium mapping and genetic correlation analysis.Nutrients for human show individual genetic characteristics.By the research results in functional and phenotypic changes of coding area and control area individual SNP information,we could assess and intervene nutrients deficiency risk.We could also build SNP genotype frequency distribution characteristics and obtain public SNP database which was highly related to nutrients deficiency risk.Then the accurate intervention of nutrients based on individual or group difference would be achieved.Therefore,the exploration of rapid,accurate and high-throughput SNP genotyping technology is necessary and urgent.Objective1.The establishment of innovative SNP high-throughput microfluidic chip methodFirst of all,the lab has collected various sources of blood and saliva samples to research the extraction process on automatic nucleic acid workstation and then compare and detect DNA concentration and purity.Secondly.the design principle of the primer was researched.Subsequently,this study used the obtained DNA above.designed and synthesized primers,developed an innovative microfluidic detection method for MDR-SNPs.2.Preliminary study on the microfluidic detection method for MDR-SNPs.Genotyping and biochemical indicator detection data of 1130 blood samples have been conducted and analyzed.MethodsDNA extraction process was established with automatic workstation,meanwhile primers were designed with competitive alleles PCR principle.Various SNPs which are related notably to micronutrient deficiency have been imported such as vitamin A,D,E,B₆,B₁₂.folic acid,calcium,iron,zinc and selenium.SNP retrieval principle is from Chinese biomedical literature database,China journal full-text database,Wanfang database,Chongqing Weijibaike technology journal full-text database,PubMed database and Web of Science.Related materials from the above database during the time from beginning to 25th,June,2017 have been searched with the key words of "single nucleotide polymorphism or SNP" and"vitamin A,D,E,B₆,B₁₂,FA,Ca,Iron,Zn,Se".At the same time,more related literatures were collected by literature tracking method.An innovative SNP microfluidic detection method was established and the following indicators were evaluated:?1?Cross-contamination prevention ability test:set mixture with the primer in odd-number reaction hole and mixture without the primer in even-number reaction hole.In addition,gel electrophoresis test was used to detect amplification products.The experiment was repeated six times.?2?Primers specific classification ability and accuracy assessment:first the 52 SNPs primer were preloaded in different reaction holes,then 156 DNA samples were preloaded in the different reaction holes,the concentration of which was 10 ng/?l.The chips will be dry after 30 min in room temperature,and then inject PCR pre-mixed solution into the sampling channel.All the genotyping results were compared with NGS genotyping results.The experiment was repeated six times.?3?Suitable DNA reaction concentration detection:first,preload the 52 SNP primers in different reaction holes,then dilute DNA sample to 1,5,10 and 15 ng/?l,respectively.The experiment was repeated six times.?4?Reproducibility test:The 52 S"NP primer were preloaded in different reaction holes,and one DNA sample was used for reproducibility test.The experiment was repeated six times.?5?Application of clinical blood sample:the method was used to randomly six clinical samples detection.The 52 SNP primers were preloaded in different reaction holes.?6?Comparison between DNA extracted from blood and saliva of the same three persons were obtained.The 52 SNP primers were preloaded in different reaction holes.?7?Next-generation sequencing method was used to verify the chip genotyping results.The amplification and sequencing of the target fragments were performed by Sangon bioengineering?Shanghai?co.,LTD.With the established high-throughput genotyping microfluidic chip detection method,the genotyping results of 1130 blood samples were conducted.The 143 SNP sites which were highly related to micronutrient deficiency such as vitamins A,D,E,B₆,B₁₂,folic acid,calcium,iron,zinc and selenium.The inclusion principle of the 143 SNPs was the same as above.People with CRP>10 mg/l were excluded from this study in the analysis of iron-related biochemical indicators.Iron reserves in vivo were calculated by the following formula:Body iron,BI,mg/kg=-[log?sTfR*1000/SF?-2.8229]/0.1207.Since data loss is less than 5%.the loss in continuous variables was simulated by using the existing original data.Q-Q diagram and Shapiro were used to test whether the data conformed to normal distribution.If not,the Warped linear mixed model?warped-LMM?was used to transform biochemical data.The distorted linear mixing model is an analytical method based on the standard mixed linear model.The phenotypic transformation is applied in biochemical index variables to fit the normal distribution better.R software package was used to analyze linkage disequilibrium among PCA.Kinship and SNPs,and to analyze the characteristics of candidate SNPs.If population structure was existed,the FaST-LMM model?Factored spectrally transformed linear mixed models?was applied in correlation analysis.First of all.using continuous variables to explore the correlation between gene polymorphism and various nutrients,then the classification variables were conducted to analyze all the phenotype and genotype data correlations.Grouping standard of SF:according to?WS/T465-2015 population iron deficiency screening method?,the standard of iron deficiency was set as SF<25 ng/ml.When CRP was<5 mg/l,SF<25 g/l,it was determined as iron deficiency group;when SF was more than 25 g/1,it was determined as normal.When CRP>5 mg/1,SF<32 g/l,the group was determined as iron deficiency group.While the normal population was defined as SF>32 g/1 Grouping criteria of sTfR:iron deficiency group sTfR<4.4 mg/l,normal population sTfR>4.4 mg/I.According to?WS/T 600-2018 population folic acid deficiency screening method),folic acid?FA?deficiency was defined as FA<4 ng/ml.Homocysteine?HCY?and vitamin B₁₂ deficiency were determined,and the normal population was defined as HCY<10 M.B,2<425 pg/ml was considered as deficiency group,and B₁₂>425 pg/ml was considered as normalChi-square test was used to analyze the distribution differences of different genotypes and alleles among ethnic groups.Anova was used to analyze the distribution of various biochemical indexes of different genes and alleles.The correlation of marked traits was determined according to 0.05 threshold value.Results1.A SNP high-throughput microfluidic chip method were established.Automated DNA extraction process is established,based on blood cells with gel,EDTA anticoagulant whole blood,centrifugal to serum saliva,blood,fresh preservation solution and oral saliva swabs taken oral mucosa cells and other sample extraction effect comparison,according to the results of 96 fresh saliva,DNA concentration was 150.02±50.97 ng/?l,OD 260/280 was 1.80±0.15,OD 260/280 was 1.50±0.21.It is ideal to obtain DNA content from fresh saliva.The design method of competitive allele specific PCR amplification was established.The standardized process of SNP microfluidic chip detection was established.The method evaluation results showed zero cross contamination between adjacent reaction holes by physical blocking technique.In this study,5 and 15 ng/?l were used as the minimum optimal DNA reaction concentration for blood and saliva samples,respectively.The precision results for the same sample repetition ranged was from 0.67%to 26.06%.Six clinical samples were screened for the nutrient deficiency risk.The color atlas intuitively showed the risk of multiple nutrient deficiency and the risk degree of mononutrient deficiency,and also showed that individuals had their own uniqueness in the risk degree of nutrient deficiency.By analyzing the three individual blood and saliva DNA samples with 52 SNPs,the results showed completely consistent,and nest generation sequencing results were completely consistent.2.Preliminary application of the SNP high-throughput microfluidic chip methodThe results of principal component analysis?PCA?revealed the existence of population genetic structure.Variance analysis results of the relationship between principal component 1 and principal component 2 was P<1.36e-14,indicating that there was a significant ethnic differences among 143 SNPs.SnpgdsPCACorr function was used to analyze the relationship between the three components of the principal component and SNP sites.The results showed that:The SNPs on chromosome 3 were significantly different from those on other chromosomes,rs2280673 on the gene RAB₆B explained validity was under 25%,rs 1799852 on the TF gene explain validity was from 25%to 50%,rs2118981 on the RBP2 gene and rs1830084 on the SRPRB gene explain validity were from 50%to 75%,rs1358024,rs1525892,rs1880669.rs3811647,rs3811658,rs6794945,rs7638018 and rs8177248 on the TF gene explain validity was more than 75%.A large number of SNPs with statistical significance?P<0.05?were obtained.ConclusionThis study has established an innovative SNP microfluidic chip detection method including?1?A fast and efficient,low cost,no trauma,suitable for large-scale epidemiological studies of automated saliva DNA extraction method,competitive allele specific PCR amplification primer design method,an innovative high-throughput genotyping microfluidic chip detection method.