A Combined Direct PCR And Magnetic Lateral Flow Platform Adapted For Multiple Sample Types And Its Application In Genotyping

The aggravating trend of aging population around the world has made the life expectancy of the population prolonged while facing the deadly threat of chronic diseases.The management of the life cycle has received much attention,including early monitoring of a large number of patients with hyperlipemia,hypertension,diabetes and other diseases,combined with follow-up and drug intervention.However,the performance of different patients and the responses to therapeutic drugs are often significantly different.This is due to the different pathogenesis between individuals and the difference in living environment and genetic background.To this end,precision medical-related genetic testing is particularly urgent in the early screening and intervention of diseases,especially to build a suitable universal,accurate,rapid,and convenient genetic screening technology platform for the large-scale population,to achieve risk stratification interventions for chronically disease patients.Existing genotyping methods,such as DNA sequencing,DNA chip,mass spectrometry and real-time PCR,are favored for their high accuracy and high throughput,but still limited by expensive equipment inputs,tedious steps and professional operational staffs.and strict requirements of template for DNA quality and quantity.Therefore,it is difficult to achieve direct amplification without DNA isolation based on the above technology platform.traditional genotyping techniques usually require sample purification via a labor-intensive,time-consuming and expensive sample preparation step,which also enhances the risk of cross-contamination between samples.This may be due to the fact that the DNA protected by the cell membrane in different samples and storage conditions can be released into the liquid environment to a different extent,resulting in a large difference in amplification efficiency.Moreover,how the subsequent detection technology platform can satisfy the accurate interpretation of the interference in the presence of impurities in the direct amplification product is a major challenge,because the hemoglobin and other substances remaining in the product will bring background in fluorescence,mass spectrometry and other technologies,even interfere with the interpretation of genotyping results.Thus,it can be seen that the processing of clinical sample is one of the main obstacles affecting the rapid PCR screening technology due to common clinical samples often contain a large number of PCR inhibitors.This shows that it is of great significance to achieve a universal direct amplification technique platform using clinical samples,especially suitable for multi-sample types(such as whole blood,dried blood spot,saliva and buccal swab).We have developed a genotyping method combined allele-specific PCR and magnetic nanoparticle based lateral flow assay using genomic DNA as template,and successfully applied MTHFR C677T genotyping in the clinic.However,how to avoid the lengthy steps of DNA extraction and the development of a direct amplification platform suitable for multiple sample types through special design has certain challenges,but also feasible.In particular,the composition of the sample pad and the binding pad used in the endpoint detection lateral flow device enables the interference substances such as hemoglobin present in the direct amplification product to be effectively filtered.At the same time,the visual interference or the interpretation of the magnetic signal can avoid the background interference of the of the optical detection of the direct amplification product,which is the original intention of this research.To overcome these challenges,we report a general approach that enables us to detect the genotype of multiple sample types(whole blood,dried blood spot,saliva and buccal swab)directly without DNA purification,and the results could be further quantitatively analyzed based on a magnetic lateral flow assay platform.Herein,the following five aspects are highlighted:1.We developed a direct PCR assay for rapid amplification.The multiple sample types could be used directly for PCR without DNA purification.We have established a new approach that makes direct PCR feasible by leveraging a simple process that enables the efficient release of nucleic acids and inhibits the natural PCR inhibitors in sample.2.We developed a magnetic lateral flow assay(MLFA)system to automate interpretation of the results through visualization or magnetic signal reader.The MLFA is derived from traditional lateral flow strips using our own synthetic gold magnetic nanoparticles as carriers,which can be harnessed to develop simple and portable devices that enable both the visual and quantitative interpretation of data for point-of-care genotyping.3.The elaborately designed direct PCR-MLFA platform was evaluated from multiple angles.The results prove that:(1)The established platform has strong anti-interference capacity.The system can accurately detect hyperlipidemia whole blood samples containing 500mg/dL triglycerides,high bilirubin blood samples containing 20 mg/dL bilirubin,and high hemolysis samples.Moreover,the platform is resistant to sample treatment solution with a pH of up to 13.0,and meets the direct amplification test of common clinical complex samples.(2)The platform has strong stability.Whole blood samples stored at room temperature,2°C-8°C and-20°C can be accurately detected for storing up to 24 hours,7 days and 3 months,respectively.In addition,three batches lateral flow system were used and the inter-batch coefficient of variation(CV value)was less than 4%for C line and less than 14%for T line,indicating the stability was good.(3)As a demonstration,we show that three genotypes of aldehyde dehydrogenase 2(ALDH2)can be identified using a small volume of sample with an accuracy of 100%and a sensitivity of 1.0×10~2 cells/?L,which are better than those of the gold standard methods.4.A clinical trial was designed to examine the accuracy of direct PCR-MLFA system and association between ALDH2 genotype and nitroglycerin efficacy.The study subjects were1018 genomic DNA samples,200 whole blood samples,200 dried blood spot samples,50saliva samples and 50 buccal swab samples.The ALDH2 genotypes were detected by direct PCR-MLFA system and the accuracy were reconfirmed with DNA sequencing.The statistical results showed that the accuracy of the established assay is 100%compared with DNA sequencing and the two methods has no significant differences.5.35 patients were followed after detecting by our direct PCR-MLFA system to evaluate the effects of nitroglycerin with different genotypes of ALDH2.The statistical analysis showed that ALDH2 genotypes were significantly associated with nitroglycerin efficacy.In conclusion,we developed an integrated strategy for miniaturizing simple process laboratory assays to shorten their complex steps and demonstrated that the entire contiguous sample-to-answer workflow could enable the genotyping of a clinical sample in less than 80min.We developed and integrated two improved technologies into one platform,a direct PCR assay suitable for multiple sample types without DNA purification and an integrated MLFA system to read the results rapidly and automatically.These contributions address several bottlenecks of current methods while providing the advantages of simplicity,cost,portability and quantitative genotyping.This technology is one step closer to realizing the ubiquitous availability of gene tests,which can ultimately aid rapid medical decisions.