Establishment Of Nucleic Acid Visual Detection Based On LFD-RPA And Its Application

Nucleic acid amplification technology(NAAT)can detect trace target molecular in samples,and has many advantages such as high sensitivity and specificity.However,traditional NAATs are limited to laboratory-based applications owing to the requirements of expensive equipment,highly trained personnel,and continuous electricity dependency,making it difficult to apply these technologies in resource-limited settings.In recent years,isothermal nucleic acid amplification technologies have garnered much attention for point-of-care applications.Recombinase polymerase amplification(RPA)is a novel isothermal technique that has been demonstrated to be ideal for resource-limited applications,and can amplify trace nucleic acids to detectable levels within 15 min over a wide temperature range.The combination of RPA and LFD(LFD-RPA)shows potential for realization of a sensitive,specific,rapid,and visual detection system with great potential applicability in point-of-care testing.RPA has been widely applied and developed since first reported,such as the detection of pathogens including hand foot mouth virus,mycobacterium tuberculosis,dengue virus and plasmodium falciparum.They have demonstrated useful features for POCT application.Yet most of these studies to date are not fully integrated systems and the operation cannot realize without the use of external power,which limit their use in field testing and on-site testing.In addition,the aerosol contamination generated by non-closed operation modes is another bottleneck make them not recommended for application.Our research is aimed at developing a sample-to-answer detection system that incorporates nucleic acid extraction,amplification,and detection steps without the need for electrical power,equipment,and other laboratory infrastructure.The system must be sensitive,specific,simple to operate,and would not generate contamination of nucleic acid aerosol,that is suitable for on-site and field application.In this study,we first ascertain the optimal reaction condition of the LFD-RPA reaction system by pre-experiments and the literature reviews.In addition,we developed an alkaline lysis combined with paper-based filtration isolation of nucleic acids method(ALP FINA)and a sealed and visual recombinase polymerase amplification test(SV RPA)device that integrated the entire LFD-RPA assay into this device.This detection system formed by ALP FINA and SV RPA can carry out nucleic acid extraction in 10-15 min and complete all process steps of NAAT in 15-20 min.More important,it can be performed without electricity infrastructure and not generate contamination of nucleic acid aerosol.We used this sample-to-answer detection system to establish a detection method for S.japonicum and adenovirus.The sensitivity and specificity were evaluated,the optimal temperature and time for RPA was determined,and the effectiveness of LFD-RPA assay was assessed with clinical samples.The main research contects are as follows:1.The development of a disposable and portable nucleic acid extraction device.In this study we developed an alkaline lysis combined with paper-based filtration isolation of nucleic acids method(ALP FINA),of which could be used for different types of samples.The device consisted of a top cover,a bottom cover,and a nucleic acid extraction membrane.Based on aline lysis method for DNA extraction,NaOH was used to break down the cell wall,denature the protein and release the genomic DNA.Then the mixture was pipetted into the DNA capture membrane to perform purify.At the same time,we designed a disposable and portable nucleic acid extraction device and the entire extraction process can be performed with just the addition of mixture of samples and NaOH,absolute ethanol,and nuclease-free water in turn.This method could complete nucleic acid extraction within 10-15 min without the use of electricity for heating and centrifugation.It is suitable for on-site and field application.2.Establishment of LFD-RPA optimal detection system.RPA primers are typically 30 to 35 bases long.Avoid 5 ’long G sequence to avoid an impact to the combination of the recombinant enzyme and primer.Recommend the’ 3 ’end with G and C bases.The free energy should be less than 4 Kcal/mol.No hairpin structure is formed below 30 degrees Celsius.LFD-RPA probes are typically 46–52 nucleotides long and at least 30 of which are placed 5’ to the THF site,and at least a further 15 are located 3’ to it.There is a biotinylation at the 5’ end of the probe.The 3’ ends of probe are blocked with a C3-spacer.3.Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum.A novel detection method based on LFD-RPA was developed to detect S.japonicum DNA in fecal samples.The SjR2-specific primers for LFD-RPA were designed according to the designing criteria of RPA primers.Several primer combinations were evaluated and one primer pair was eventually identified.The result shows that the LFD-RPA assay targeting SjR2 could detect 5 fg S.japonicum DNA,which was identical to qPCR and real-time RPA assay,and showed no cross-reaction with other parasites.The detection could be finished within 15–20 min at a wide temperature range(25–45 °C),and the results could be visualized by naked eye.The diagnostic validity of LFD-RPA assay was further assessed with 14 fecal samples of infected patients diagnosed by Kato-Katz method and 31 fecal samples of healthy persons,and compared with that of Enzyme-linked immunosorbent assay(ELSIA)and Indirect Hemagglutination Assay(IHA).The LFD-RPA assay showed 92.68 % sensitivity,100 % specificity and excellent diagnostic agreement with the gold standard Kato-Katz test(κ = 0.947,Z = 6.36,P < 0.001),whereas ELISA showed 85.71 % sensitivity,93.55 % specificity,and substantial diagnostic agreement(κ = 0.793,Z = 5.31,P < 0.001),and IHA showed 78.57 % sensitivity,83.87 % specificity,and moderate diagnostic agreement(κ = 0.600,Z = 4.05,P < 0.001),indicating that the LFD-RPA was much better than the traditional methods.4.Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual for the detection of adenovirus types B and E(Adv-B and Adv-E).The results showed that Adv-B and Adv-E could be simultaneously detected with the LFD-RPA assay and showed no cross reaction with other species of adenovirus and unrelated virus.The LFD-RPA system had high sensitivity that the detection limit was 10 copies/μl.Although the sensitivity is slightly lower than qPCR(AdvB: 5 copies/μl,AdvE: 5 copies/μl),it is good enough to meet the clinical demands,and its simplicity and rapidity is much better than qPCR.The assay could be performed at a wide range of temperatures from 20 to 45 °C.It means that the assay could be performed without electrical power for heating.Meanwhile,the LFD-RPA assay could amplify the target DNA to detectable levels within 10-15 min,which was much shorter than that of other detection methods such as PCR.When applied to the detection of throat swab specimens of 20 patients infected with adenovirus as well as 10 healthy volunteers,both the sensitivity and specificity reached 100%.5.The development of a sealed LFD-RPA device.We developed a portable and sealed device that integrated the entire LFD-RPA assay into it.The operations were performed by changing the pressure that managed RPA reaction,delivery of reaction medium,and LFD detection to be performed in the same sealed space without the need to open the lid,which could effectively prevent contamination of nucleic acid aerosol.6.The development of a sample-to-answer detection system.In this study,we integrated ALP FINA and SV RPA into a sample-to-answer detection system that could carry out all three nucleic acid extraction,amplification,and detectin steps within 30-40 min at ambient temperatures.The characteristics of this system render it suitable for use in health departments with basic facilities,especially for field testing and on-site testing.