| BackgroundTetanus is a specific infection in which Clostridium tetanus invades the human body through skin or mucosal wounds,and its spores develop into proliferators under anoxic conditions,producing toxins and causing muscle spasms.Tetanus toxin mainly attacks motor neurons in the nervous system,so this disease is characterized by clenching teeth,paroxysmal spasm and tonic spasm.Tetanus is rare in developed countries and urban environments,but it is more common in developing countries and after natural disasters.The vaccination of tetanus vaccine has effectively reduced its incidence in developed countries,but due to the low vaccination rate in developing countries and poor areas,tetanus infection is still an important public health problem.It is of great significance for the treatment of tetanus infection to identify the pathogenic bacteria of trauma infection at an early stage and take targeted treatment measures.Traditional laboratory detection of pathogenic bacteria is cumbersome and time-consuming,and the culture,purification and identification of pathogens often take about one week,and it is difficult to use for rapid detection in remote areas for the technical difficulty and low sensitivity.With the rapid development of molecular biology technology,real-time fluorescent PCR,gene chips and biosensors are used for the detection of pathogenic microorganisms.These methods have high sensitivity and specificity,and also reduced the detection time.However,molecular detection techniques can only be carried out in large and medium-sized hospitals or research institutions,and it is difficult to be applied to the diagnosis and treatment of patients in remote areas.The detection of tetanus bacteria urgently needs a new set of highly sensitive,specific and rapid detection methods which are simple,practical and convenient.Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)and CRISPR Associated Proteins(Cas)systems(CRISPR/Cas)is an adaptive immune system formed by most bacteria and archaea in the process of resisting the invasion of virus foreign substances such as phage.The research results confirm that the cross-integration of CRISPR/Cas system and other technologies shows the unique advantages of both sensitivity and specificity in disease diagnosis and treatment.Among them,the accessory cleavage activity of proteins such as Cas12 and Cas13 can prevent CRISPR/Cas from target amplification in vitro and signal amplification,thus shortening the detection time of biological targets.However,when the sample had low target concentration,the number of activated Cas proteins was limited and cannot generate a large number of detectable signals in a short period of time to meet the requirements of rapid testing.Recombinase Polymerase Amplification(RPA)is a amplification technique that uses single-stranded DNA binding protein,recombinase(T4Uvs X),and strand-substituted DNA polymerase(Bsu)to amplify a target exponentially at a constant temperature.RPA technology has many advantages,such as simple primer design,simple operation process,reaction temperature from 37℃～42℃,and rapid amplification within 20min.However,the combination of primers or probes with the target sequence can still tolerate a certain degree of mismatch,and there is no thermal cycle to avoid the combination between primers,so it is difficult to eliminate some non-specific amplification.In summary,we propose the following hypothesis,combining the CRISPR/Cas system with RPA technology,which can make up for each other’s technical deficiencies and achieve the combined effect of rapid,high sensitivity and high specificity,and apply it to the detection of Clostridium tetani molecular markers,so as to innovatively construct a programmable,rapid,simple,high sensitivity and high specificity assay that can be used without the limitation of environment and laboratory equipment.Objectives1.Establish a rapid,sensitive and specific detection method for Clostridium tetani TetX gene based on CRISPR/Cas12a gene editing technology,and to construct a combined CRISPR/Cas12-RPA detection system combined with RPA amplification technology to further improve the specificity and sensitivity of the reaction and to validate its detection performance.2.Construct the clinical standard detection PCR method and use both PCR and the assay constructed in this study to detect clinical standard strains,compare the consistency of the results of the two methods and evaluate the sensitivity and specificity of the new assay,so as to provide a reliable,rapid and convenient detection method for the diagnosis of tetanus in remote areas with scarce medical resources.Methods1.Establishment of CRISPR/Cas12a detection system1)Construction of CRISPR/Cas12a detection system.The conserved and specific target gene fragments were screened,and the crRNA was designed,and the CRISPR/Cas12a detection system was initially constructed,which verified the feasibility of the detection method.2)Optimization of reaction conditions and performance verification.The reaction conditions of CRISPR/Cas12a detection system are optimized to achieve the best detection performance.The sensitivity and specificity of the detection system were verified.2.Construction and application of CRISPR/Cas12a-RPA assay system1)Constructed the CRISPR/Cas12a-RPA assay system and optimized the crRNA and time of reaction.The sensitivity and linearity of the reaction system were verified after doubling dilution of standards,and the limit of detection(LOD)was calculated.2)The performance of the detection system is verified,including sensitivity,specificity,repeatability and anti-interference.A PCR method was constructed for methodological comparison.Clostridium tetanus was detected,and the consistency of detection results and sensitivity of the two methods were compared.Results1.CRISPR/Cas12a assay system was constructed to detect Clostridium tetani nucleic acid fragments,and the performance of the reaction system was optimized when the concentration of Cas12a protease was 30 nM,the concentration of crRNA was 30 nM,and the concentration of fluorescent reporter probe was 1μM.2.The CRISPR/Cas12a-RPA detection system was constructed,and multiple crRNA sequences were designed for the target sequences,and the optimal crRNA for the reaction was optimized,and the best detection performance of the reaction system was achieved when crRNA1 was used.3.The performance of CRISPR/Cas12a-RPA detection system should be validated,and its sensitivity,specificity,reproducibility and interference resistance were evaluated.The best linearity of detection was achieved when the standard plasmid was of low to medium copy number(concentration of 10~0～10~4copies/μL),and the minimum detection limit could reach 1copy/μL based on linearity calculation.4.The PCR method was established as a clinical standard molecular assay,and the standard strains were detected by both PCR assay and CRISPR/Cas12a-RPA assay and then compared there’s detection performance.The results showed that both methods could detect the standard strains of C.tetani containing TetX gene,and the LOD of the assay established in this study was better than that of the PCR method.Conclusions1.A fluorescence detection system based on CRISPR/Cas12a was successfully established and the reaction conditions were optimized.CRISPR/Cas12a system achieved the best performance when the concentration of Cas12a protease was 30nM,the concentration of crRNA was 30nM and the concentration of fluorescence reporter probe was 1 m.2.The CRISPR/Cas12a-RPA detection system was successfully constructedfor rapid,sensitive and specific detection of Clostridium tetani standard plasmids.The assay can be completed within 90 min with an LOD of 1 copy/μL,and detected standard strains of Clostridium tetani containing TetX gene successfully.3.Compared with the clinical standard molecular detection method PCR,the CRISPR/Cas12a-RPA detection system can be carried out at a constant temperature of 37℃,and the reaction LOD is lower.Combining this system with a portable and small instrument such as a blue light meter may enable immediate visualization of tetanus detection. |
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