A Study Of Legionella Pneumophila,haemophilus Influenzae And Klebsiella Pneumoniae Highly Sensitive On-site Detection Technique Based On CRISPR/Cas13a System

Objective: Respiratory infectious diseases caused by pathogens such as bacteria,viruses,mycoplasma,and chlamydia have been a public health problem that has caused widespread social concern.In this study,we combined Recombinase aided amplification(RAA),CRISPR(clustered regularly interspaced short palindromic repeats)fluorescence and immunochromatographic techniques for the detection of three respiratory pathogens,Legionella pneumophila,Haemophilus influenzae and Klebsiella pneumoniae,with the aim of achieving accurate,efficient,specific and sensitive field detection.Methods:1.Search the literature and patents to identify the highly conserved and stable genes of Legionella pneumophila,Haemophilus influenzae and Klebsiella pneumoniae as the target genes for the assay,and synthesize the target gene sequences as positive references for backup.2.Designing specific target sequences(protospacer)of Legionella pneumophila,Haemophilus influenzae and Klebsiella pneumoniae in the target gene sequences based on CRISPR/Cas13 a detection principle and synthesizing the sequences into cr RNA(CRISPR-RNA).3.Establishment of CRISPR/Cas13 a fluorescence detection system and screening of Legionella pneumophila,Haemophilus influenzae and Klebsiella pneumoniae by CRISPR fluorescence method with relatively superior cr RNA.4.The primers for each bacterium were designed according to RAA primer design principles,the relatively better primers for each bacterium were screened by DNA gel electrophoresis experiments.5.Evaluation of the specificity,detection limit of CRISPR fluorescence assay and immunochromatographic assay for each bacterium.Results:1.For Legionella pneumophila,the target gene was identified as Mip,and the relatively better specific primer sets and cr RNAs were screened as F6R3,cr RNA10.CRISPR/Cas13 a fluorescence assay and immunochromatographic assay have good specificity and no cross-reactivity with other ten respiratory pathogens,with detection limits of 1 copy/μL and 0.5 copies/μL,respectively.2.For Haemophilus influenzae,the target gene was determined to be Omp6,and the relatively better specific primer set and cr RNA were screened as F5R4 and cr RNA1 respectively.The specificity of CRISPR/Cas13 a fluorescence assay and immunochromatographic assay was satisfactory,and there was no cross-reactivity with other ten respiratory pathogens,and the detection limits of both assays reached 1 copy/μL.3.For Klebsiella pneumoniae,the target gene was identified as pho E,and the relatively better specific primer set and cr RNA were screened as F2R6,cr RNA1.CRISPR/Cas13 a fluorescence assay and immunochromatographic assay have good specificity and no cross-reactivity with other ten respiratory pathogens,with detection limits of 0.5 copies/μL and 1 copy/μL,respectively.Conclusion:1.Based on the CRISPR/Cas13 a fluorometric detection system,it can specifically,sensitively and rapidly detection of respiratory pathogens(Legionella pneumophila,Haemophilus influenzae and Klebsiella pneumoniae).2.Based on CRISPR/Cas13 a immunochromatographic detection system,the detection of respiratory pathogens(Legionella pneumophila,Haemophilus influenzae and Klebsiella pneumoniae)has the characteristics of rapid,sensitive,specific,portable and intuitive results,which is expected to achieve on-site detection.