Establishment And Application Of A Detection Method For Respiratory Pathogens In Children Based On LAMP Coupling CRISPR/Cas12a

Objective:This research established a new respiratory pathogen detection method based on the loop-mediated isothermal amplification(LAMP)technology combined with CRISPR/Cas12 a technology.The LAMP-CRISPR/Cas12 a method was applied to detect two pathogens,mycoplasma pneumoniae(MP)and streptococcus pneumoniae(SP),in nasopharyngeal swab(NPS)and bronchoalveolar lavage fluid(BALF)samples from patients with lower respiratory tract infections.The results were compared with clinical microbiological diagnostic results to explore the application value of this method in the diagnosis of respiratory tract infections.Method:We collected a total of respiratory samples from pneumonia patients with confirmed pathogen diagnosis who were admitted to the pediatric respiratory department of the First Bethune Hospital of Jilin University from January 2021 to March 2021.After purifying the nucleic acid,the LAMP-CRISPR/Cas12 a detection method was used to detect MP and SP.This detection method first uses LAMP technology to rapidly amplify pathogenic nucleic acid at a constant temperature,providing the sensitivity of the detection method.After that,CRISPR/Cas12 a technology is combined,which can specifically recognize the target sequence in LAMP products,providing the specificity of the detection method.Finally,after the activation of Cas12 a enzyme,the fluorophore-quencher probe is cleaved,outputting fluorescence and making the results of the detection method visible.The detection method result is judged based on the amplification curve.The consistency of the results were compared with clinical pathogen diagnosis results that included serum pneumoniae chlamydia antibody testing,blood culture,BALF culture,and BALF nucleic acid detection.Any one of the above pathogen detection results being positive indicates a diagnosis of MP or SP infection.Meanwhile,the sensitivity and specificity of the detection method were evaluated using the existing clinical pathogen diagnosis methods as the standard,and the consistency of the results from upper and lower respiratory tract samples were compared.We selected three samples with inconsistent results from two types of detection using second-generation sequencing technology for further testing.Result:A total of 20 respiratory samples were collected in this study,including 13 BALF samples,4 NPS samples,and 3 dual samples.The clinical pathogen diagnosis results showed 14 cases(70%)with a single detection of MP,1 case(5%)with a single detection of SP,2 cases(10%)with MP and SP,1 case(5%)with cytomegalovirus,and2 cases(10%)with MP and Hemophilus influenzae.The LAMP-CRISPR/Cas12 a detection method results showed 7 cases(35%)of MP,7 cases(35%)of SP,1 case(5%)of MP and SP,and 5 cases(25%)of negative results.The detection of MP had a specificity of 100% and a sensitivity of 42.86%,which was highly consistent with the results of clinical nucleic acid detection.Whereas the detection of SP had a sensitivity of 100% and a specificity of 72% and was consistent with the results of clinical nucleic acid detection.The consistency comparison between upper and lower respiratory samples showed that the results of MP detection were consistent,with 2negative cases and 1 positive case.For SP detection,2 cases had consistent results(1positive and 1 negative),and 1 case had inconsistent results(NPS positive,BALF negative).The results of three samples tested using second-generation sequencing technology show that one sample was positive for SP,which validates the actual positive result detected by this detection method but not detected by clinical pathogen diagnosis.Another two samples were positive for MP,suggesting false negatives detected by this detection method but detected by clinical microbiology.Conclusion:LAMP-CRISPR/Cas12 a detection method can rapidly amplify common pathogenic bacteria nucleic acid in respiratory infection samples.Compared with conventional pathogen detection methods,this technology has the advantages of accuracy,speed,simplicity,and lower requirements for experimental temperature and instruments.Our study showed the LAMP-CRISPR/Cas12 a detection method has high sensitivity and specificity for detecting SP,therefore it can be used for nucleic acid detection of clinical samples of SP.At the same time,it has relatively high specificity for detecting MP,but the sensitivity of MP detecting is poor.Therefore,the clinical pathogen diagnose of MP needs to be combined with serological antibody results which have higher sensitivity.In our study,the LAMP-CRISPR/Cas12 a detection method results are consistent with clinical pathogen detection results,at the same time,in the co-detection of upper and lower respiratory tract samples,this detection method also demonstrated potential for identification and detection of colonizing bacteria in upper respiratory tract samples.Therefore,the LAMP-CRISPR/Cas12 a detection method is expected to become an important tool for the point-of-care detection of respiratory tract infection pathogens in children.