Integration Of Multiplex PCR And CRISPR-Cas Allows Highly Specific Detection Of Multidrug-resistant Acinetobacter Baumannii

ObjectiveThe increase of bacterial resistance to antibiotics poses a major public health challenge.Multidrug-resistant Acinetobacter baumannii is considered to be one of the most difficult Gram-negative resistant bacteria to control and treat.In clinical practice,bacterial identification and phenotypic antibiotic susceptibility tests need at least 48 hours,which delays the timely anti-infection of patients.Therefore,a point-of-care testing that can quickly identify resistant genes is highly desirable so that antibiotic treatment can be administered more effectively,reducing morbidity and preventing the rapid spread of resistant bacteria.Based on this,this project developed a rapid detection method for multidrug resistant Acinetobacter baumannii in combination with multiplex PCR and CRISPR-Cas array.MethodsThe housekeeper gene and fourβ-lactamase resistant genes of Acinetobacter baumanmannei were simultaneously amplified by multiplexed PCR.The single fluorescent signal output of the multiplexed PCR product was performed by using the indiscriminately single-stranded DNase activity of LBacas12a.The reporter and RNA concentration,incubation temperature and time were optimized.In addition,DNA template and a series of primer pairs of different complementary lengths were designed to simulate the generation of primer dimer,and the specificity of CRISPR-Cas12a system for primer dimer was proved by combining agarose gel electrophoresis,real-time quantitative PCR and fluorescence spectroscopy detection techniques.ResultsBased on the optimized experimental conditions,the multiple detection method could complete the genotypic antibiotic susceptibility test of Acinetobacter baumannii within 2 hours,and the detection limit was 50CFU/m L.In the range of bacterial concentration of 10~2-10~5CFU/m L,the fluorescence signal showed a logarithmic correlation with bacterial concentration(R~2>0.98).In addition,the selected five genes were not detected in E.coli,K.pneumoniae,P.aeruginosa and S.aureus by using this method,indicating that this method has good specificity.ConclusionBy combining multiplex PCR and CRISPR-Cas array,the rapid detection of multidrug resistant Acinetobacter baumannii was achieved in this project.The resistance mechanism of multidrug resistant Acinetobacter baumannii is complex.It has been reported that the production ofβ-lactamase is the most common mechanism of CRAB.The four drug resistance genes selected in this project have a clear correspondence with the drug resistance of Acinetobacter baumannii,which can be used to guide clinical drug use and control nosocomial infection as early and efficiently as possible.Given the generality and universality of the CRISPR-Cas12a platform,this study will further promote its application in the diagnosis and treatment of multidrug resistant bacteria.