A Multiplex PCR Assay Mediated By Universal Primers For The Detection Of Bacterial Meningitis

Part Ⅰ A novel method for efficient screening of universal primersObjective:To design a novel efficient method for screening universal primers.Method:Three forward primers and three reverse primers were connected with the 5’ and 3’ends of human β-actin gene fragment, respectively. And the resultant sequence was used to constructed plasmid template by pMD19-T vector. Through the constructed plasmid template, the PCR amplification sensitivity of nine pairs of primers was detected. The method included primer design, the choice of inserted sequence, restriction enzyme digestion, transformation, plasmid extraction, and sensitivity detection.Results:By discriminating PCR amplication sensitivity, highly sensitive universal primers were obtained.Conclusion:The method is simple, efficient and fast, thus serving as a highly efficient screening of universal primers.Part Ⅱ Screening universal primers for bacterial meningitisObjective:To screen one universal primer used in a multiplex PCR system for detecting human bacterial meningitis.Method:Three pairs of primers were screened out by comparing the specificity of nine pairs of universal primers. The experiments for their PCR amplification sensitivity were carried out according to the novel method for efficient screening of universal primers.Results:UP1, UP2, and UP3 are highly specific, with a sensitivity of 10~2,10~6,10~3, respectively.Conclusion:The screened UP1 primer can be rightly prioritized to apply to the construction of multiplex PCR system.Part Ⅲ The construction and evaluation of multiplex PCR detection system for bacterial meningitisObjective:To develop a modified universal primer-based multiplex PCR assay for the six common bacteria associated with human bacterial meningitis.Method:It consisted of the construction of plasmid templates, the establishment of multiplex PCR system, and the detection of clinical specimens. In the first part, the interest amplicon was cloned into the pMD19-T vector by double enzyme digestion and ligation reaction, and then transformed into DH5a competent bacterial cells. The recombinant plasmids were verified by colony PCR and sequencing. The second part included the design of specific primers and chimeric primer, the establishment of amplification system and cycling conditions, the detection of sensitivity and specificity of UP-M-PCR system. Lastly, the constructed UP-M-PCR was used to detect the clinical specimens.Results:Chimeric primer and UP-M-PCR had no non-specific products. The sensitivity of UP-M-PCR for S. agalactiae, L. monocytogenes, S. pneumoniae, N. meningitis, H. influenza, and S. aureu, was 10~2,10~3,10,10~2,10~2, and 10~4, respectively. Three samples were successfully detected by using UP-M-PCR system.Conclusion:Our proposed UP-M-PCR system has high specificity and sensitivity, serving as a potential tool to diagnose bacterial meningitis.