[Objective]To establish a duplex PCR assay for the simultaneous detection of Klebsiella pneumoniae and Streptococcus.[Methods]Two pairs of specific primers were synthesised by selecting the conserved fragments of Klebsiella pneumoniae KP-Khe gene and Streptococcus EF-Tu gene respectively,and the dual PCR reaction system was optimized to explore its specificity,optimal annealing temperature,primer concentration and bacterial nucleic acid concentration.The method was used to test and verify the clinical samples.[Results]The duplex PCR assay was able to effectively amplify 489 bp and 197 bp specific fragments of Klebsiella pneumoniae and Streptococcus,with the optimal annealing temperature of 58℃,the optimal primer concentration of 12 ng/µL,and the optimal template concentration of 3 ng/µL.The method was applied to 15 clinical samples detection,of which 2 samples were positive for Klebsiella pneumoniae and 1 sample was positive for Streptococcus,which was consistent with the isolation and identification results.[Conclusion]The duplex PCR technique established in this study can provide a convenient and rapid detection method for clinical diagnosis of Klebsiae pneumoniae,streptococcus and mixed infection,which has certain application value.
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