日勾维多细菌源菌双重PCR快速检测方法建立OACSTPCD

A rapid detection method for Pluralibacter gergoviae was established,and this method was used to quickly and effectively detect Pluralibacter gergoviae in samples.Four house-keeping genes(gyrB,infB,atpD and ropB)were selected as target genes,and 4 pairs of primers were designed.The bacterial suspension was used as amplification,and the 4 pairs of primers could specifically bind with the Pluralibacter gergoviae to produce bright DNA bands.Among the four pairs of primers,only gyrB-F/gyrB-R and ropB-F/ropB-R obtained a small amount of non-specific DNA bands when Escherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa,Enterobacter cloacae and Burkholderia cepacia were used as DNA templates.Two pairs of specific primers gyrB-F/gyrB-R and ropB-F/ropB-R(insert)were used to establish the rapid detection method of duplex-PCR for Pluralibacter gergoviae in rinsing cosmetics.The specificity of duplex-PCR was improved by optimizing the annealing temperature of amplification.When the annealing temperature was 60℃,using the primer pairs gyrB-F/gyrB-R and ropB-F/ropB-R(insert)to amplify 4 strains of Pluralibacter gergoviae,two specific target DNA bands were obtained,but Escherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa,Enterobacter cloacae and Burkholderia cepacia would obtain non-specific DNA bands.When the annealing temperature was optimized and set at 65℃,the specific target DNA bands could be amplified when 4 strains of Pluralibacter gergoviae were used as DNA templates.When Escherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa,Enterobacter cloacae and Burkholderia cepacia were used as DNA templates,no DNA bands were detected.When the concentration of Pluralibacter gergoviae in the sample reached 4.4 CFU/mL,duplex-PCR was able to detect Pluralibacter gergoviae in the sample with high sensitivity.Duplex-PCR method could be used to quickly detect the Pluralibacter gergoviae in the rinsing cosmetics such as shower gel and shampoo.