Establishment of a qPCR method to detect Staphylococcus xylosus and its application / 中国实验动物学报

ABSTRACT

Objective To establish and evaluate a method for rapid and sensitive S.xylosus detection using qPCR(real-time quantitative PCR).Methods A gehM gene fragment was selected as the target for S.xylosus.A set of specific primers was synthesized and a qPCR method was established to detect S.xylosus.A S.xylosus standard strain and other non-target strains were chosen for analysis.DNA of S.xylosus was diluted 10-fold to determine its sensitivity.Clinical samples were tested,and positive products were sequenced.The result were compared with those of bacterial culture.Results S.xylosus had a specific amplification curve,whereas other non-S.xylosus species did not,indicating that the primers were specific for S.xylosus.Sensitivity was 100 fg/μL DNA.Repeatability within and between groups was less than 3％.A total of 60 clinical samples were analyzed,of which five samples had a typical S curve.qPCR products were sequenced and BLAST searched.The similarity of the gene sequences was 99.63％,indicating that the sample was positive for the S.xylosus gehM gene with a positivity rate of 8.3％.However,the positivity rate of bacterial culture was 6.7％.The positivity rate of qPCR was slightly higher than that of the culture.Conclusions The established qPCR method is rapid with high sensitivity and specificity,and can be used to detect S.xylosus.