Staphylococcus aureus DNA in human venous blood detected by real-time quantitative PCR assay / 中华临床营养杂志

ABSTRACT

Objective To establish a rapid real-time quantitative polymerase chain reaction (RQ-PCR)assay in quantifying and detecting Staphylococcus aureus DNA from human venous blood samples,so as to quantificationally evaluate the systemic infection caused or deteriorated by intestinal bacteria translocation.Methods Totally 26 clinical blood samples and 15 simulation blood samples were detected.The primers and TaqMan probe were designed targeting the highly conserved house-keeping femA gene of Staphylococcus aureus,and a 20 μl RQ-PCR amplification reaction system was established.The standard curve was built based on the recombinant plasmid DNA containing the amplicon of the target gene,and genomic DNA was extracted using QIAamp DNA Blood Mini Kit.Results The specificity of primers and probe was excellent,the detecting limit was 100 copies/μl (103 CFU/ml),the sensitivity was 99.7％,and the specificity was 94.6％.The correlation coefficient of the standard curve was between 0.9918 and 0.9997.For samples with different Staphylococcus anreus concentrations,the average accuracy of the RQ-PCR assay was (96.25 ± 2.26) ％ ; the intra-and interassay coefficients of variation were (8.06 ±0.07)％ and (10.01 ±4.40)％,respectively.The average recovery rate of Staphylococcus aureus DNA in blood samples was (111.72 ± 20.72) ％.In clinical blood samples,the positive rate of Staphylococcus aureus DNA was 15.4％ (4/26),while the blood culture of these samples all produced negative result for Staphylococcus aureus.Conclusion RQ-PCR assays is a rapid,sensitive,and specific method with good repetitiveness and can be used in the quantitative detection of Staphylococcus aureus in whole blood samples.