基于16S rDNAs的快速检测血液细菌污染的荧光定量PCR研究

Rapid FQ-PCR screening for bacterial contamination of blood products based on 16S rDNAs

目的探讨快速筛选血液及血液制品中细菌污染的方法。方法对20余种常见致病性细菌的16SrDNAs进行多序列比对,设计扩增细菌16SrDNAs基因的荧光定量PCR(FQ-PCR)通用引物。以12种致病菌、2种真菌、1种支原体、1种病毒和人基因组DNA为模板,验证通用引物检测细菌的特异性。通用引物扩增金黄色葡萄球菌16SrDNA靶片段,构建标准质粒pMDT-Bfr,并建立FQ-PCR定量标准曲线。分别以金黄色葡萄球菌和大肠埃希菌作为革兰阳性和革兰阴性细菌代表菌,以其不同浓度的DNA为模板进行FQ-PCR反应,检验该方法的敏感性。抽提梯度稀释的金黄色葡萄球菌和大肠埃希菌模拟血液细菌污染标本的总DNA作为FQ-PCR反应模板,检验基于16SrDNAs的FQ-PCR作为血液细菌污染检测方法的敏感性。结果设计的FQ-PCR通用引物有较高的特异性,仅能检测出细菌的16SrDNAs;用该方法检测革兰阳性和革兰阴性菌,对于浓度在103拷贝/μl以上的16SrDNAs基因都可以准确定量;以该方法检测血液细菌污染模拟标本,灵敏度可达到105CFU/ml。结论初步建立了以16SrDNAs作为FQ-PCR靶基因快速检测血液细菌污染的方法。该方法特异性强、灵敏度高、成本低廉,具有很好的研究价值与应用前景。

Objective To establish the rapid screening method for bacterial contamination of blood and related products.Methods Multiple sequences of 16S rDNAs from over 20 bacteria were aligned,and the universal primers were designed for fluorescence quantitative PCR（FQ-PCR）assay.Various genomic DNAs from 12 kinds of common pathogenic bacteria,2 kinds of fungi,1 kind of mycoplasma,1 kind of virus and human genomic DNA were used as templates of routine PCR to confirm the specificity of the universal primers.Genomic DNA of Staphylococcus aureus was extracted,and the target 16S rDNA fragment was amplified by PCR with universal primers to construct standard plasmid pMDT-Bfr.The standard quantitation curve was then established by using of the dilution gradients of pMDT-Bfr.To determine the sensitivity of FQ-PCR assay,dilution series of DNAs of Staphylococcus aureus and E.coli as representative strains for gram-positive and gram-negative bacteria,respectively,were first detected by FQ-PCR with SYBR Green I.To further verify the sensitivity of established FQ-PCR in the screening of bacterial contamination,DNAs extracted from blood samples spiked with series diluted Staphylococcus aureus and E.coli were used as templates.ResultsThe amplified products only have high homology with bacterial 16S rDNAs.The sensitivity of the FQ-PCR assay was 103 copies/μl of 16S rDNAs for both gram-positive and gram-negative bacteria.For the blood samples of artificial contamination,bacteria could be detected at concentrations of ≥105 CFU/ml.The above results suggest that FQ-PCR targeting the 16S rDNAs has high specificity and sensitivity.Conclusions The FQ-PCR assay targeting 16S rDNAs gene used to detect bacterial contamination of blood exhibits perfect application prospect in the future,because it is rapid,affordable,high sensitive and specific.