Cross-priming amplification targeting the coagulase gene for rapid detection of coagulase-positive Staphylococci
Cross-priming amplification targeting the coagulase gene for rapid detection of coagulase-positive Staphylococci
复制标题
针对凝固酶基因的交叉引发扩增，用于快速检测凝固酶阳性葡萄球菌
DOI：
10.1111/jam.12836
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发表时间：
2015-07-01
影响因子：
4
通讯作者：
Zhao, Y. -L.
中科院分区：
文献类型：
--
作者：
Qiao, B.;Cui, J. -Y.;Zhao, Y. -L.
目的建立和评价交叉引物扩增（CPA）结合免疫印迹检测凝固酶阳性葡萄球菌（包括金黄色葡萄球菌）的方法。方法和结果根据金黄色葡萄球菌凝固酶基因的4个序列设计了24套交叉引物和检测引物。筛选出最特异的引物对用于下一步的扩增和相互作用。在共计53种金黄色葡萄球菌和非金黄色葡萄球菌中评价了专属性。对于金黄色葡萄球菌的12个亚种，在BioHisperidExpress试剂条上始终观察到两条表示阳性的红线。相比之下，在所有非金黄色葡萄球菌样本中仅检测到一条显示阴性结果的信号线。CPA对基因组DNA的检测限为3627 fg，比PCR和环介导等温扩增的灵敏度分别提高了100倍和10倍。对金黄色葡萄球菌纯培养物和奶粉的检出限分别为134 CFU/反应和5237 CFU/100 g奶粉。CPA方法还成功应用于评价318份日常食品样品中金黄色葡萄球菌的污染情况。结论CPA是一种非常灵敏、快速的方法，可以通过简单的实验室仪器检测金黄色葡萄球菌。该研究的意义和影响这是首次应用免疫印迹CPA检测凝固酶阳性葡萄球菌（包括金黄色葡萄球菌）的报告。
AimsTo develop and evaluate cross-priming amplification (CPA) combined with immuno-blotting for the detection of coagulase-positive Staphylococci including Staphylococcus aureus.Methods and ResultsTwenty-four sets of cross and detection primers were designed according to four sequences of coagulase gene in Staph.aureus. The most specific primer pair was screened out for the next amplification and interaction. The specificity was evaluated in a total of 53 species of Staph.aureus and non-Staph.aureus. Two red lines indicating positive were always observed on the BioHelix Express strip for 12 subspecies of Staph.aureus. In contrast, only one signal line showing negative results was detected in all of non-Staph.aureus samples. The limit of detection (LOD) of CPA was 3627fg for the genomic DNA, which is about 100 and 10 times sensitive than those of PCR and loop-mediated isothermal amplification respectively. For the pure culture of Staph.aureus and milk powders, the LODs of CPA were about 134CFU per reaction and 5237CFU per 100g of milk powder respectively. The CPA method was also successfully applied to evaluate the contamination of Staph.aureus in 318 samples of daily food.ConclusionsCPA is a very sensitive and rapid method to detect Staph.aureus by simple laboratory instrument.Significance and Impact of the StudyIt is the first report on the application of the CPA with immuno-blotting for detection of coagulase-positive Staphylococci including Staph.aureus.