Qiao B, Cui J-Y, Sun L, Yang S, Zhao Y-L
School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
School of Life Science, Shandong Province Key Laboratory of Applied Mycology, Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural University, Qingdao, Shandong Province, China.
J Appl Microbiol. 2015 Jul;119(1):188-95. doi: 10.1111/jam.12836. Epub 2015 May 29.
To develop and evaluate cross-priming amplification (CPA) combined with immuno-blotting for the detection of coagulase-positive Staphylococci including Staphylococcus aureus.
Twenty-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 3·6 ± 2·7 fg 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 1·34 CFU per reaction and 5·2 ± 3·7 CFU per 100 g of milk powder respectively. The CPA method was also successfully applied to evaluate the contamination of Staph. aureus in 318 samples of daily food.
CPA is a very sensitive and rapid method to detect Staph. aureus by simple laboratory instrument.
It is the first report on the application of the CPA with immuno-blotting for detection of coagulase-positive Staphylococci including Staph. aureus.
开发并评估交叉引物扩增（CPA）结合免疫印迹法用于检测包括金黄色葡萄球菌在内的凝固酶阳性葡萄球菌。
根据金黄色葡萄球菌凝固酶基因的四个序列设计了24组交叉引物和检测引物。筛选出最具特异性的引物对用于后续扩增和相互作用。在总共53种金黄色葡萄球菌和非金黄色葡萄球菌中评估其特异性。在BioHelix Express试纸条上，12个金黄色葡萄球菌亚种始终观察到两条指示阳性的红线。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（LOD）为3·6±2·7 fg，分别比PCR和环介导等温扩增灵敏约100倍和10倍。对于金黄色葡萄球菌纯培养物和奶粉，CPA的LOD分别约为每个反应1·34 CFU和每100 g奶粉5·2±3·7 CFU。CPA方法还成功应用于评估318份日常食品样本中金黄色葡萄球菌的污染情况。
CPA是一种通过简单实验室仪器检测金黄色葡萄球菌的非常灵敏且快速的方法。
这是关于CPA结合免疫印迹法用于检测包括金黄色葡萄球菌在内的凝固酶阳性葡萄球菌的首次报道。
