Objective To construct reference standards for detection and quantification of Klebsiella pneumoniae(K.pneumoniae)with SYBR Green I-based real-time PCR assay.Methods Primers were designed based on the published sequence of the phoE gene of K.pneumoniae.The standard was prepared by cell culture,PCR and T-A clone methods,and was identified by colony PCR and DNA sequencing.Results The standard curve showed a very good linear negative regression between threshold cycle(Ct)and Log starting quantity of copy number.The detection range was from 5.2 to 5.2×106 copies per reaction,and the detection limit was 6 copies per reaction.The coefficients of variance(CVs)of three parallel experiments were in the range of 0.05%-0.91%.Conclusion The reference standards have high stability and reproducibility.They can be used in the quantitative detection of K.pneumoniae.
Fei-Long Sun1,2,Min Jin3,Zhi-Gang Qiu3,Zhi-Qiang Shen3,Xin-Wei Wang3,Jun-Wen Li31.School of Life Science and Technology,Xi’an Jiaotong University,Xi’an 710049
【目的】结合纳米技术建立检测大肠杆菌(Escherichiacoli)O157∶H7高灵敏检测技术。【方法】采用化学共沉淀法制备出核心粒径约为10nm的免疫纳米磁颗粒,柠檬酸钠还原法制备粒径约为20nm的免疫胶体金。压电免疫传感器通过金黄色葡萄球菌蛋白A(Protein A from Staphylococcus aureus SPA)法将抗体固定于石英晶振上,两种免疫纳米颗粒借助不同的抗体连接于传感器上对检测频率信号进行放大。【结果】SPA在石英晶振上的最佳固定浓度和时间为1.2mg/mL和40min,抗体的最佳固定浓度和时间为1.0mg/mL和60min。压电免疫传感器通过两种免疫纳米颗粒的放大作用,使其对大肠杆菌O157∶H7的检测限从104cfu/mL提高到101cfu/mL。【结论】免疫纳米颗粒强化对压电免疫传感器的检测频率信号具有很好的放大效应,可以明显提高其检测灵敏度。
