实时荧光定量聚合酶链反应技术检测解脲脲原体生物群的研究

目的建立解脲脲原体生物群Taqman荧光定量聚合酶链反应(PCR)检测方法。方法根据解脲脲原体生物群MBA基因差异,分别设计并合成引物和探针。优化引物和探针浓度及试验条件,并进行试验的灵敏度、特异性和重复性评价。结果生物1群最适缓冲体系:25μL反应体系中包含2.5U Taq酶,Mg2+2.5mmol/L,上、下游引物0.1pmol/L,TaqMan探针0.2pmol/L,模板DNA 2μL。生物2群最适缓冲体系:25μL反应体系中包含2.5UTaq酶,Mg2+2.5mmol/L,上、下游引物0.2pmol/L,TaqMan探针0.3pmol/L,模板DNA2μL。PCR反应条件:95℃2min;95℃10s;55℃(检测荧光信号)20s,循环40次。该方法线性范围在1.0×102～8 copy/mL之间,检测限达到100～200copy/mL,特异性达到100%,CV值为2.5%。结论 本研究建立的TaqMan荧光PCR检测解脲脲原体生物群线性范围宽、灵敏度高、特异性及重复性好,能够快速进行解脲脲原体生物群检测。

Study on the detection of ureaplasma urealyticum biovars by real-time fluorescence quantitative PCR

Objective To establish Taqman fluorescence quantitative PCR method for detecting ureaplasma urealyticum（UU） biovars.Methods Based on differences in MBA genes of UU,primers and probes which were designed.We optimized the concentration of primers and probes and test conditions,and assessed the sensitivity,specificity and reproducibility of the tests.Results Taqman fluorescence quantitative PCR buffer systems for biovar 1 were as follow：25 μL reaction systems contained 2.5 U Taq enzyme,the level of Mg2＋was 2.5 mmol/L,each of the upstream and downstream primer was 0.1 pmol/L,Taqman probe was 0.2 pmol/L,the template DNA was 2 μL.Optimum buffer systems of biovar 2 were as follow：25 μL reaction system contained 2.5 U Taq enzymes,the level of Mg2＋was 2.5 mmol/L,each of the upstream and downstream primers was 0.2 pmol/L,Taqman probe was 0.3 pmol/L,the template DNA was 2 μL.There were the PCR reaction conditions,step l：95 ℃,2 min;step 2：95 ℃,10 s;55 ℃（detection point of fluorescent signal）,20 s,40 cycles.The linear range of the test ranges were from 1.0×102 copy/mL to 2.0×108 copy/mL.The sensitivity of detection reached 100-200 copy/mL,the specificity reached 100%,and CV value was 2.5%.Conclusion The new established Taqman fluorescent PCR method for detecting UU biovars,which has a wide linear dynamic range,a high sensitivity and specificity,and a good reproducibility,can be used to test quickly UU biovars.