猪链球菌核酸检测量值溯源研究及方法评估

Assessment of traceability meastwement for Streptococcus su/s nucleic acid detection YANG Ting-ting,

目的针对病原微生物检测中急需解决的计量标准和量值溯源问题，建立猪链球菌核酸量值荧光定量PCR方法，研制猪链核酸检测标准物质，探索等效一致的核酸测量技术。方法根据已报告的DNA序列设计并合成种特异性16SrRNA引物，建立荧光定量WaR方法，摸索最佳上、下游引物配比浓度，利用已知核酸浓度的标准品作标准曲线并获得0值与样本起始拷贝数的关系，对方法的特异性、灵敏度、稳定性和不确定性进行评估，并进行DNA标准品效期实验。结果建立的猪链球菌DNA实时定量PCR方法，最佳引物浓度为上游0．6μmoL／L，下游0．3μmol／L。方法的灵敏度为18．6DNA拷贝数，DNA最佳检出浓度为1．86×102—1．86×104拷贝／10vL反应体系。0值只与模板的DNA起始浓度有关（F：597．60，P〈0．01），与不同的实验时间及实验人员（F=0．60、1．90，P〉0．05）无关。0值的不确定度为0．46％～5．40％。DNA标准品4oC可保存半年以上，而一60℃保存则可达1年以上。结论建立的荧光定量PCR方法特异性及稳定性良好，可用于猪链球菌核酸量值测定；制备的核酸标准物质具备国际计量标准，且稳定性好，可作为猪链球菌核酸检测的量值溯源和传递示范的标准物质。

Objective By establishing quantitative PCR （QPCR） assay for Streptococcus suis DNA detection and preparing reference materials in order to research the measurement traceability technology aiad metmlogical standard to ap- ply on pathogenic micwbes nucleic acid inspection. Methods According to reported Streptococcus suis DNA sequence, 16S rRNA specific primers were designed and synthesized and the concentration ratio of forward to reverse primer was tested and employed for QPCR. A standard curve was drawn according as the concentration of reference DNA materials. From there, the relation between Ct （Cycle threshold） value and sample DNA copies was demonstrated. The specificity, sensitivity, stability and uncertainty of the assay were evaluated, and the useful-life of the reference material was tested. Results The QPCR assay for the Streptococcus su/s DNA detection was established and Streptococcus suis DNA from clini- cal samples was quantitatively analyzed. There was no in-specific amplification. The primer concentration ratio was 0.6 tu＇nol/L （Fw） to 0.3 tmml/L （Re）. The sensitivity was 18.6 DNA copies. Reaction system was good for 1.86 x 102- 1.86 x 104 DNA copies/10 p.L detection. Experiment data from different experimenters and different time were analyzed by mixed effective model. It was suggested that the variety of Ct value was associated with the initial template concentra- tion （ F = 597.60, P 〈 0.01）. There were no significant differences with time or experimenters （ F = 0.60, 1.90, P 〉 0.05）. The uncertainty of Ct value was 0.46%-5.40%. The useful-life of DNA reference material was at least half a year for stored at 4~C or one year for - 60 ~C. C_.ondusiom This QPER assay with its well specificity and stability could be used as Streptococcus su/s DNA quantitative detection. The prepared DNA reference material provided with inter- national metrical standard with its good useful-life could be used as a certified reference material for the demonstration of measurement traceability and tranffer in Streptococcus su/s DNA quantitative analysis.