Abstract
Objective By establishing quantitative PCR (QPCR) assay for Streptococcus suis DNA detection and preparing reference materials in order to research the measurement traceability technology and metrological standard to apply on pathogenic microbes 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 suis DNA detection was established and Streptococcus suis DNA from clinical samples was quantitatively analyzed. There was no in-specific amplification. The primer concentration ratio was 0.6 μmol/L (Fw) to 0.3 μmol/L (Re). The sensitivity was 18.6 DNA copies. Reaction system was good for 1.86 × 102-1.86 × 104 DNA copies/10 μL 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 concentration ( 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℃ or one year for - 66℃. Conclusions This QPCR assay with its well specificity and stability could be used as Streptococcus suis DNA quantitative detection. The prepared DNA reference material provided with international metrical standard with its good useful-life could be used as a certified reference material for the demonstration of measurement traceability and transfer in Streptococcus suis DNA quantitative analysis. Key words: Streptococcus suis; Quantitative PCR; Measurement traceability
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