Establishment of Fluorescence Quantitative PCR Assay in Detection of Shigella Based on Internal Reference

Abstract:

According to Shigella gene sequences published by Genbank，we selected specific target genes，designed specific primers and probes and optimized the reaction system. An internal amplification control（IAC） was added to reaction system. This IAC was detected by TaqMan probes labeled with different fluorophore. 5~50 CFU/25 g artificially contaminated sample was added to evaluate the performance of reaction system. The assay was could be used reliably for detection of Shigella with a sensitivity of 1pg/uL. For the 10 -fold dilutions bacteria DNA extracted by cooking water，the lowest detection limit was 9 ×103 cfu/mL. But for the plasmid with ipaH，the lowest detection limit can reach 103 copies /uL. The standard curve of ipaH and ipaH-IAC was established，and the quantification was linear between Ct and template copy number（R2=0.999）. While the initial sample amount of artificially contaminated bacteria was 10 CFU/25 g mutton，the Shigella could be detected after 6 hours culture. The ipaH-IAC fluorescence quantitative PCR assay was developed. It could not only be applied for detection of Shigella in food，but also monitor the PCR reaction system and prevent the false negatives. Therefore，the ipaH-IAC fluorescence quantitative PCR assay further ensures the reliability of the results and is conducive to the realization of standardized quantitative PCR method for Shigella in samples.