ABSTRACT
Objective To develop a sensitive,specific, simple and rapid quantitative real-time PCR (Q-PCR) assay for detection of Staphylococcus aureus with SmartCycler.Methods According to the nuc gene sequences specific to S.aureus, a pair of primers and one TaqMan probe were designed. An internal amplification control (IAC) which is a chimeric double-stranded DNA constructed from a fragment of the Listeria monocytogenes hly gene flanked by the nuc-specific target sequences was added to the reaction system. This IAC was detected using a second TaqMan probe labeled with a different fluorophore. The performance of the nuc-IAC Q-PCR was evaluated using artificially contaminated drinking water and commercial UTH whole milk samples spiked with ATCC 6538.Results The nuc-IAC assay could be used reliably for detection with a sensitivity of 5 copies of linear plasmid DNA per reaction, 10 fg of genomic DNA in 62.5% of the reactions or 50 cfu/ml S.aureus cells with 50% probability. The quantification was linear (r~2≥0.998) over a 6-log dynamic range, with a PCR efficiency over 0.967. The 5×10~2 CFU per 25 ml mimic sample of drinking water or milk could be detected by this assay consistently and quantifiably.Conclusion The nuc-IAC Q-PCR assay for S.aureus is developed. It could not only be applied for the quantitative detection of S.aureus, but also prevent the false negatives and underestimations of contamination loads due to PCR failure.
ABSTRACT
<p><b>OBJECTIVE</b>To express the domain III of DENV II envelop protein in Escherichia coli, obtain the purified recombinant protein and identify its immunoreactivity.</p><p><b>METHODS</b>Suckling mice were inoculated with live DENV II in the brain. The total RNA was extracted from the brain of the infected mice, and the envelope protein DNA fragment was amplified by RT-PCR and ligated into pMD 18-T to construct pMD 18-T-DV2-E. The domain III DNA fragment of the envelope protein was amplified by PCR with pMD 18-T-DV2-E as the template and cloned into pET-32a(+) to construct the expression plasmid pET-32a(+)-DV2-E-DIII. The recombinant plasmid was transformed into E.coli BL21(DE3) and induced by IPTG, and the expressed products were analyzed by SDS-PAGE and Western blotting.</p><p><b>RESULTS</b>After RT-PCR amplification, a specific DNA fragment of about 1.5 kb was obtained and ligated into pMD 18-T to construct pMD 18-T-DV2-E. With pMD 18-T-DV2-E as the template, the domain III DNA fragment about 320 bp in length was amplified and the expression plasmid pET-32a(+)-DV2-E-DIII was successfully constructed. After induction with IPTG, a specific soluble protein with a relative molecular mass of 29000 was obtained and the expression product accounted for 52.50 percent; of the total protein of the cell lysate. Western blotting demonstrated reactivity of the recombinant protein with His-Tag McAb and DENV (Type I-IV) McAb.</p><p><b>CONCLUSION</b>The recombinant plasmid can be highly expressed in E.coli BL21(DE3) in a soluble form and the recombinant protein can react with DENV (Type I-IV) McAb.</p>