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| Establishment and Application of Real-time TaqMan-quantitative PCR Assay for Detection of Mycoplasma suis (Eperythrozoon suis) |
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Abstract Abstract: The Taqman Fluorescent probes and primers were designed and synthesized according to the conserved part of hypothetical functional protein gene of Mycoplasma suis (Eperythrozoon suis) available in GenBank，and then reaction parameters were optimized and standard carve was established based on the quantitative 10×series of dilutions recombinant M.suis plasmid （pGEX-T/M.suis）to develop a real-time TaqMan-fluorescent-quantitative PCR assay (FQ-PCR) of Mycoplasma suis. The sensitivity, specificity and repetition assay of FQ-PCR were tested, and 24 heparinized-blood samples taken from suspicious infected pigs basing on the clinical signs and microscope check were detected by using the established FQ-PCR assay comparing with the routine PCR method. The results indicated the cycle numbers of standard carve had a good linear relation with the template concentration, and the correlation coefficient was 0.998. The developed FQ-PCR assay could detect 1.0×101 copy/μL of plasmid DNA and its sensitivity was 100 times higher than that of the routine PCR. The sensitivity assay exhibited that the positive carves could be achieved from the pGEX-T/M.suis recombinant plasmid, whereas the negative carve were displayed from the genomic DNA of the other 8 kinds of pathogenic microorganism acting as the control. Three pGEX-T/M.suis recombinant plasmids samples with different concentrations were examined using the FQ-PCR repeated 2 times and the results indicated that the FQ-PCR was reproducible. The FQ-PCR was used to detect the DNA of 24 suspected heparinized-blood samples and 20 positive samples were displayed, which showed the better sensitivity than that of the routine PCR , with 19 positive samples from the same 24 suspected samples.
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Received: 22 April 2007
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