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| Establishment of a SYBR Green Ⅰ qRT-PCR for Rapid Detection of Mycoplasma mycoides subsp. capri |
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Abstract Mycoplasma mycoides subsp. capri (Mmc) causes Mycoplasma pneumonia of goats and sheep (MPGS). The existing methods for Mmc detection are time-consuming and unquantifiable, but not suitable for accurate and rapid detection of Mmc. In order to establish a SYBR Green I real-time PCR assay for detecting Mmc. A pair of primers was designed based on the hypothetical protein gene (MLC_1770) gene of Mmc. The recombinant plasmid of Mmc-1770 was constructed as positive control for standard curve development, and the specificity, sensitivity and reproducibility of the assay were evaluated. The result showed that the correlation coefficient (R2) of standard curve was 0.999 and the amplification efficiency (E) was 101.3%. There was no cross reactions with Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma ovipenumoniae (Mo), Pasteurella multocida (Pm), Escherichia coli (Ec), Staphylococcus aureus (SA) and Orf virus (ORFV), indicating a good specificity. The sensitivity of this assay was 226 copies/μL which was 100 times higher than that of the conventional PCR. The coefficients of variation between the intra-groups assay were 0.54%~0.79%, and the inter-groups assay was 0.70%~0.96%. The 95 clinical samples were tested with this assay, the Mmc positive rate was 11.6% (11/95). The assay could be used to detect Mmc rapidly in clinical samples with strong specificity, high sensitivity and reliable reproducibility.
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Received: 21 April 2017
Published: 01 November 2017
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