Brucella viable counting method based on PMA-qPCR technology
A technology of brucella and enumeration method, applied in the field of enumeration of brucella live bacteria
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Embodiment 1
[0049] 1. According to the Brucella outer membrane protein gene BCSP31 (GenBank accession number: M20404.1) sequence posted by NCBI, a pair of specific primers were designed using PremierPrimer 5, and the upstream primer sequence was: 5'-AAACATCAAATCGGTCGC-3'( SEQ ID No.1), the downstream primer sequence is: 5'-CCGCCCACAAAGAAATAG-3' (SEQ ID No.2), the length of the amplified fragment is 200bp, synthesized by Changchun Kumei Biotechnology Co., Ltd.
[0050] 2. PMA treatment conditions
[0051] Set the following 3 groups of processing methods:
[0052] (1) Take 500 μL each of 7 samples of Brucella attenuated strain S2 heat-killed bacteria liquid, 1 sample without PMA was used as a control, and the remaining 6 samples were respectively added with PMA to make the final concentration 15 μg / mL. The samples were exposed for different times, and the optimal exposure time was determined by qPCR detection.
[0053] (2) Take 500 μL each of 7 copies of Brucella attenuated strain S2 heat...
Embodiment 2
[0060] Sensitivity Analysis of PMA-qPCR Detection Method
[0061] Treat the Brucella S2 bacterial liquid with a known concentration with PMA at a final concentration of 15 μg / ml, and the exposure time is 10 minutes, extract genomic DNA, dilute it by 10 times, and perform PMA-qPCR detection, while ordinary PCR is used as a control. Sensitivity of the analytical method.
[0062] Through the comparative experiment of PMA-qPCR and common qPCR to different concentrations of Brucella S2, it was found that ( figure 2 -A), when the bacterial solution concentration is 10 3 ~10 8 Within the range of CFU / mL, the CT values of Brucella S2 treated with PMA and untreated were basically consistent, and both had a good linear relationship (R 2 are 0.986 and 0.991 respectively), and the sensitivity is 10 3 CFU / mL, and by ordinary PCR ( figure 2 -B) detect its sensitivity at 10 5 CFU / mL, it can be seen that the sensitivity of PMA-qPCR to detect Brucella S2 is 100 times that of ordinary...
Embodiment 3
[0064] Specific Analysis of PMA-qPCR Detection Method
[0065] Escherichia coli, Salmonella, Yersinia enterocolitica, Vibrio parahaemolyticus, RNase-free water were used as controls, Brucella suis attenuated strain S2, Brucella bovis 2308 and A19 strains, sheep The 16M and M5 strains of Brucella sp., and the Brucella sp. ovis epididymis were used as representatives of different Brucella species to conduct PMA-qPCR experiments and ordinary PCR experiments to analyze the specificity of the method.
[0066] The results of the specificity experiment took each non-brucella and RNase-free water as templates as controls ( image 3 -A), the results showed that the amplification was negative, and the reaction product was subjected to melting curve analysis ( image 3 -B), when the Tm value shows a single melting peak at 87.87-88.36°C, there is no primer dimer and non-specific amplification, indicating that the primer specificity is good, and ordinary PCR also confirms that the PMA-qPC...
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