PMA-qPCR for detecting live bacteria of Shaanxi kiwifruit cancer dominant pathogen
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(1. College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, Shaanxi 710119, China; 2. Shaanxi Kiwifruit Engineering Technology Research Center, Xi’an, Shaanxi 710404, China; 3. Shaanxi Rural Science and Technology Development Center, Xi’an, Shaanxi 710054, China)

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    Abstract:

    Combining Propidium Monoazide Bromide (PMA) with real-time quantitative (qPCR) to detect the number of the live bacteria of kiwifruit cancer dominant pathogen in Shaanxi province. The optimal condition for PMA-qPCR to distinguish between live and dead ulcer bacteria was determined by optimizing the optimum concentration of PMA, incubation time and light exposure time. The results showed that all Psa were killed when treated by boiling water bath at 98.3 ℃ for 13 min. When the concentration of dead Psa was 1×107 CFU/mL, the suitable concentration of covalent crosslinking between PMA and dead Psa was 105 μg/mL, the best incubation time was 8min and the best light exposure time was 20 min. Under this condition, no DNA amplification of the dead bacteria, and no effect on the DNA amplification of live bacteria. The linear regression equation established based on the Psa plasmid standard was Y=-3.220 4x+37.73, for which R2=0.995 5. It can detect the Psa in the 6.39×102 copies/μL at minimum. The established PMA-qPCR method can detect 2.38×102 copies/μL Psa at minimum. By using the sample of artificially infected branches, the minimum detection limit was 6.30×104 CFU/mL,consistent with the detection results of colony counting method.

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肖妍,刘芸宏,高贵田,等. PMA-qPCR方法检测陕西猕猴桃溃疡菌优势病原菌活菌的研究[J].食品与机械英文版,2019,(4):48-53.

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  • Received:November 28,2018
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  • Online: November 26,2022
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