PMA-qPCR for detecting live bacteria of Shaanxi kiwifruit cancer dominant pathogen

Authors

XIAO Yan, College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, Shaanxi 710119, China
LIU Yunhong, College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, Shaanxi 710119, China
GAO Guitian, College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, Shaanxi 710119, China; Shaanxi Kiwifruit Engineering Technology Research Center, Xi’an, Shaanxi 710404, China
CAO Fan, College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, Shaanxi 710119, China
MA Yanping, College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, Shaanxi 710119, China
ZHAO Wuqi, College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, Shaanxi 710119, China
LEI Yushan, Shaanxi Kiwifruit Engineering Technology Research Center, Xi’an, Shaanxi 710404, China; Shaanxi Rural Science and Technology Development Center, Xi’an, Shaanxi 710054, China

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.

Publication Date

4-28-2019

First Page

48

Last Page

53,59

DOI

10.13652/j.issn.1003-5788.2019.04.009

Recommended Citation

Yan, XIAO; Yunhong, LIU; Guitian, GAO; Fan, CAO; Yanping, MA; Wuqi, ZHAO; and Yushan, LEI (2019) "PMA-qPCR for detecting live bacteria of Shaanxi kiwifruit cancer dominant pathogen," Food and Machinery: Vol. 35: Iss. 4, Article 9.
DOI: 10.13652/j.issn.1003-5788.2019.04.009
Available at: https://www.ifoodmm.cn/journal/vol35/iss4/9

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