乳品中大肠杆菌PMA-qPCR活菌检测方法的建立

doi:10.16431/j.cnki.1671-7236.2016.02.030

Abstract

Abstract: In order to detect viable E.coli in milk,a new PMA-qPCR method was established.The influences of different PMA concentration,dark incubation time,exposure time on dead bacteria inhibition effect were determined by detection of the cell numbers of viable and heat-killed E.coli suspensions at concentration of 1×10⁸ CFU/mL through fluorescence quantitative PCR (qPCR) method.The results showed that qPCR assay could specifically detect E.coli,and the viable E.coli must be exposed to 90 ℃ for 30 s in water bath to be lethal.The best treatment was 10 μg/mL PMA with 15 min of dark incubation time and 10 min of exposure time.This treatment could inhibit dead cell signals to a largest extend,while had little impact on aviable cells.The stability of PMA-qPCR assay was kept while the concentration of bacteria was more than 1×10⁸ CFU/mL.The regression equation of standard curve was y=-3.356x+47.413,R²=0.9989,the lowest detection limit was 10³ CFU/mL.The result of adding assay was agreed with the actual situation.This study laid a foundation for using of PMA-qPCR to detect the viable E.coli in food.

Key words: E.coli; PMA; qPCR; counting

CLC Number:

GAI Dong-xue, REN Hong-lin, LU Shi-ying, HU Pan, MENG Xian-mei, LIU Xi, SONG De-gang, JIN Wen, ZHANG Song, CHANG Jiang, LIU Yan-yan, LIU Zeng-shan. Establishment of PMA-qPCR Assay for Detection of Viable E.coli in Milk[J]. , 2016, 43(2): 493-498.

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Establishment of PMA-qPCR Assay for Detection of Viable E.coli in Milk

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