Rapid Detection and Quantification of Viable Cells of Pectobacterium brasiliense Using Propidium Monoazide Combined with Real-Time PCR

文献类型： 外文期刊

第一作者： Li, Junhui

作者： Li, Junhui;Chen, Ruxing;Wei, Xinchen;Shi, Yanxia;Xie, Xuewen;Chai, Ali;Fan, Tengfei;Li, Baoju;Li, Lei;Yang, Ruwei;Xie, Hua

作者机构：

关键词： Pectobacterium brasiliense; bacterial soft rot; PMA-qPCR; detection; viable cell

期刊名称：MICROORGANISMS （ 影响因子：4.5； 五年影响因子：4.8 ）

ISSN：

年卷期： 2023 年 11 卷 11 期

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收录情况： SCI

摘要： Pectobacterium brasiliense (Pbr) has caused significant economic losses in major vegetable production areas in Northern China by causing bacterial soft rot in cash crops such as potatoes and cucumbers. This study aimed to establish a PMA-qPCR detection method for Pbr by screening specific and sensitive primers based on the glu gene and the conserved region of the 23S rRNA gene. Based on the optimized PMA pretreatment conditions, a standard curve was designed and constructed for PMA-qPCR detection (y =-3.391x + 36.28; R-2 = 0.99). The amplification efficiency reached 97%, and the lowest detection limit of viable cells was approximately 2 x 10(2) CFUmL(-1). The feasibility of the PMA-qPCR method was confirmed through a manually simulated viable/dead cell assay under various concentrations. The analysis of potato tubers and cucumber seeds revealed that nine naturally collected seed samples contained a range from 10(2) to 10(4) CFUg(-1) viable Pbr bacteria. Furthermore, the system effectively identified changes in the number of pathogenic bacteria in cucumber and potato leaves affected by soft rot throughout the disease period. Overall, the detection and prevention of bacterial soft rot caused by Pbr is crucial.

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