Rapid Detection and Quantification of Viable Cells of Using Propidium Monoazide Combined with Real-Time PCR.
Junhui Li, Ruxing Chen, Ruwei Yang, Xinchen Wei, Hua Xie, Yanxia Shi, Xuewen Xie, Ali Chai, Tengfei Fan, Baoju Li, Lei Li
Author Information
Junhui Li: State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Ruxing Chen: State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Ruwei Yang: Comprehensive Experimental Farm, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China.
Xinchen Wei: State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Hua Xie: Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China.
Yanxia Shi: State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Xuewen Xie: State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Ali Chai: State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Tengfei Fan: State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China. ORCID
Baoju Li: State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Lei Li: State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China. ORCID
() 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 by screening specific and sensitive primers based on the 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 = 0.99). The amplification efficiency reached 97%, and the lowest detection limit of viable cells was approximately 2 × 10 CFU·mL. 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 to 10 CFU·g viable 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 is crucial.
