Development of a QPCR detection approach for pathogenic burkholderia cenocenpacia from fresh vegetables

Authors: LIU, AIXIN - Mississippi State University; PHILLIPS, KATE - Mississippi State University; JIA, JIAYUAN - Mississippi State University; DENG, PENG - Mississippi State University; Zhang, Dunhua; CHANG, SAM - Non ARS Employee; LU, SHIEN - Mississippi State University

Submitted to: Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/3/2023
Publication Date: 7/6/2023
Citation: Liu, A., Phillips, K., Jia, J., Deng, P., Zhang, D., Chang, S., Lu, S. 2023. Development of a QPCR detection approach for pathogenic burkholderia cenocenpacia from fresh vegetables. Food Microbiology. 115:104333. https://doi.org/10.1016/j.fm.2023.104333.
DOI: https://doi.org/10.1016/j.fm.2023.104333

Interpretive Summary: The gram-negative bacterium, Burkholderia cenocepacia, exists in diverse natural environments and is recognized as a dangerous opportunistic pathogen, namely in immunosuppressed individuals. Research has found that the sources of the bacterium are often nosocomial (associated with hospital exposure) but can also include crop plants and agricultural, urban, and suburban soils. While a serious issue, methods for the surveillance of this bacterial pathogen are underdeveloped. Conventional identification methods, including a combination of selective media and biochemical analysis, often lead to erroneous results. Genetic-based molecular approaches such as polymerase chain reaction (PCR) and multi-locus sequence typing (MLST) also have challenges because of the bacterium’s diverse and large genome size. In this study, we identified unique genome loci of this bacterium utilizing genome-wide comparative methods. Specific primers were designed for quantitative PCR assay for effective detection of this bacterium in fresh agricultural products. The high sensitivity and specificity developed in this study makes it a promising technique for future surveillance study on this bacterium.

Technical Abstract: The natural environment serves as a reservoir for Burkholderia cepacia complex organisms, including the highly transmissible opportunistic human pathogen B. cenocepacia. Currently, there is a lack of an effective and quantitative method for B. cenocepacia detection. A quantitative real-time PCR (qPCR) detection method for B. cenocepacia bacteria was established in this study and validated using artificially inoculated produce samples. Genome wide comparative methods were applied to identify target regions for the design of species specific primers. Assay specificity was measured with 12 strains of closely related Burkholderia bacteria and demonstrated the primer pair BCF6/R6 were 100% specific for detection of B. cenocepacia. The described qPCR assay evaluated B. cenocepacia with a 2 pg µl-1 limit of detection and appropriate linearity (R2=0.999). In 50 samples of experimentally infected produce (lettuce, onion, and celery), the assay could detect B. cenocepacia as low as 2.6 X102 cells in each sample equal to 1 gram. The established qPCR method quantitatively detects B. cenocepacia with high sensitivity and specificity, making it a promising technique for B. cenocepacia detection and epidemiological research on B. cepacia complex organisms from fresh vegetables.