Application of qPCR Method for Investigation of Plant Colonization by Human Pathogen Bacteria
Chapter from the book: Vucelić-Radović, B et al. 2019. Application of Molecular Methods and Raman Microscopy/Spectroscopy in Agricultural Sciences and Food Technology.
The consumption of vegetables is very important for prevention cardiovascular diseases and it is recommended by WHO. The fresh vegetables are essential for healthy nutrition and provide minerals and vitamins. The vegetables are mostly consumed raw and it is very important to avoid its microbiological contamination during the production chain. The disease which are caused by human pathogen bacteria are very big problem for public health. These bacteria are able to contaminate fresh vegetables in any part of chain of food production.
The Salmonellosis is the usual foodborne infection which is caused by bacteria Salmonella spp. According to U.S. Public Health Service (2009), Salmonella is in the second place as causer of foodborne disease in the USA. Approximately, there are 40 000 cases of Salmonellosis in the USA per year (DFBMD 2009).
The most common serovars which are found worldwide are Salmonella enteritidis and Salmonella typhimurium but others serovars are limited to specific regions in the world (OIE 2005).
Today, there is lot of methods for detection human pathogen bacteria in food. The conventional methods are generally timeconsuming. The PCR is much faster and with qPCR we can get results only in few hours.The PCR allows increasing speed, sensitivity, specificity of detection human pathogen bacteria in fresh vegetables.
The aim of this study is application of the qPCR method for detection of Salmonella enterica subsp. Welteweden and Salmonella typhimurium LT2 in wheat seedlings.
Bacterial strains used in this experiment were Salmonella enterica subsp. Welteweden and Salmonella typhimurium LT2. The model plant was wheat. The bacterial suspension applied for inoculation seeds was ≈ 108 CFU. The inoculated plants left to grow in phitochamber for 3 weeks. The standard PCR was done for Salmonella strains. The primers for Salmonella were: rfbJ; fliC; fijB; invA, hilA. It was done cloning for getting plasmid with invA gene which was used for preparing standards for qPCR. Isolation Salmonella DNA from plants was done using kit. The sequencing of invA isolated from plant samples also was done.
The qPCR was done for DNA samples isolated from wheat root, shoot and substrate liquid inoculated with Salmonella strains. The Fluorescence In Situ Hybridization was done for inoculated plant samples. It was used specific probes for detection Salmonella by CLSM.
The results show that both investigated Salmonella strains were able to colonize wheat plants. The number of Salmonella DNA copies was 4.01 × 106 per 1 g root (S. enterica) and 3.32 × 107 per 1 g root (S. typhimurium).