Development and evaluation of a multiplex real-time PCR (qPCR) assay targeting ttrRSBCA locus and invA gene for accurate detection of Salmonella spp. in fresh produce and eggs

Safe water is a global concern, and methods to accurately monitor quality of water are vital. To assess the risks related to bacterial pathogen load in Lake Vomb that provides drinking water to the southern part of Sweden, this study combined molecular analyses of enterobacteria and bacterial pathogens in water using quantitiative real-time PCR with hydrodynamic modeling and quantitative microbial risk assessment (QMRA). A real-time PCR assay to detect enterobacteria was set up by primers targeting ssrA. Between February 2015 and May 2016, presence of ssrA gene copies as well as Campylobacter spp., Salmonella spp., and EHEC O157 DNA was analyzed by real-time PCR at several locations in the catchment of Lake Vomb and its tributaries Björkaån, Borstbäcken, and Torpsbäcken. Björkaån had the highest detected concentrations of the ssrA gene and, according to the results of hydrodynamic modeling, contributed most to the contamination of the water intake in the lake. None of the water samples were positive for genes encoding EHEC O157 and Campylobacter spp., while invA (Salmonella spp.) was present in 11 samples. The QMRA showed that the suggested acceptable risk level (daily probability of infection <2.7 × 10−7) is achieved with a 95% probability, if the Salmonella concentrations in the water intake are below 101 bacteria/100 mL. If a UV-disinfection step is installed, the Salmonella concentration at the water intake should not exceed 106 bacteria/100 mL.

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Evaluation of real-time PCR coupled with immunomagnetic separation or centrifugation for the detection of healthy and sanitizer-injured Salmonella spp. on mung bean sprouts

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2016.01.013 ◽

2016 ◽

Vol 222 ◽

pp. 48-55 ◽

Cited By ~ 17

Author(s):

Qianwang Zheng ◽

Marta Mikš-Krajnik ◽

Yishan Yang ◽

Sang-Myun Lee ◽

Seung-Cheol Lee ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Mung Bean ◽

Immunomagnetic Separation ◽

Salmonella Spp ◽

Mung Bean Sprouts ◽

Bean Sprouts

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Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

Applied and Environmental Microbiology ◽

10.1128/aem.04137-13 ◽

2014 ◽

Vol 80 (10) ◽

pp. 3086-3094 ◽

Cited By ~ 115

Author(s):

Hyatt C. Green ◽

Richard A. Haugland ◽

Manju Varma ◽

Hana T. Millen ◽

Mark A. Borchardt ◽

...

Keyword(s):

Real Time ◽

Surface Waters ◽

Real Time Pcr ◽

Fecal Pollution ◽

Qpcr Assay ◽

Taqman Assay ◽

Reference Database ◽

Quantitative Real Time Pcr ◽

Ambient Surface

ABSTRACTQuantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genusBacteroidesare among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

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Evaluation of Applied Biosystems MicroSEQ® Real-Time PCR System for Detection of Salmonella spp. in Food

Journal of AOAC International ◽

10.1093/jaoac/94.4.1106 ◽

2011 ◽

Vol 94 (4) ◽

pp. 1106-1116 ◽

Cited By ~ 9

Author(s):

Priya Balachandran ◽

Yanxiang Cao ◽

Lily Wong ◽

Manohar R Furtado ◽

Olga V Petrauskene ◽

...

Keyword(s):

Real Time ◽

Study Design ◽

Real Time Pcr ◽

Detection System ◽

Peanut Butter ◽

Reference Method ◽

Black Pepper ◽

Salmonella Spp ◽

Culture Methods ◽

Significant Difference

Abstract Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time-to-results compared to traditional culture methods. In this study, the MicroSEQ® real-time PCR system was evaluated for detection of Salmonella spp. in 10 different food matrixes following the AOAC Research Institute's Performance Tested MethodSM validation program. In addition, the performance of the MicroSEQ system was evaluated for the detection of Salmonella in peanut butter as a part of the Emergency Response Validation Program sponsored by the AOAC Research Institute. The system was compared to the ISO 6579 reference method using a paired-study design for detecting Salmonella spp. in raw ground beef, raw chicken, raw shrimp, Brie cheese, shell eggs, cantaloupe, chocolate, black pepper, dry infant formula, and dry pet food. For the peanut butter study, the system was compared to the U.S. Food and Drug Administration's Bacteriological Analytical Manual procedures using an unpaired-study design. No significant difference in performance was observed between the MicroSEQ Salmonella spp. detection system and the corresponding reference methods for all 11 food matrixes. The MicroSEQ system detected all Salmonella strains tested, while showing good discrimination against detection of an exclusivity panel of 30 strains, with high accuracy.

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