Rapid and Sensitive Detection of Viable but Non-culturable Salmonella Induced by Low Temperature from Chicken Using EMA-Rti-LAMP Combined with BCAC

O157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodology fails to identify O157 E. coli once it enters VBNC status. Crossing priming amplification (CPA) is a novel, simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to firstly develop and apply a CPA assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were rfbE, stx1, and stx2, and evaluated for its effectiveness in detecting VBNC cell of E. coli O157:H7 with detection limits of pure VBNC culture at 10 3 , 10 5 , and 10 5 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 10 3 , 10 5 , and 10 5 CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting E. coli O157:H7 in VBNC state from food samples. In conclusion, this is the first development of PMA-CPA assay on the detection of VBNC cell, which was found to be useful and a powerful tool for the rapid detection of E. coli O157:H7 in VBNC state. Undoubtedly, the PMA-CPA method can be of high value to the food industry owing to its various advantages such as speed, specificity, sensitivity, and cost-effectiveness.

Section: Methodsmentioning

confidence: 99%

Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli

Zhou

Wang

et al. 2020

“…As described by Chen et al (2019) more than 10 or 1% of S. enterica cells were induced into a VBNC state without nutrients at 4° or −20°C, respectively. In our study, S. enterica entered into a VBNC state under starvation condition at 4° or −20°C (groups 1, 2, 5, 7, 9, 10, 13, and 14).…”

Section: Resultsmentioning

confidence: 92%

Reduction, Prevention, and Control of Salmonella enterica Viable but Non-culturable Cells in Flour Food

Huang

Bai

et al. 2020

The processing and storage conditions of flour food inevitably pose environmental stress, which promote bacteria to enter a viable but non-culturable (VBNC) state. The existence of VBNC cells causes false-negative detection in traditional culture-based detection methods, resulting in food quality and safety issues. This study aimed at investigating the influence factors including nutrition, acid, salt, and temperature for the entry into a VBNC state of Salmonella enterica and an efficient detection method. During induction with multi-stress conditions, nutrition starvation antagonizes with lowlevel acidity. Besides, high-level acidity was considered as an inhibitor for VBNC induction. Four inducers including nutrition starvation, salt stress, low-level acidity, and low temperature were concluded for a VBNC state. In addition, the keynote conditions for S. enterica entering a VBNC state included (i) nutrient-rich acidic environment, (ii) oligotrophic low-acidity environment, and (iii) oligotrophic refrigerated environment. Based on the keynote conditions, the environmental conditions of high acidity (1.0% v/v acetate) with low temperature (−20 • C) could successfully eliminate the formation of S. enterica VBNC cells in flour food. In addition, combining with propidium monoazide pretreatment, PCR technology was applied to detect S. enterica VBNC cells. The sensitivity of the PMA-PCR technology was 10 5 CFU/ml in an artificially simulated food system. The results derived from this study might aid in the detection and control of VBNC state S. enterica in flour food products.

“…The PMA reagent was used at the concentration of 5 μg/mL. Subsequently, the detection samples mixed with PMA were incubated in the dark at room temperature for 10 min before the tubes were placed horizontally on ice exposed to a halogen lamp (650 W) at a distance of 15 cm for 15 min to complete the combination of DNA and PMA ( Chen et al, 2020 ). The mixed samples were centrifuged at 10,000 rpm for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Study on the Viable but Non-culturable (VBNC) State Formation of Staphylococcus aureus and Its Control in Food System

Huang

Mao

et al. 2020

A Viable but non-culturable (VBNC) state is a bacterial survival strategy under reverse conditions. It poses a significant challenge for public health and food safety. In this study, the effect of external environmental conditions including acid, nutrition, and salt concentrations on the formation of S. aureus VBNC states at low temperatures were investigated. Different acidity and nutritional conditions were then applied to food products to control the VBNC state formation. Four different concentration levels of each factor (acid, nutrition, and salt) were selected in a total of 16 experimental groups. Nutrition showed the highest influence on the VBNC state formation S. aureus, followed by acid and salt. The addition of 1% acetic acid could directly kill S. aureus cells and inhibit the formation of the VBNC state with a nutrition concentration of 25, 50, and 100%. A propidium monoazide-polymerase chain reaction (PMA-PCR) assay was applied and considered as a rapid and sensitive method to detect S. aureus in VBNC state with the detection limit of 104 CFU/mL.