Direct Detection of Viable but Non-culturable (VBNC) Salmonella in Real Food System by a Rapid and Accurate PMA-CPA Technique

Abstract

Salmonella enterica is a typical foodborne pathogen with multiple toxic effects, including invasiveness, endotoxins, and enterotoxins. Viable but nonculturable (VBNC) is a type of dormant form preserving the vitality of microorganisms, but it cannot be cultured by traditional laboratory techniques. The aim of this study is to develop a propidium monoazide-crossing priming amplification (PMA-CPA) method that can successfully detect S. enterica rapidly with high sensitivity and can identify VBNC cells in food samples. Five primers (4s, 5a, 2a/1s, 2a, and 3a) were specially designed for recognizing the specific invA gene. The specificity of the CPA assay was tested by 20 different bacterial strains, including 2 standard S. enterica and 18 non-S. enterica bacteria strains covering Gram-negative and Gram-positive isolates. Except for the two standard S. enterica ATCC14028 and ATCC29629, all strains showed negative results. Moreover, PMA-CPA can detect the VBNC cells both in pure culture and three types of food samples with significant color change. In conclusion, the PMA-CPA assay was successfully applied on detecting S. enterica in VBNC state from food samples.

A novel fluorescence cross-priming amplification based on universal molecular beacon for rapid and specific detection of Salmonella enterica in food samples

“…A previously published LFD-CPA assay for detecting Salmonella in food reported that the LOD of CPA was 10 times lower than that of real-time PCR, it might result from the primers they used are not optimal. Furthermore, LOD of established assay is signi cantly higher to that reported in published literature amounting to 10 3 CFU/mL by Liu et al (Ou, et al, 2021), 62 CFU/mL by Wang et al (Wang, et al, 2018). The current MB-CPA assay showed better sensitivity than the CPA assays which performed with single and double cross-priming principle.…”

“…However, 5 to 7 days is required for the conventional detection method with requiring expensive laboratory instruments and specialized expertise (Shang & Zhang, 2021). Recently, numerous molecular methods, such as polymerase chain reaction (PCR), real-time quantitative PCR(q-PCR), loopmediated isothermal ampli cation (LAMP), cross-priming ampli cation (CPA), recombinase polymerase ampli cation (RPA), polymerase spiral reaction (PSR), have been used for Salmonella detection results from their comparative advantages over standard methods (Hirose, et al, 2002;Momin, et al, 2020;Ou, et al, 2021;Ren, et al, 2020;Techathuvanan, Draughon, & D'Souza, 2010;Woods, et al, 2008). However, PCR-based methods inherently require an accurate instruments, along with skilled operators, making cumbersome, time-taking process and inability in resource de cient establishments.…”

Biofilm formation of two genetically diverse Staphylococcus aureus isolates under beta-lactam antibiotics

“…VBNC formation has also been previous reported to be responsible for food poisoning and spoilage ( Deng et al, 2015a ; Liu et al, 2016b , c ; Xu et al, 2022 ). Despite incapable of growing on medium plates, VBNC cells can solely express toxins or produce spoilage substances ( Liu et al, 2017d ; Zhou et al, 2020 ; Jiang et al, 2021 ; Ou et al, 2021 ). Biofilm and VBNC formation are both significant concerns in food safety, and their formation is also closely relevant to the incubation conditions.…”

A triton X-100 assisted PMAxx-qPCR assay for rapid assessment of infectious African swine fever virus

“…This makes the assay scalable with a possibility of deployment to places with limited resources, such as pig farms. Additionally, PMA assays are said to be capable of detecting live but unculturable pathogens [according to the manufacturer’s instructions, and other literature ( Zhong and Zhao, 2018 ; Ou et al, 2021 ; Zhao et al, 2022 )]. Lastly, considering time, and labor costs of cell culture, it is more convenient to include the PMAxx-qPCR in routine diagnosis to detect infectious ASFV.…”