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

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.


INTRODUCTION
During food processing, food is frequently contaminated by foodborne bacteria, including Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157 (Kirk et al., 2015;Sharma et al., 2019). S. enterica is a typical foodborne pathogen with multiple toxic effects, including invasiveness, endotoxins, and enterotoxins (Eng et al., 2015). Various serotypes of Salmonella are implicated in foodborne infections and contaminate food products, including eggs, milk, poultry, meat, and vegetables. It is the main cause of human gastrointestinal and other related diseases (Bao et al., 2017a,b;Wen et al., 2020). Recently, studies confirmed that S. enterica is capable of entering into the viable but non-culturable state (VBNC) state under an adverse environment, which could include low-temperature, salt stress, and nutrient starvation (Roszak et al., 1984;Chmielewski and Frank, 1995;Gupte et al., 2003;Zeng et al., 2013;Morishige et al., 2017;Highmore et al., 2018). VBNC cells cannot be detected by traditional culture-based methods (Xu et al., 2011a;Lin et al., 2017;Xie et al., 2017a). Therefore, it is urgent to develop a rapid and sensitive assay to detect S. enterica, especially in the VBNC state.
Cross Priming Amplification (CPA) is a novel isothermal method relying on five primers (2a/1s, 2a, 3a, 4s, and 5a) to amply the target nucleotide sequences . It does not require any special instrumentation and presents high rapidity, specificity, and sensitivity. Recently, CPA assays have been used for the detection of E. coli O157:H7, Listeria monocytogenes, Enterobacter sakazakii, Yersinia enterocolitica, and other pathogens (Wang et al., 2014;Zhang et al., 2015;Wang et al., 2018;Xu et al., 2020). Therefore, CPA is a potentially valuable tool for the rapid detection of foodborne pathogens, and the combination of propidium monoazide (PMA) may achieve the detection of VBNC state (Xu et al., 2010;Wang et al., 2011;Xu et al., 2011c).

Bacterial Strains
To standardize and evaluate the reaction system of CPA assay, non-S. enterica bacteria strains, including various species of Gram-negative and Gram-positive non-target strains, were used in this study ( Table 1). Two standard S. enterica ATCC14028 and ATCC29629 were used as positive controls. All strains used in this study had been preliminarily identified in the Lab of Clinical Microbiology, Zhongshan Supervision Testing Institute of Quality and Metrology.
All bacteria were prepared for genomic DNA isolation after incubation in trypticase soy broth (TSB, Huankai Microbial, China) at 37 • C at 200 rpm overnight. The genomic DNA was isolated by Bacterial DNA extraction Kit (Dongsheng Biotech, Guangzhou, China) according to the manufacturer's protocol.
The concentration and quality of the DNA were measured using Nano Drop 2000 (Thermo Fisher Scientific Inc., Waltham, MA, United States) at 260 and 280 nm. The isolated DNA was stored at −20 • C for further use.

CPA Detection System Design
As a species specific gene in Salmonella, invA has been selected, and its specificity in Salmonella has been previously confirmed. The CPA primers were specifically designed for invA gene in S. enterica using Primer Premier 5, including five primers recognizing five distinct regions in the gene open reading frame sequence ( Table 2). All primers were assessed for specificity by BLAST against the sequences in Genebank.
The PCR reaction was conducted to serve as a control in a total 25 µL volume with 12.5 µL 2× Taq PCR Master Mix (Dongsheng Biotech, Guangzhou, China), 3 µM each of forward and reverse primers, 2 µL of DNA template and the total volume was added up to 25 µL with nuclease-free water. The amplification procedure included a 5-min denaturation at 95 • C, 32 cycles of amplification at 95 • C for 30 s, 52 • C 30 s, 72 • C for 35 s, and final amplification at 72 • C for 5 min. The PCR products were detected by electrophoresis on 1.5% agarose gels.   and 5a, 1 µL mixture chromogenic agent (mixture with calcein and Mn 2+ ), 1 µL template DNA, and a volume of up to 26 µL of nuclease-free water. The mixed chromogenic agent consists of 0.13 mM calcein and 15.6 mM MnCl 2 ·4H 2 O. And mixed reaction solution was incubated at 65 • C for 60 min and heated at 80 • C for 2 min to terminate the reaction. Nuclease-free water substituted target DNA was used as a negative control. Subsequently, the amplified products were analyzed by electrophoresis on 1.5% agarose gels and observed the color change by naked eyes.

CPA Detection System Optimization
The specificity of CPA was evaluated by amplifying the genomic DNA extracted from 2 standard S. enterica strains and 18 non-S. enterica strains.
To determine the sensitivity of the CPA assay, serial 10-fold dilutions of the genomic DNA of S. enterica ATCC14280 were prepared and used in the reaction. The sensitivity of the CPA method was compared with the PCR method. All the tests were performed in triplicate.

Application of CPA Assay in Food Products
The application of CPA assay in the detection of S. enterica was conducted in three rice products (Cantonese rice cake, steamed bread, and rice noodle purchased from Guangzhou Restaurant, Guangzhou, China). Different concentrations (from 10 8 CFU/mL to 10 CFU/mL) of S. enterica ATCC14028 were applied to contaminate food samples. Subsequently, genomic DNA was extracted from the contaminated food samples and subjected to CPA and PCR methods in triplicate .

VBNC State Induction
The VBNC state of S. enterica was induced by oligotrophic medium (sterile saline) at a low temperature. The bacterial overnight culture (∼10 8 CFU/mL) was washed three times and resuspended by sterile saline and then stored at −20 • C. The culturable cell number was measured by plate counting method, and viable cells were determined by LIVE/DEAD R BacLight kit TM (ThermoFisher scientific, United States) with a fluorescence microscope after the cells were no longer culturable. The culturable and viable cell enumerations were performed every three days.

PMA-CPA Detection System
The PMA-CPA were developed to detect the VBNC cells of S. enterica with the observation of color change. The PMA-CPA was further applied in the detection of VBNC cells in contaminated rice food products (Cantonese rice cake, steamed bread, and rice noodle from Guangzhou Restaurant, Guangzhou, China).

Development of CPA Assay
The CPA assays for the detection of invA gene were set up using S. enterica ATCC14028. The products were analyzed by 1.5% FIGURE 3 | Sensitivity of the CPA assay in food samples of S. enterica with invA genes by 1.5% agarose gel electrophoresis (A) and agent mixture (B); M-DNA marker; lane 1-8, 10 7 CFU/mL, 10 6 CFU/mL, 10 5 CFU/mL, 10 4 CFU/mL, 10 3 CFU/mL, 10 2 CFU/mL, 10 CFU/mL negative control. agarose gel electrophoresis, and the bands were observed under UV light (Figure 1A). The results of electrophoresis revealed that the amplicons of CPA are of various sizes, showing as a ladder pattern on agarose gel instead of a single band. The fluorescent dye (MgCl 2 and calcein) changes the reaction system from orange to green in the reaction system ( Figure 1B).
The evaluation of the specificity of CPA assay was performed in 2 S. enterica strains and 18 non-S. enterica reference strains. Results were recorded by 1.5% agarose gel electrophoresis and color change. Ladder pattern bands and orange to yellow color changes were only observed in the 2 S. enterica strains (Figure 2). It indicated that only the target S. enterica strains were

Detection of S. enterica in Food Products
The CPA assays were applied in the detection of S. enterica in three food samples (Cantonese rice cake, steamed bread, and rice noodle). The homogenized rice products (9 mL) were inoculated with 1 mL 10-fold serial dilution of pure S. enterica culture. The concentration of artificial contamination food samples ranging from 10 8 CFU/mL to 10 CFU/mL with 10-fold series dilutions was applied. All three food samples were included for the application. As expected, the LOD shows an insignificant difference among food samples, and the results are identical. Only the samples with a concentration higher than 10 3 CFU/mL were able to be detected (Figure 3). Thus, the detection limit of the CPA assay was 10 3 CFU/mL.

Detection of VBNC Cells by PMA-CPA Assay
The PMA-CPA assay was established using VBNC cells of S. enterica. The PMA dye was added to either pure culture or flour samples with a final concentration of 5 µg/mL. After incubation at room temperature for 10 min in dark, the samples were exposed to a 650 W halogen lamp with a distance of 15 cm for 5 min, which inactivates unbinding PMA molecules rather than PMA-DNA molecules. All the dying process was performed in an ice bath to prevent DNA damage. Results showed that PMA-CPA can detect the VBNC cells both in pure culture and food samples with significant color change from orange to yellow (Figure 4). The samples with dead cells remain orange in both pure culture and food samples.

DISCUSSION
Salmonella enterica is a common foodborne pathogen that may cause serve illness. The generation of S. enterica VBNC state occurs during the chlorination of wastewater or food (Oliver et al., 2005;Zeng et al., 2013). Non-ionic detergents and sanitizers can also induce S. enterica into VBNC state (Morishige et al., 2013;Purevdorj-Gage et al., 2018;Robben et al., 2018). Furthermore, a multi-stress environment in complex components of food and storage conditions may induce the VBNC state formation of foodborne pathogens (Lin et al., 2016;Miao et al., 2016;Xu et al., 2016a,b).
Various molecular techniques have been developed to identify microbes. PCR is a mature method to detect foodborne microbes, but it has low sensitivity and complex variable temperature programs (Kim et al., 1999). RT-PCR has been used to detect microbes compared with the method in ISO 21872-1:2007. RT-PCR achieved higher sensitivity but with long pre-enrichment and 24 h of complicate procedure. Although RT-PCR is able to show results via a digital curve, the sensitivity of this method is limited and the procedure is complicated (Xu et al., 2008a;Miao et al., 2018;Xu et al., 2018;Zhao et al., 2018a,b). RT-LAMP or other isothermal amplification has been used reported to replace RT-PCR to increase the detection limit (Xu et al., 2008b;Xu et al., 2009;Liu et al., 2018a,b;Lv et al., 2020). However, sophisticated equipment with these technologies has brought certain difficulties to rapid on-site testing (Xu et al., 2011b;Xu et al., 2012b). CPA technique is a new strategy to achieve rapid detection and can be performed under constant temperature using a simple water bath. Furthermore, with the improvement of fluorescence dye, results can be identified by the color change in the reaction tube by the naked eye. For VBNC and dead cells, they are both nonculturable. However, VBNC cells differ from dead cells in their intact cell membrane and thus could be differentiated via PMA, which is capable of differentiating viable and dead cells.
Therefore, the developed CPA method can successfully detect the S. enterica with high rapidity and sensitivity and can identify the VBNC cells in food samples when combining with PMA.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
JL conceived of the study and participated in its design and coordination. AO and KW performed the experimental work and collected the data. YY and XG organized the database. LC and LQ performed the statistical analysis. AO wrote the manuscripts. All authors contributed to manuscript revision, read, and approved the submitted manuscript.