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Salmonella belongs to one of the most common zoonotic pathogens causing a notable number of foodborne outbreaks and product recalls. In the European Union, 94, confirmed salmonellosis cases were reported in [ 1 ].
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The most important food vehicles for foodborne Salmonella outbreaks were eggs and egg products, pig meat and products thereof, as well as bakery products [ 1 ]. Especially bakery products pose a direct risk to the consumers as no further decontamination steps take place before consumption. In order to improve food safety related to Salmonella , it is important to carry out preventive quality controls based on standard detection technologies. According to several governmental regulations or recommendations, Salmonella must not be detectable in 25 g of food.
Hitherto, the culture-based detection method is considered as the ideal way for detection of microorganisms. However, this method also shows limits and disadvantages such as skill level required, antimicrobial effects, matrices that inhibit detection completely e. For instance, 3 to 4 days are required to obtain a negative result and more than 5 days to confirm a positive one ISO ; [ 3 ].
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Further, viable but nonculturable VBNC cells cannot be detected. An alternative approach is polymerase chain reaction PCR , a powerful method for the detection of microorganisms in various matrices. PCR is rapid, sensitive and allows specific detection of the target microorganism based on the amplification of their DNA.
The biggest drawback of the classical PCR is that this method cannot differentiate between live and dead populations or extracellular DNA. By applying a photo-reactive dye to the sample prior to the nucleic acid extraction, the dye can enter compromised cells and can covalently link to the nucleic acid through a photo-activation step, with the result that the amplification of the nucleic acid by molecular tools is inhibited.
However, vPCR seems to have specific advantages and shortcomings.
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The success of vPCR depends on several factors such as dye type and concentration incubation conditions temperature and time , light source, microorganism and matrices as well as PCR amplification conditions e. In addition, membrane integrity of the microorganisms affects the vPCR efficiency. Many efforts were made in recent years to optimize the vPCR protocols regarding those critical points.
These authors have shown that the expulsion of EMA applied in low concentration from living Salmonella enteritidis cells is not a passive process. Despite many efforts, even using well-optimized vPCR procedures and in spite of the expected full neutralization of nucleic acids of dead microorganisms, it is not unusual to obtain a partial signal reduction. In this sense, recent investigations demonstrated that, at least for Legionella and Salmonella , a fraction of DNA remains inaccessible to vPCR due to their interaction with the tube wall [ 6 ] and in turns generates false positive results.
Based on the cumulated knowledge of the recent years regarding optimization of vPCR protocols, we developed a robust vPCR protocol suitable to exclusively detect live Salmonella in several matrices. Our protocol was verified in 33 food samples, which were artificially contaminated with heat killed Salmonella enterica cells.
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Further, it was simulated that food samples are contaminated with Salmonella by adding live and dead cells and PCR detection was carried out after enrichment. Salmonella enterica subsp. Cells were harvested from the agar plates and suspended in 10 ml phosphate buffered saline PBS, pH 7.
The cell density was adjusted to an OD of 0. Thereafter, the suspension was transferred in a new reaction tube GenIUL. The primers and TaqMan probe according to Cheng et al. Water PCR grade was used as a non-template control.
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The real-time PCR efficiency was calculated using the slope of the standard curve, which was generated using 10 fold serial dilutions of Salmonella DNA extracted from a pure culture with known concentration. To obtain a standard curve, cycle threshold C t values were plotted against the corresponding log 10 cell count. Serial dilutions of live Salmonella cells 5. As controls, samples containing either only 5. The experiments were conducted by two independent assays, each performed in duplicate. During a 2-month period, a total of 33 food samples and 6 peptone water controls obtained for routine quality controls were subjected to Salmonella detection according to the reference culture method ISO , [ 3 ] and other routine tests S1 Table in a local accredited laboratory Barcelona, Spain.
Ground pork meat and a ready-to-eat salad mix Cichorium intybus var. Then, 1 ml of live cells 5. A linear regression analysis was performed by plotting the C t values against the respective log 10 live Salmonella cells Fig 1 , left. The control samples contains either 5. Despite the presence of high dead cell concentration, the linear regression curve of PEMAX treated samples did not lose the linearity, showing a correlation coefficient R 2 of 0.
The PCR efficiency of this experiment was As depicted in the linear regression data Fig 1 , the quantification limit of the PCR reaction corresponds to 5. At this gene copy level, the mean C t values were The next tenfold serial dilution step showed no PCR signal. For this reason, C t values between 35 and 39 should be considered below the practical quantification limit.
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FEB of 33 food samples and 6 peptone water negative controls were artificially contaminated with 5. The goal of this experiment was to examine the applicability of vPCR in food samples. Results are presented in Table 1. Once the PEMAX-qPCR procedure has been applied, it was able to completely eliminate the fluorescence signal of 23 samples containing dead Salmonella cells; in 2 samples C t values were over 37 and in 8 samples C t values were between 35 and Additionally, samples without the addition of dead Salmonella also showed positive PCR signal in 8 cases, although cultural enrichment results showed negative results.
Based on these results, C t values over 35 might be background noise and cannot be assessed reliable as positive. By setting the limit of detection to C t 35, all contaminated FEB samples and controls would be assessed as negative. The aim of this study was to demonstrate that PEMAX treatment can fully suppress PCR signals of dead cells in food samples without showing any negative effect on the detection on live cells after enrichment.
The results are summarized in Table 2. However, enrichment at In addition, the C t values for the particular food samples varied heavily, resulting in the high standard deviation of the given C t values. For both enrichment temperatures, salad showed higher C t values than meat or the control. In theory, vPCR is a rapid, sensitive, and reliable method to simply detect live cells in a sample by molecular methods.
However, the practical experience from many researchers raised doubts about their effectiveness [ 12 , 15 , 16 ]. Despite their huge potential, incomplete suppression of PCR signals resulting in false positive results, in particular with high background of dead cells [ 17 — 19 ], has been one of the major limitations in the spread of this technique and their application in routine quality control. To address this issue, researchers around the world have made efforts for improving the effectiveness of vPCR methodology in order to detect only live cells in the sample.
Factors such as the ratio between viable and dead bacterial cells, organic material composition and concentration have been highlighted as potential inhibitors for the vPCR procedure, DNA extraction and qPCR [ 11 , 15 ]. With the aim to overcome false-positive results, we developed an improved vPCR protocol.
In the present work, the improved vPCR protocol was tested in various experiments to demonstrate the applicability of this in real food samples. In these studies, we combined several improvements suggested in the literature in conjunction with our previous vPCR works regarding Salmonella in pure cultures [ 6 ].
Our focus was to keep the protocol as simple as possible and suitable for adaption in the routine quality control. In most of the vPCR experiments, heat treatment was used to obtain dead bacteria cells [ 4 , 17 — 19 ], therefore we also chose this method to inactivate the cells. Nevertheless, thermally-treated bacteria might differ in their performance compared to bacteria treated with other inactivation processes.
Our killing condition could successfully kill the cells without releasing DNA as the signal of live and dead cells were similar Fig 1. In the case of heat treatment, cell membrane becomes compromised and viability dyes can easily penetrate into the cell.