Introduction

Listeria are a common bacterial food contaminant found mainly in cheese and meat products [1,2,3,4,5,6,7,8]. These bacteria pose a serious health hazard. Listeria monocytogenes is considered the most threatening food born infection. Classical microbiology uses enrichment techniques like colony counting to determine the bacterial load in food samples. Such methods are recommended and certified by the International Organization for Standardization (ISO). Specific culture plates for Listeria are available, but often they do not detect Listeria other than L. monocytogenes. Less selective plates can lead to unclear results as, e.g., it is reported for some Bacillus cereus strains on the chromogenic isolation medium ALOA [9].

Many PCR and qPCR systems are available specifically for Listeria monocytogenes and Listeria species (spp.) [10,11,12,13,14]. Specific qPCR protocols have also been published for other Listeria strains [15]. Multiplex qPCR systems that detect also other bacteria species beside Listeria have also been published, making qPCR more efficient and allowing easy screening for unexpected bacteria in food [16,17,18,19]. Recently, a multiplex qPCR system was published that enables the detection of six of the most common strains of Listeria at once [20]. Other techniques, like MALDI-TOF was also applied to identify Listeria spp. [21]. Unfortunately, MALDI-TOF is not capable to identify specific bacteria in bacterial mixes. However, subtyping of Listeria strains from clones is possible [22].

The detection of any Listeria in food is interpreted as prerequisite also for the growth of pathogenic listeria and has serious implications. Such food is considered a possible health hazard for consumers. Production must be improved immediately to eliminate this risk. It is, therefore, important to exclude false negative results when using PCR systems with limited specificity.

We, therefore, decided to develop an efficient multiplex PCR system detecting the most prevalent listeria strains like Listeria monocytogenes, innocua, ivanovii, welshimeri and seeligeri using specific primers and probes. In addition, the system should detect Listeria spp covering 15 other available Listeria strains as to be: Listeria costaricensis, Listeria thailandensis, Listeria grayi, Listeria rocourtiae, Listeria marthii, Listeria weihenstephanensis, Listeria fleischmannii, Listeria riparia, Listeria aquatica, Listeria floridensis, Listeria grandensis, Listeria cornellensis, Listeria booriae, Listeria newyorkensis, Listeria goaensis. This was achieved using additional primers and probes, detecting all these strains in one single detection channel.

Materials and methods

Bacterial strains

For the experiments the following strains from German Collection of Microorgansims and Cell Cultures (DSM) were used: Listeria seeligeri DSM 20,751, Listeria innocua DSM 20,649, Listeria costaricensis DSM 105,474, Liseria thailandensis DSM 107,638, Listeria grayi DSM 20,601, Listeria rocourtiae DSM 22,097, Listeria marthii DSM 23,813, Listeria weihenstephanensis DSM 24,698, Listeria fleischmannii DSM 24,998, Listeria riparia DSM 26,685, Listeria aquatica DSM 26,686, Listeria floridensis DSM 26,687, Listeria grandensis DSM 26,688, Listeria cornellensis DSM 26,689, Listeria booriae DSM 28,860, Listeria newyorkensis DSM 28,861, Listeria goaensis DSM 29,886 and Listeria sp. DSM 29,997. Additionally, the strain from the American Type Culture Collection (ATCC) Listeria monocytogenes ATCC 7644 and strains of Listeria ivanovii and Listeria welshimeri identified by the National Reference Centre for Enteropathogenic Bacteria and Listeria (NENT) were utilized.

The strains Candida albicans ATCC 10,231, Escherichia coli ATCC 11,775, Enterococcus faecalis ATCC 19,433, Salmonella enteritidis ATCC 13,076, Staphylococcus aureus ATCC 25,923 and Bacillus cereus DSM 2302 were used as negative controls. These were complemented with additional strains listed in Table 4

Listeria monocytogenes, innocua, ivanovii, welshimeri and seeligeri and the non-target Bacteria strains were enriched in tryptic soy broth (TSB) by incubation at aerophilic conditions for about 24 h, but at least until an obvious turbidity was visible. Afterwards, these listeria strains were transferred on separate blood agar plates and the other bacteria on plate count agar plates. The remaining listeria species were plated on blood agar as described in the instruction for first-time application from the DSM. All plates were incubated at aerophilic conditions for about 24 h, but at least until single colonies reached a sufficient size. Listeria rocourtiae, Listeria weihenstephanensis, Listeria riparia, Listeria aquatica, Listeria floridensis, Listeria grandensis, Listeria cornellensis, Listeria booriae, Listeria newyorkensis, Listeria goaensis and Listeria sp. were incubated at 30 °C, the others at 37 °C.

Bacterial lysis and DNA extraction

For DNA extraction 100 µl PCR-water was transferred to a 1.5 ml Tube. With a one-way inoculation loop, a single colony was picked from an agar plate and added to the water in the tube. To lyse the bacteria a shaking and heating step in a heating block at 300 rpm and 95 °C was applied for 5 min. From each sample strain, DNA from several colonies were extracted individually in this way.

Primers and probes

Primers and probes of the multiplex PCR system named AllList, were taken from previous publications or developed in this work (see Table 1). All primers and probes were synthesized by Microsynth AG, Balgach, Switzerland. Labelling of probes with fluorescent markers was done according to the recommendations of the Rotorgene 6000 manual and are listed in Table 1.

Table 1 Overview of the primers and probes used for the multiplex qPCR system AllList for the simultaneous determination of 21 Listeria strains
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Primer development for the Listeria spp. system

A series of primers and probes targeting the gene for the bifunctional UDP-N-acetylglucosamine diphosphorylase/glucosamine-1-phosphate N-acetyltransferase (GlmU, Reference CP044432.1:206,781–206,942 Listeria monocytogenes strain FDA00009448 chromosome, complete genome) in all the tested Listeria strains were developed. Beside targeted selection of possible sequences, it was mainly a “try and error” approach leading to the successful amplification without cross reactions to other bacteria strains. The selected sequences, primer and probe positions are listed in Table 1. The label FAM was taken as summary channel for all Listeria spp amplicons.

Real-time PCR procedure

5 μl DNA extracts were added to 20 μl of reaction mix containing Sensifast Probe no ROX Kit (Bioline, Meridian Bioscience, United Kingdom), and all primers and probes of AllList (primers and their concentrations are listed in Table 1). PCR was performed on a Rotorgene 6000 real-time PCR system (Corbett, Australia) according to the following cycling protocol: Initial step of 5 min at 95 °C; followed by 40 cycles of 5 s at 94 °C and 15 s. at 60 °C and 7 s. at 72 °C.

Validation data to determine the performance characteristics of AllList

DNA-dilution rows were used to assess the performance characteristics of AllList (Tables 2 and 3). We estimate the amount of DNA from one clone to be easily sufficient to produce a positive signal by PCR.

Table 2 Multiplex serial dilution of DNA standard rows for AllList in the range from 0.2 to 100 ng DNA per assay
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Table 3 Performance of AllList
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Comparing of results from classical microbiological ISO-method with AllList using proficiency trial samples

Template DNA from stored proficiency tests were analyzed by AllList. The Listeria strains were chosen by the proficiency test provider and preferred Listeria monocytogenes. However, this is a stress-test for the method, as it does not use DNA from enriched clones as done for the validation of this method, but often from enrichment broth directly spiked with low concentrations of Listeria.

Comparing three methods

Food samples were analyzed by routine cultural ISO-method and AllList. AllList was applied after enrichment and cultivating on culture plates (clone picking). In addition, as a confirmation method the clones were analyzed by MALDI-TOF.

Results and discussion

AllList is based on the published PCR system for the six most common Listeria strains [1]. For the detection of the other Listeria strains several primer pairs were developed at the locus for Listeria spp. (GlmU or ribose-phosphate diphosphokinase gene). The sequences of the primers and probes were adapted to run under the same conditions. No special primer design software was used for this purpose, as the behavior is not precisely predictable and has to be evaluated with empirical data. The primers and probes that performed best (amplification efficiency and specificity) were used in multiplex format and tested with DNA from reference bacterial strains.

AllList is designed for screening of single colonies. Therefore, only one bacteria species is expected and competition by another template DNA does not take place. For this application competition between the PCR-systems does not occur. The applied DNA dilution row takes this in account (Table 2). The results are compiled in Table 3. As all dilutions revealed a positive signal at 0.2 ng/µl we assume that the LOD can be estimated to be at least at this level. From experience, the amount of DNA from one clone can be expected to be much higher. The performance characteristics presented here confirm these expectations. The estimated maximal measurement uncertainty of ± 18% is acceptable but does not include the isolation of the DNA. However, these data serve to characterize the PCR system AllList and has no impact on the final qualitative results.

The specificity of AllList was tested using the DNA from a wide range of bacteria (see Table 4). No false positive signals were observed and only the target species and strains gave a positive signal.

Table 4 Specificity testing results for target and non-target bacteria strains
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Proficiency test samples from the Health Protection Agency (HPA, UK) over a period of 1 year, were analyzed in parallel with classical microbiological methods and AllList. In total 36 samples were analyzed and most of the samples contained several species at once, predominantly Listeria monocytogenes with another strain (Table 5). The resulting match between AllList and the spiked bacteria according to the proficiency test was 48 detects of 52 spikes, which is 92%. We believe this to be a high degree of confirmation considering the prolonged storage of the sample material (isolated DNA) and that the sample material was not from a picked clone but directly from an enrichment broth.

Table 5 Compilation of the results from proficiency test program of Public Health England (PHE)
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Table 6 shows the results of routine samples which were analyzed by classical microbiology (ISO method), MALDI-TOF and AllList direct from enrichment broth and after cultivating on culture plate followed by clone picking and AllList. The ISO-method often gave unclear results, while AllList and MALDI-TOF did not. After enrichment AllList was sometimes positive for Listeria but after growing on the culture plate the Listeria was lost. This may indication false negative results by cultivation methods. The results of AllList match the results of MALDI-TOF better than the ISO-method. The influence of the matrices is comparable to the ISO method, as the sample is first cultivated on ISO recommended plates.

Table 6 The 8 samples of prepared green salads were analyzed
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Conclusion

AllList was developed to enable the fast and accurate determination of 5 listeria strains and the Listeria species in general including a total of 15 Listeria strains. This method was challenged using DNA from reference strains, proficiency test samples and routine samples. In all settings the performance was satisfying. The tests were performed over a period of 1 year by several technical assistants, demonstrating the robustness of the method. First attempts were made to use AllList directly after enrichment without cultivation on cultivating plates. However, in this case the LOD needs to be determined in future work. In short, AllList can characterize the most common listeria clones and extends the range of detectable Listeria to all strains of the species.