Same-day PCR testing of Salmonellain meat: From research to routine application at slaughterhouses

Due to the very short shelf-life of fresh (especially ground) meat, slaughterhouses benefit from faster screening tests to dispatch Salmonella-free meat as soon as possible after slaughter. An increasing number of European countries require that the meat is tested as free for Salmonella before it is imported. This is currently the case for Sweden and Finland, which have a special agreement with the European Commission for import of fresh meat.

This was the background for collaboration between Danish scientists and the two major Danish slaughterhouses. The research project aimed at reducing the time of testing from 24 – 28 hours (when the project started in 2006) to obtaining results within the same day as the samples are collected. Slaughterhouses have a two shift working day of 16 hours, which makes it feasible to design a faster test, while control laboratories usually have an eight hour working day.

In Denmark, major slaughterhouses test around 100,000 meat and carcass swab samples annually from slaughtered pigs and cattle. This testing scheme is part of the companies’ quality control programme. In addition, many environmental, veterinary, blood and meat juice (exudate) samples are tested as part of the national surveillance program¹. Most meat samples are expected to be Salmonella-negative due to the very low prevalence of Salmonella in Danish pig, cattle and poultry herds.

One day vs. same-day PCR

At the time of the project start, most one-day testing methods were mainly based on real-time PCR, where a preliminary culture enrichment of approximately 18 hours is included, before the meat samples are treated and tested in PCR. We aimed at reducing this time of enrichment to approximately eight hours, but improve the detection limit of PCR in order to compensate for this shorter enrichment time. Previous comparative studies on detection chemistries of real-time PCR indicate that it is possible to improve the detection limit of PCR by applying advanced fluorescence oligo-nucleotide probes^2,3. The developed test is based on a short pre-enrichment (8 – 12 hours, depending of the sample material), a one-hour DNA purification step using paramagnetic beads, and a real-time PCR assay taking an additional hour to perform^4,5. To further simplify the test, the magnetic purification step has recently been replaced with an easy-to-perform boiling step, which has also substantially reduced the cost of testing. The new and simplified protocol has undergone the same strict validation and ringtrial examination as the previous protocol⁶.

The test is an open-formula, noncommercial, non-royalty protocol that can be set up by any laboratory. However, those laboratories that lack the facilities or expertise to prepare the PCR test, or are interested in a more quality-assured test, can purchase the Salmonella-12 PCR master mixture through the Danish company DNA Technology (www.dna-technology.dk).

Validation and ring-trials

The test that is currently used on about 100,000 samples in some of the major Danish slaughterhouses has gone through several comparative (in-house) and collaborative (ring-trial) validations. The participating laboratories have included a range of control and end-use laboratories.

The final protocols have been approved for meat testing (fresh meat and carcass swab samples) by the Nordic Validation Organisation for Validation of Alternative Methods (NordVal, www.nmkl.org), which is part of the Nordic Committee for Food Analysis (NMKL). The NordVal approvals⁷ are automatically accepted by all the Nordic food authorities (Sweden, Iceland, Finland, Denmark and Norway). Fulfilment of EU requirements Some years ago, meat exporters, kit manufacturers and authorities requested that the Commission accept the use of alternative methods, which could release meat for export within 24 hours. The EU Commission responded by seeking advice from the Scientific Committee on Veterinary Measures relating to Public Health (SCVPH). In June 2002, the SCVPH issued an opinion on criteria for evaluation of methods for Salmonella detection. Consequently, in June 2003 the EU Commission issued a decision on authorisation of certain alternative methods to be used in microbiological testing of meat intended for Finland and Sweden.

The requirements for approval of the alternative methods for Salmonella control of meat for Finland and Sweden are still based on advice from the SCVPH, but are more specific in the Commission Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs, which is an implementing rule of the recent hygiene legislation, entered into force in January 2006. It is here stated that alternative methods to be authorised for special guarantees for Salmonella control must have been validated against the most recent editions of the ISO 6579 or NMKL 71 and, if a proprietary method, certified by a third party in accordance with the protocol set out in standard EN ISO 16140 or other international similar accepted protocols. AFNOR, AOAC – OMA, MicroVal and NordVal are considered to be using such similar international protocols, even though they are not mentioned in the EU regulation.

Challenges ahead

While it was possible to speed up testing of meat products, veterinary samples such as faeces continue to be a challenge, mainly due to the complex sample matrix⁸. Efforts continue to eliminate the PCR inhibitory substances, while keeping the protocol simple and non-complex for routine laboratories. Here lies the real challenge for scientists that intend to develop tests for control laboratories located at production lines.

Another challenge is enumeration of contamination level. The first question any food producer asks when facing a test positive sample is “how contaminated were the samples?” Besides providing a general picture of the hygienic level of production environment and personnel, enumeration can point towards a possible breach in critical production points to help food producers with establishing integrated tracing systems⁹.

Existing enumeration methods using culture cultivation (such as the traditional Most Probable Number methods) are costly and time consuming, which is why only few samples (if any) are tested by this method. The European Committee for Standardisation (CEN) is currently working on a draft protocol for a Salmonella mini-MPN (Most Probable Number), based on Medium-Solid Rappaport-Vassiliadis (MRSV) agar. In the meantime, novel samples preparation techniques¹⁰ in combination with real-time PCR is catching up and overtaking this development by being a cheaper and faster alternative. Furthermore, studies indicate that it is possible to apply quantitative PCR even to culture-enriched samples¹¹, making this solution even more attractive¹².

In general, the real advantage of this type of PCR testing is screening of large number of Salmonella-negative samples, relieving laboratory resources to focus on source tracing of few positive samples.

References

1. Wegener HC, Hald T, Lo Fo Wong D, Madsen M, Korsgaard H, Bager F, Gerner-Smidt P, Mølbak K. (2003). Salmonella control programs in Denmark. Emerg Infect Dis 9:774-80
2. Reynisson E., Josefsen M. H., Krause M., Hoorfar J. (2006). Novel probes to improve the detection limit of real-time PCR, using Salmonella as a model. Journal of Microbiological Methods 66:206-216
3. Josefsen M.H., Löfström C., Sommer H.M., Hoorfar J. (2009). Diagnostic PCR: Comparative sensitivity of four probe chemistries. Molecular and Cellular probes 23:201-203
4. Josefsen M.H., Krause M., Hansen F., Hoorfar J.(2007). Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella in meat. Applied and Environmental Microbiology 73:3040-3048
5. Löfström C., Krause M., Josefsen M.H., Hansen F., Hoorfar J. (2009). Validation of a same-day real-time PCR method for screening of meat and carcass swabs for Salmonella. BMC Microbiology 9:85. http://www.biomedcentral.com/logon/logon. asp?msg=ce
6. Löfström C., Hansen F., Mansdal S., Hoorfar J. Salmonella detection in meat: Comparative and collaborative validation of a non-complex and cost-effective pre-PCR protocol. Journal of AOAC International (in press)
7. Qvist S. (2011). International validation, ring-trial and standardizationof rapid methods.In: Rapid Detection, Characterization and Enumeration of Food-Borne Pathogens. Hoorfar J. (ed.). American Society for Microbiology. Washington, D.C., USA. Pp. 157-161
8. Löfström C., Hansen F., Hoorfar J. (2010). http://orbit.dtu.dk/getResource?recordId=258507&obj ectId=1&versionId=1Validation of a 20-h real-time PCR method for screening of Salmonella in poultry fecal samples. Veterinary Microbiology 144: 511-514
9. Hoorfar J., Wagner M., Jordan K., Bouquin S.L., Skiby J. (2011).Towards biotracing in food chains. International Journal of Food Microbiology 145:S1-S4
10. Löfström C, Schelin J, Norling B, Vigre H, Hoorfar J, Rådström P. (2011). Culture-independent quantification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR. International Journal of Food Microbiology 145:S103-S109
11. Krämer N., Löfström C., Vigre H., Hoorfar J., Bunge C., Malorny B. (2011). A novel strategy to obtain quantitative data for modelling: Combined enrichment and real-time PCR for enumeration of salmonellae from pig carcasses. International Journal of Food Microbiology 145:S86-S95
12. Hoorfar J., Christensen B.B., Pagotto F., Rudi K., Bhunia A., and Griffiths M. (2011). Future trends in rapid methods: Where is the field moving, and what should we focus on? In: Rapid Detection, Characterization and Enumeration of Food-Borne Pathogens. Hoorfar J. (ed.). American Society for Microbiology. Washington, D.C., USA. Pp. 413-420

About the Author

Jeffrey Hoorfar is professor of food microbiology at the National Food Institute, Technical University of Denmark. He has led two major European research projects on PCR testing (FOOD-PCR I and FOOD-PCR II). His long collaboration with the food industry has given him insight into the challenges of using new tests in real-life situations. By launching one of the largest European projects in food safety, he has taken PCR further into predictive modelling, risk assessment and source-attribution studies.