Analytical and Bioanalytical Chemistry

, Volume 398, Issue 2, pp 963–972 | Cite as

GC/MS detection of central nervous tissue as specified BSE risk material in meat products and meat and bone meals: thermal stability of markers in comparison with immunochemistry and RT-PCR

  • Ernst Lücker
  • Wolfgang Biedermann
  • Thomas Alter
  • Andreas Hensel
Original Paper

Abstract

Methods for the detection of central nervous tissue (CNT) are urgently needed in food control as a means for controlling strict adherence to both food labeling and banning of specified BSE risk material. Here, we report data on heat stability of the CNT markers neuron-specific enolase (NSE) in western blotting, glial fibrillary acidic protein (GFAP) in an enzyme linked immunoassay, mRNAGFAP in a real-time PCR assay, and several fatty acids (C22:6, C24:0-OH, C24:1ω9/ω7, C24:1ω9-OH/ω7-OH, and C24:0) in gas chromatography mass spectrometry (GC/MS). The sample matrix, a standard material of emulsion-type sausage with varied contents of CNT (brain), was heat-treated in three studies: (1) routine meat technological heat treatment with low (85 °C, 30 min), medium (115 °C, 30 min), and high (133 °C, 30 min, 3 bar) heating of 72 anonymous samples from a blind trial; (2) heat treatment under experimental conditions (100, 110, …, 200 °C, 45 min); and (3) fractionized heating of central nervous system (up to three times) under moderate routine technological conditions (85, 100, and 115 °C, 30 min). The markers of the immunochemical methods showed a low GFAP or very low NSE temperature stability at medium and high temperature conditions. The real-time PCR assay gave inconsistent, non-quantitative results, which indicated an uncontrollable matrix effect. The relevant GC/MS markers (C24:0-OH, C24:1ω9/ω7, and C24:1ω9-OH/ω7-OH) proved to be extremely stable. Neither meat and bone meal conditions (133 °C) nor experimental heating (up to and above 140 °C) showed any reduction of GC/MS CNT quantification. On the contrary, a slight but significant increase was noted over a certain temperature range (120–140 °C) for most fatty acids, possibly due to an improved extractability of the fatty acids. We conclude that a quantitative approach is highly unreliable when using immunochemical methods; moreover, these methods might be basically prone to false-negative results depending on heat treatment and matrix composition. Therefore, antibodies with higher affinity to heat-treated CNT marker epitopes are needed. Relevant amounts of CNT (≥0.5%) in low- and medium-heated products would still be reliably detectable by the GFAP ELISA, which justifies its use as a screening method in official food control. The results obtained by the real-time PCR assay were contradictory to recently published data, indicating a need for further protocol optimization and collaborative trials. Up to date, the analytical approach using GC/MS is the only valid procedure as pertaining to heat stability and quantitative analysis; consequently, it should be recommended as the reference procedure in official food control for CNT detection in heat-treated meat products.

Figure

The introduction of central nervous tissue from bovines into the food chain probably caused a new variant of Creutzfeldt-Jacob disease in humans. Analytical control of meat products by immunochemical CNT detection can be hindered by so far unknown severe heat induced losses. In contrast the CNT-specific fatty acids detected by GC/MS turned out to be remarkably stable up to temperatures of 160 °C

Keywords

Bovine spongiform encephalopathy Central nervous tissue Specified risk material GC/MS ELISA Western blot RT-PCR Fatty acids NSE GFAP 

Notes

Acknowledgements

The authors would like to thank Mrs. Kisslinger and Mrs. Pilz for their valuable technical assistance, Dr. C. Krex for the scientific assistance in parts of the repeated heating experiments, as wells as Prof. Dr. Hans Otto Honikel and his co-workers at the Max-Rubner-Institute for the provision of standard material from an externally controlled blind trial. This work was financially supported by the German Federal Ministry of Food, Agriculture and Consumer Protection (BMELV) through the Federal Office for Agriculture and Food (BLE), grant number 514-33.34/03 HS 011.

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Ernst Lücker
    • 1
  • Wolfgang Biedermann
    • 1
  • Thomas Alter
    • 2
  • Andreas Hensel
    • 3
  1. 1.Institut für LebensmittelhygieneUniversität LeipzigLeipzigGermany
  2. 2.Institut für LebensmittelhygieneFreie Universität BerlinBerlinGermany
  3. 3.Bundesinstitut für RisikobewertungBerlinGermany

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