Saturday, July 17, 2010

Methods for the detection of central nervous tissue (CNT) are urgently needed in food control due to TSE prion disease

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

Journal Analytical and Bioanalytical Chemistry

Publisher Springer Berlin / Heidelberg

ISSN 1618-2642 (Print) 1618-2650 (Online)

Category Original Paper

DOI 10.1007/s00216-010-3956-5

Subject Collection Chemistry and Materials Science

SpringerLink Date Tuesday, July 13, 2010

Ernst Lücker1 , Wolfgang Biedermann1, Thomas Alter2 and Andreas Hensel3

(1) Institut für Lebensmittelhygiene, Universität Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany (2) Institut für Lebensmittelhygiene, Freie Universität Berlin, Königsweg 69, 14163 Berlin, Germany (3) Bundesinstitut für Risikobewertung, P. O. Box 330013, 14191 Berlin, Germany

Received: 10 May 2010 Revised: 18 June 2010 Accepted: 20 June 2010 Published online: 13 July 2010


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, C24:1?9/?7, C24:1?9-OH/?7-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, C24:1?9/?7, and C24:1?9-OH/?7-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

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Variant Creutzfeldt–Jakob disease Ironside JW., Haemophilia. 2010 Jul;16 Suppl 5:175-80



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