Nel Haagsma

Evolution and recombination of bovine DNA repeats

The history of the abundant repeat elements in the bovine genome has been studied by comparative hybridization and PCR. The Bov-A and Bov-B SINE elements both emerged just after the divergence of the Camelidae and the true ruminants. A 31-bp subrepeat motif in satellites of the Bovidae species cattle, sheep, and goat is also present in Cervidae (deer) and apparently predates the Bovidae. However, the other components of the bovine satellites were amplified after the divergence of the cattle and the Caprinae (sheep and goat). A 23-bp motif, which as subrepeat of two major satellites occupies 5% of the cattle genome, emerged only after the split of the water buffalo and other cattle species. During the evolution of the Bovidae the satellite repeat units were shaped by recombination events involving subrepeats, other satellite components, and SINE elements. Differences in restriction sites of homologous satellites indicate a continuing rapid horizontal spread of new sequence variants.

Determination of chloramphenicol in swine muscle tissue using a monoclonal antibody-mediated clean-up procedure

A rapid and specific clean-up procedure based on immunoaffinity chromatography for the high-performance liquid chromatographic determination of chloramphenicol (CAP) in swine muscle tissue at the 10 micrograms kg-1 level is described. For preparation of the columns, monoclonal antibodies against CAP were coupled to cyanogen bromide-activated Sepharose. After immunosorption of CAP from the aqueous meat extract, CAP was eluted with methanol. Mean recoveries from spiked swine muscle tissue were 70 +/- 6% (10-50 micrograms kg-1 spiking levels). The loss of CAP can be completely attributed to the extraction; no loss was observed during the antibody-mediated clean-up.

Monoclonal antibody-mediated clean-up procedure for the high-performance liquid chromatographic analysis of chloramphenicol in milk and eggs

A simple, rapid and specific sample preparation method based on antibody-mediated clean-up for the determination of chloramphenicol (CAP) in milk and eggs was developed. Skimmed milk and centrifuged egg homogenates were filtered and directly applied to immunoaffinity columns which were prepared by coupling monoclonal antibodies against CAP to a carbonyldiimidazole-activated support. Using a 0.2 M glycine, 0.5 M NaCl (pH 2.8) solution as an eluent, the immunoaffinity columns can be used more than 30 times without a decrease in column capacity. In subsequent high-performance liquid chromatographic analysis, no matrix interferences were observed. Good recoveries were obtained at spiking levels of 1-100 micrograms kg-1. Due to the high specificity of the clean-up procedure, the limit of detection can be lowered by increasing the test portion. Concerning milk, the limit of detection was successfully lowered to 20 ng kg-1 by increasing the test portion to 11 (recovery 99%). The method was applied to eggs produced by hens treated with CAP. The results are compared with those obtained by solid-phase extraction using silica gel.

Species identification by oligonucleotide hybridisation: the influence of processing of meat products

We have evaluated the influence of meat processing on the results obtained with a species identification test by DNA oligonucleotide hybridisation. Freezing and thawing of meat did not cause a substantial reduction in the hybridisation signal. Heating of meat at 100°C or 120°C, however, led to signal reduction caused by DNA degradation, but identification was still possible. The signal is highly influenced by the kind of tissues processed. Four extensively processed products with 50 g kg−1 species admixtures were tested. Admixtures could be detected in three products but no hybridisation signal was observed with corned beef. We conclude that the quantification of admixtures by hybridisation is not better than with most alternative methods of species identification, as the strength of the signal depends on factors such as tissue origin and sample processing. © 1999 Society of Chemical Industry

Thin-layer chromatographic screening method for the tranquillizers azaperone, propiopromazine and carazolol in pig tissues

A procedure is described for the detection of azaperone, propiopromazine and carazolol in pig muscle, liver and kidney tissue. The method comprises extraction from an alkaline tissue homogenate with diethyl ether, followed by cleaning up and concentration of the extract on a silica gel solid-phase extraction column. Two-dimensional thin-layer chromatography on a silica plate was used for the detection of the tranquillizers. Detection levels were 25 micrograms kg-1 for propiopromazine, 50 micrograms kg-1 for azaperone (or its metabolite azaperol) and 125 micrograms kg-1 for carazolol. In pigs treated with the usual doses the presence of propiopromazine and azaperol could be established in kidney tissue 8 h after administration, whilst in injection sites all three tranquillizers could be detected.

Rapid species identification in meat by using satellite DNA probes

A fast procedure for species identification in heated meat is described. Deoxyribonucleic acid (DNA) was isolated from meat samples by an alkaline extraction method and hybridized to a conjugate of a specific oligonucleotide and alkaline phosphatase. The oligonucleotide probes are based on satellite DNA, tandem-repeated sequences, which are highly specific for species. Probes are developed for the identification of meat from cattle, sheep/goat, horse, deer, pig, chicken and turkey. Differentiation between closely related species like chicken and turkey is possible. Admixtures of 1–5% of meat of one species in another could be detected. The complete assay of up to 50 samples takes 4 h. Heated meat samples could be analysed.

Rapid sample preparation method for the determination of chloramphenicol in swine muscle by high-performance liquid chromatography.

A simple and rapid sample preparation method for the determination of chloramphenicol in swine muscle tissue at the 10 micrograms/kg level is described. The method comprises sonication-aided extraction with ethyl acetate, addition of hexane to the extract and cleaning up and concentration of the extract on a small column packed with silica gel. Analysis was performed by high-performance liquid chromatography on a ChromSep column with ChromSpher C8 using acetonitrile-sodium acetate buffer as the mobile phase. Detection was performed at 280 nm. Mean recoveries from spiked muscle samples were 79 +/- 3% (10-50 micrograms/kg). The distribution of chloramphenicol in different muscle and fatty tissues from a pig to which a single dose of chloramphenicol was administered was also investigated.

Analysis of chloramphenicol residues in swine tissues and milk: comparative study using different screening and quantitative methods.

Both screening and quantitative methods for chloramphenicol residues in swine tissues and milk were compared, using samples from animals treated with chloramphenicol. For screening purposes a previously developed streptavidin-biotin enzyme-linked immunosorbent assay and a commercially available immunochemical card test were used. For quantitative purposes two previously developed high-performance liquid chromatographic procedures were applied using antibody-mediated clean-up and solid-phase extraction. Some improvements in both methods were also described. The results obtained with the screening tests and those obtained with the quantitative methods correspond well with each other. Using a combination of these methods, an effective control of residues of chloramphenicol can be performed in milk from the 1 microgram/kg level and in swine tissues from the 10 micrograms/kg level.

An enzyme-linked immunosorbent assay for the determination of chloramphenicol using a monoclonal antibody Application to residues in swine muscle tissue

ZusammenfassungEs wird eine kompetitive Methode „ELISA” (enzyme-linked immunosorbent assay) mit monoklonalen Antikörpern zur Bestimmung von Chloramphenicol-Rückständen (CAP) in Schweinefleisch beschrieben. Für Standardlösungen wurde als Nachweisgrenze 25 ng ml−1 festgestellt (=2.0 ng CAP). Die sehr große Spezifität dieser monoklonalen Antikörper für CAP drückt sich durch die unwichtigen Kreuzreaktionen mit anderen antimikrobiellen Substanzen und mit strukturell verwandten Verbindungen aus. Zur Probenaufarbeitung wurde das gleiche Schnellverfahren benutzt wie bei der Hochleistungs-Flüssigchromatographie. Rückstände > 5 μg kg−1 können im Schweinefleisch leicht bestimmt werden. Bei Zusatzmengen von 10, 15, 25 und 50 μg CAP pro kg sind die Wiederfindungsraten gut. Die Ergebnisse mit der ELISA-Methode wurden mit HPLC bestätigt.SummaryA competitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody is described for the determination of chloramphenicol (CAP) residues in swine muscle tissue. The limit of detection of standard solutions is established to be 25 ng ml−1 = 2.0 ng CAP per well). The very high specificity of the monoclonal antibody for CAP is expressed by the insignificant cross-reactivity with other antimicrobial agents and with structurally related compounds. By means of the rapid sample preparation method described earlier for the CAP determination using high-performance liquid chromatography (HPLC), residue levels of CAP in swine muscle tissue above 5 μg kg−1can be easily quantitated. The muscle samples show good recovery percentages at 10–50 μg kg−1 spiking levels. The results obtained by the ELISA method were confirmed by HPLC.

Simultaneous determination of malachite green and its metabolite leucomalachite green in eel plasma using post-column oxidation

A rapid HPLC method with solid-phase extraction (SPE) clean-up for malachite green (MG) and leucomalachite green (LMG) in eel plasma was developed. MG and LMG were extracted with a buffered methanolic solution. The extract was subjected to aromatic sulphonic acid SPE. MG and LMG were eluted from the SPE column with methanol after a treatment with ammonia gas. The reconstituted eluate was analyzed on a Chromspher B column with acetonitrile-ion-pair buffer (pH 4.0) (6:4, v/v) as the mobile phase and detection at 610 nm after post-column oxidation with PbO2. The average recoveries for MG and LMG over the linear range of applicability (20-2500 ng/ml) were 82 +/- 1% and 83 +/- 1%, respectively. The limits of quantification were 5.0 micrograms/l for MG and 0.9 micrograms/l for LMG.