A Strategy for Molecular Species Detection in Meat and Meat Products by PCR-RFLP and DNA sequencing using mitochondrial and chromosomal genetic sequences
Dietrich Maede
Landesamt fuer Verbraucherschutz Sachsen-Anhalt, Freiimfelder Str. 66/68, D-06112 Halle, Germany
Fon ++49 345 5643 222
Fax ++49 345 5643 439
Email: dietrich.maede@hal.lav.ms.lsa-net.de
Abstract:
Molecular species detection in food has become common in the last 10 years. The methods are sensitive enough to detect small, but relevant, amounts of one species in composed food. We have developed a strategy for detecting different animal species in food by molecular means. This strategy uses a combination of published PCR systems and new developed PCR primer systems for the detection of porcine, bovine, ovine, avian, cervine and equine DNA by PCR followed by restriction analysis (PCR-RFLP). In some cases, analysis is completed by DNA sequencing. The species detection system includes an amplification control and so is in accordance with the relevant food standards.
Keywords: species detection - meat - meat products - PCR - PCR-RFLP - DNA sequencing
Introduction
Abbreviations:
PCR - polymerase chain reaction, RFLP - restriction fragment length polymorphism,
REase - restriction endonuclease, bp - base pairs
Molecular methods for species detection are widely applied in food diagnostics. These methods, based on Polymerase Chain Reaction and subsequent restriction analysis or DNA sequencing, allow a sensitive and specific detection of all relevant animal species [1, 2, 3, 4, 5, 6, 7, 8, 9, 10].
Species detection strategies can be separated into two major principles. The first includes methods based on universal genetic elements located in the mitochondrial Cytochrome b (cyt b ) Gene [3, 4, 5, 6, 10]. The cyt b - gene is present in multiple copies per cell; so very sensitive species detection methods can be designed based on this gene. The mitochondrial nucleic acid sequences share homologies between different vertebrate species. This allows us to locate PCR primers in the conserved regions of cyt b spanning over a variable region of the gene. With one set of PCR primers, cyt b derived DNA fragments of all relevant species can be generated [3, 5]. In this system, the PCR product of all species has the same length. The determination of the animal species is done by restriction analysis of the PCR products which leads to different fragment sizes in different species. Another system used one unique forward primer and species adapted reverse primers located in different positions in the cyt b - gene [6]. The amplified PCR products differ in their length; the length in base pairs (bp) is specific for the species from which the PCR product was generated.
The second principle is that species detection is based on the amplification of specific chromosomal genetic elements [2, 11, 12, 13], which target either low copy number genes or repetitive genetic elements. Chromosomal detection systems can be used for quantification [13] if the copy number of the target genes is stable.
According to European standards in molecular microbiology [14] and molecular detection of genetically modified organisms, [15] it is necessary to verify the PCR products generated. Reliable methods used to verify PCR products are restriction analysis with at least two restriction endonucleases (REases), probe hybridisation, or DNA sequencing [16].
Sensitive molecular species detection relies on the ability to amplify the nucleic acids extracted. Amplification can be inhibited by co-extracted food ingredients [17] so all molecular detection systems should include an amplification control [14, 15, 16]. DNA amplification can also be influenced by DNA deterioration due to food processing [1, 18]. In species detection, the amplification of universal genetic elements [3] allows one to check for inhibition and for deterioration of DNA as well.
The previously published papers are focussed on one methodology for species detection. In this work we describe a combination of mitochondrial and chromosomal detection systems. Using this combination of methods, it is possible to detect all relevant species. Routine analysis using this strategy has been used for more than five years.
Materials and methods
Analytical scheme
A flowchart of the general strategy is diagrammed in Figure 1. The application of primer systems and REases depends on the expected animal species of the product, based on the food labelling or on food standards and guidelines. If unexpected results in the first evaluations of data occur, the strategy is extended by using additional primer systems or REases. The analysis is completed by DNA sequencing. In case of ambiguous RFLP results, sequence data are compared with sequences of public nucleic acid databases. Sequencing is further applied to authenticate the meat of exotic animals.
The system is based on the detection of the mitochondrial cyt b - gene using universal PCR primers cytb1 and cytb2 [3]. We found that in the presence of porcine DNA, bovine and avian DNA is amplified with decreased efficiency. For several species, additional primer systems need to be applied. Either specific chromosomal systems, e.g. for cattle and sheep, or the cyt b derived primers with altered sequence in case of the presence of Equidae, Cervidae and poultry (Table 1) are necessary. Species determination is done by restriction analysis. The primers cytb1/2 [3], mel1/2 [19], cerv1/2 and equus1/2 share the same position in the cyt b - gene. The PCR product generated with these primer systems has the same length of 359 bp. The RFLP pattern of the species adapted primers corresponds to that of the cytb1/2 primers.
Place Figure 1 here:
Flowchart of the strategy for molecular species detection in meat and meat productsDNA extraction
DNA is extracted using a modified CTAB protocol [20, 21, 22]
PCR Primers and general reaction conditions
. Briefly, 100 g of food products were homogenised using a Grindomix® grinder (Retsch, Germany). From the resulting homogenised sample, all DNA extraction was done in duplicates. Two portions of each 200 mg of homogenised sample were used for DNA extraction. If pieces of meat were to be analysed, 2 x 200 mg of material were taken from the inside without homogenisation. Clean instruments were used for each sample to prevent cross contamination. 1.5 mL CTAB-extraction buffer (c(CTAB) = 20 g/L, c(NaCl) = 1.4 mol/L, c(TRIS) = 0.1 mol/L, c(Na2EDTA) = 0.02 mol/L, pH 8.0) and 10 µL Proteinase K solution (c = 20 mg/mL) were added to the sample. The samples were incubated at 60 °C under current agitation for at least 3 h, normally overnight, until animal tissue was completely digested. In some cases, particles of spices still remained visible after digestion, without having had an influence on the following analysis. Samples were centrifuged for 10 min at 13000xg. The supernatant was transferred into a new vial, 0.75 mL of chloroform was added and shaken vigorously. The samples were centrifuged at 13000xg for 5 min, and the upper phase was transferred into a new vial. The volume of the solution was determined. The amount of two volumes of the transferred solution of CTAB precipitation buffer (c(CTAB) = 5 g/L, c(NaCl) = 0.04 mol/L) was added and incubated 60 min at room temperature without agitation. The samples were centrifuged for 15 min at 13000xg, then the supernatant was discarded, and the pellet was resuspended in 350 µl NaCl solution (c(NaCl) = 350 mol/L). 350 µL of chloroform was added; the samples were vortexed and centrifuged for 10 min at 13000xg. The upper phase was transferred into a new vial; 0.6 volumes of isopropanol were added for nucleic acid precipitation. After 20 min incubation at room temperature the samples were centrifuged 10 min at 13000xg. The supernatant was discarded, the pellet was washed with 500 µL ethanol solution (c = 70%) and resolved in 200 µL 0.1 TE buffer (c(TRIS) = 1 mmol/L, c(Na2EDTA) = 0.1 mmol/L, pH 8.0) as stock solution. 5 µL of a 1 to 10 dilution of the DNA stock solution was used as template for PCR.All the PCR primers that were used are listed in Table 1. The primers were all synthesised by TIB MOLBIOL (Berlin, Germany), reactions were carried out on ABI 9700 thermal cycler (Applied Biosystems, Darmstadt, Germany) using the 9600 mode for ramping times. The reactions were run in duplicate in a reaction volume of 25 µL. Cycling conditions are given in Table 2. 5 µL of the PCR products are separated on a 2% agarose gel in TAE buffer using a 100 bp fragment length marker (MBI Fermentas, St. Leon-Roth, Germany).
Place Table 1 here:
PCR primer names and sequencesPlace Table 2 here:
Reaction conditions of the PCR systemsAmplification control and pig detection
The first reaction carried out was the Universal Mitochondrial Detection System. This system was used as amplification control to verify the ability to amplify the DNA extracted. The system was also used for the detection of pork and food products containing porcine entrails. The PCR was done using the PCR primers cytb1 and cytb2 targeting the mitochondrial cytochrome b gene. The resulting PCR product has a length of 359 bp. The reaction conditions were as follows: 2.5 µL 10x PCR Buffer containing c(MgCl2) = 15 mmol/L, resulting in a final concentration of c(MgCl2) = 1.5 mmol/L, (Qiagen, Hilden Germany), c (dATP, dCTP, dGTP, dTTP) = 200 µmol/L each (Roche Diagnostics, Mannheim, Germany), 0.5 µmol/L of each primers cytb1 and cytb2, 0.625 U thermostable Taq DNA polymerase (Hot Star Taq Polymerase, Qiagen, Hilden, Germany), and 5 µL of a 1:10 dilution of the extracted DNA, representing 10 to 50 ng DNA.
The detection of porcine DNA was accomplished by RFLP of the cytb1/2 PCR product with REases AluI und HinfI (table 3)
Cattle, sheep and goat detection
For detection of bovine DNA, the primers MM1 and MM2 were used. This primer pair specifically amplifies bovine DNA resulting in a product with a length of 270 bp. Ovine and caprine DNA was amplified with the primers MM1 and MM3 which results in a PCR product of 218 bp. The primer concentration was 0.5 µmol/L for each primer. The other reagents were used in the same concentrations as described above.
Poultry, horse, and deer detection
The poultry detection system used the primer pair mel1 and mel2 [19]. These oligonucleotides were located at the same positions as cytb1/2 but their sequences were adapted to Aves. Deer detection was done using the adapted oligonucleotides cerv1 and cerv2; in the horse adapted system the PCR primers equus1 and equus2 were used. Each oligonucleotide was used in the concentration of 0.5 µmol/L; the other reagents were the same as described above. The PCR products of the primers mel1/2, cerv1/2 and equus1/2 are of 359 bp length.
Verification of PCR products by RFLP
PCR products are verified by restriction analysis and DNA sequencing. Restriction analysis was done with the following conditions: To 10 µL of the PCR product 1 µL of the REase (i.e. 5 to 10 units), and 2 µL of the buffer supplied with the enzyme was added. Water was added up to a final volume of 20 µL. The reaction assays were incubated for 3 h at 37 °C for all REases except for TaqI; TaqI assays were incubated at 65 °C. The restriction fragments were separated on 3% agarose gel in TAE buffer using a 50 bp fragment length marker (MBI Fermentas, St. Leon-Roth, Germany). Species specific restriction fragments of cytb1/2 PCR, mel1/2 PCR, cerv1/2 PCR, and equus1/2 PCR are listed in Table 3. Restriction fragments of the cattle specific MM1/2 PCR and sheep/goat specific M1/3 PCR are summarised in table 4.
Place Table 3 here:
Restriction fragments in the published mitochondrial Cytochrome b sequences according GenBank
Place Table 4 here:
Restriction fragments of the cattle specific MM1/2 PCR and sheep/goat specific MM1/3 PCRVerification of PCR products by DNA sequencing.
As an alternative to RFLP, DNA sequencing of the PCR product was performed. PCR products were sequenced directly after purifying them using QIAquick® PCR Purification Kit (Qiagen, Hilden, Germany). If by-products were visible in the agarose gel, the band of the expected length was excised with a sterile scalpel; DNA was extracted from the agarose block with the QIAquick® Gel Extraction Kit (Qiagen, Hilden, Germany).
After purification, the PCR products were directly sequenced using the above mentioned oligonucleotides as sequencing primers with the BigDye Terminator V 1.1 Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany); the analysis was carried out on an automated DNS sequencer (ABI Prism 310 Genetic Analyzer, Applied Biosystems, Darmstadt, Germany). Resulting nucleic acid sequences were aligned with the help of Sequence Navigator software (Applied Biosystems, Darmstadt, Germany). The sequences were compared to sequences in GenBank® using the computer algorithm BLAST 2 [23].
In general, DNA sequencing was done in products consisting of only one species which could not clearly be verified by RFLP. In products consisting of two species, DNA sequencing was done after restriction analysis. This was applicable in species which differ in their RFLP pattern: The PCR product of one species was cut with an enzyme which does not cut the PCR product of the other species. The undigested PCR product of 359 bp was excised from the gel and could be sequenced. The sequence reaction was not affected by DNA of the other species present in the food item.
Results and discussion
Amplification control
Animal species in food products are detected using a combination of methods. The system is able to detect small amounts of one species as long as the DNA is not deteriorated [1], and no inhibition of the enzymatic reaction has occured [17]; therefore, a requirement of molecular diagnostics is that one must verify the amplificability of the DNA extracted. To test the amplificability, the cytb1/2 primer system [3] is used. Due to the location in conserved regions of cyt b, these oligonucleotides generate PCR products of all relevant species. The cytb1/2 PCR system is very robust and sensitive. Non-amplification of the DNA extracted can be either the result of the presence of co-extracted inhibiting substances or the result of deteriorated DNA by food processing. PCR inhibition is not considered as being an important problem in species detection. PCR inhibition was not monitored during the last years when the CTAB extraction protocol was applied [20, 21, 22].
Deterioration of DNA which could occur in sterilized meat products is reported by non-amplification of the extracted DNA [3, 18, 25]. It should be noted that care must be taken during DNA extraction and reaction set up to prevent contamination with DNA from the environment. This could result in false positive reactions. To monitor proper analysis, we perform one negative extraction control according to [14] every five samples. These controls should be negative in the cytb1/2 assay. It should be mentioned that some commercially available Taq DNA polymerase preparations were contaminated with DNA. This resulted in false positive reactions which were detectable in a negative PCR control [14] (data not shown).
Cytochrome b based systems
Detection of pig
The cytb1/2 system serves both as amplification control and detection system for the species Sus scrofa. REases AluI and HinfI are used for the detection of porcine DNA. Even in the presence of 1% pork in meat products the characteristic porcine fragments are detectable (data not shown). HinfI does not cut cytb1/2 derived PCR products of domestic pigs [3]. The reported HinfI restriction site in wild boar [3] does not occur frequently and therefore, this system is not applicable for differentiation between domestic pigs and wild boars. This corresponds to the findings of [24].
Application of the cytb1/2 primer system as universal species detection system
The cytb1/2 system is used to identify unknown samples. Due to the primer location in a conserved region, the PCR of all species results in a detectable PCR product which can be further analysed, either by RFLP or by DNA sequencing.
Restriction analysis (RFLP) of universal PCR products provides several advantages in species diagnostics when used for verification of PCR products: It is fast and does not require the use of specific equipment. Furthermore, non-expected species in food products can be detected by RFLP by the presence of additional DNA fragments.
In Table 3 a compilation of theoretically occurring restriction sites is given. The compilation is based on mitochondrial DNA sequences in public nucleic acid databases (GenBank). In some species, especially sheep and deer, some of the restriction sites are altered, forming a different RFLP pattern [3]. It is, therefore, necessary to use at least two REases.
RFLP does not always lead to unambiguous results, this requires further analysis. We used DNA sequencing to complete the analysis. Direct sequencing of the cytb PCR product, and comparison with sequences in nucleic acid databases, is (except for the limitations mentioned below) the most comprehensive method for species detection or species identification. Sequence information of the cyt b - gene from nearly all mammals is available because this gene is widely used for phylogenetic analysis. We applied DNA sequencing to composed meat products after restriction analysis. In this case, a food sample consisted of two species. One was pig and the other species was not clearly attributable. After digest with AluI, the uncut fragment was excised from the gel and sequenced. The other species turned out to be fallow deer.
Species adapted Cytochrome b primer systems for detection of poultry, horseflesh and venison
Using the universal cytb1/2 primer set, competition of primer binding sites in mixtures can occur leading to superior amplification of one species compared to the other species present in the food item. In the presence of pork, we found that small amounts of some species, e.g. chicken, turkey, beef, roe deer, do not generate detectable PCR products in composed meat products. This can be attributed to a competition in PCR with the cytb1/2 primers. Porcine DNA is amplified with more efficiency which can lead to non-detection of minor amounts of other species present.
There are several ways to overcome the competitive conditions leading to non-detection of one species in the presence of another species. Matsunaga et al. [6] used a system with one single universal forward primer and several species specific reverse primers in a multiplex PCR system. In this system, differentiation between species is achieved by analysing the different lengths of PCR products. In our hands, the system was not sensitive enough. We attribute this to the high length of the PCR products. The length of PCR products should not exceed 400 bp because of degradation of DNA during food processing [1, 18, 25]. This is especially important in the investigation of heated meat products in which DNA is degraded to fragments of 100 to 200 bp of length. This system according to [6] does not possess a verification system as required in food analysis [14, 15, 16].
We, therefore, decided to modify the cytochrome b derived primers making them more specific. The primers mel1 and mel2 are avian adapted primers [19]. The advantage of this system is that primer binding sites are the same as the cytb1/2 primers and so the restriction sites are the same (see Table 3). Poultry is determined by digestion with RsaI which results in typical fragments. No other animals used for food production have the same RsaI pattern as turkey and chicken. Turkey is verified by its PstI restriction site. Chicken can be further specified by HinfI or HaeIII. The mel1/2 system is appropriate for detection of goose and duck as well.
We introduced two species adapted primer sets for amplification of cervidae (cerv1 and cerv2) and equidae (equus1 and equus2). Further characterisation of the PCR product is done with the REases HaeIII, TaqI, RsaI or HinfI.
Using the species adapted primer systems, 1% and less of chicken, turkey and venison is detectable. The equus1/2 system was applied using material from a ring test organised by the Federal Institute for Risk Assessment (BfR) (formerly BgVV). Using DNA from samples supplied in this study, 0.1 % horsemeat is detected as displayed in figure 2.
Place Figure 2 here:
Detection of horsemeat in pork using the oligonucleotides equus1/2 and REase HinfISpecific PCR systems based on chromosomal sequences for detection of cattle, sheep and goat
For detection of bovine and ovine DNA, the specific primer systems MM1/MM2 and MM1/MM3 are used. This system combines one forward primer MM1 with specific reverse primers MM2 resp. MM3. The primers MM1 and MM2 allow a detection of at least 1% beef (Figure 3). The verification of the PCR product is done using the REases HaeIII and AluI. Digest with HaeIII results in fragments of 211 and 59 bp; digest with AluI results in fragments of 136, 102, 32 bp (data not shown).
Chromosomal genetic elements in species that are less commonly used for consumption are not that widely sequenced in contrast to mitochondrial sequences; data of chromosomal genes in public databases is available only for commercially important species. Sequence data from the majority of wildlife species is still lacking which can lead to PCR positive results in non-target, but related, species. A practical evaluation of species specific primers is necessary as well as further characterisation of the PCR products.
Related species like buffalo and red deer are amplified with the cattle system, because of the comparably high sequence homology at the primer binding sites (data not shown). Differentiation is done by RFLP as described (table 4).
It shall be mentioned that care has to be taken when establishing reaction conditions for the MM1/MM2 (cattle detection system) and MM1/MM3 (sheep/goat detection system). The annealing temperature should be optimised with a lot of precision, as a reduced annealing temperature will result in sheep being detected with the cattle system and vice versa. To verify the specificity in every reaction, ovine DNA serves as one of the negative controls in the MM1/MM2 system, whereas bovine DNA is used as negative control in the MM1/MM3 system. Ovis aries and Capra hircus do not differ in primer binding sites. The two differences in the PCR product can only be found by DNA sequencing. Another system for the detection of caprine DNA is published [26]. PCR based detection of beef using the specific primer system described is very sensitive. As seen in Fig. 3, 1% beef in pork is detected with high efficiency. Minor contaminations of a species in food should not lead to inadequate legal consequences. Adventitious traces of DNA cannot be excluded in the production of meat products. To differentiate traces from technologically required ingredients, a positive result with a specific detection system requires further analysis. In our laboratory, detection of beef with the primer systems described without a corresponding declaration is verified either by a quantitative analysis [13] or a commercial ELISA test (ELISA-TEK Cooked Meat Species Test Kit, Transia, Ober-Mörlen, Germany). According to the manufacturer, the ELISA system has a sensitivity of about 1%. Quantitative analysis or ELISA tests ensure that the proportion of beef or beef derived material in most food products is relevant.
Place Figure 3 here:
Detection of beef in pork using the oligonucleotides MM1/2 and REase HaeIIIApplication of the molecular detection strategy in proficiency studies
The methods have been tested regularly in commercially organised proficiency studies (LVU, Herbolzheim, Germany) using tinned sausage as test material. The results are displayed in table 5. There was neither a false positive nor a false negative result using the combination of primer systems described. The results demonstrate that sensitive and specific species detection in food is possible using the combination of methods. We have more than five years of experience with these systems in routine diagnostics. Up to now, species detection was possible in each food product from which amplificable DNA was extracted. It should be mentioned that in several samples, DNA sequencing was needed to complete the analysis. This is the case when restriction sites are altered in the amplified region which occurs from time to time.
Place Table 5 here:
Results of proficiency studies for species detection in tinned sausageAcknowledgements
I would like to thank Dr. Regine Stark and Mrs. Stefa Kahle for making this work possible and also for their critical and helpful suggestions. I acknowledge the excellent technical assistance of Christine Degner, Edith Ronniger, Marion Grosser, Ilona Balogh, Sigrid Niendorf, Birgit Jahn and Katja Trübner (LAV Sachsen-Anhalt) for their excellent technical assistance over the years.
References
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG., Smith JA., Struhl K (1988): Current Protocols in Molecular Biology. John Wiley & Sons, Inc. ISBN 0-471-50338-X
- Rogers S, Bendich A (1985): Plant Mol Biol 5:69-76
22. Anonymus (2005) Foodstuffs . Methods of analysis for the detection of genetically modified organisms and derived products - Nucleic acid extraction. International Standard EN ISO 21571:2005
23. Altschul, SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DL (1997): Nucleic Acids Res 25:3389-3402
24. Butschke A (2003): Untersuchungen zur Differenzierung der domestizierten und der Wildform von Sus scrofa in Lebensmitteln. Thesis. TU Berlin. URL:
25. Poser R, Detsch R, Fischer K, Müller WD, Behrschmidt M, Schwägele F (2000) Fleischwirtschaft 8/2000:87-89
26. Altmann K, Binke R, Schwägele F (2004): Fleischwirtschaft 2/2004:115
Figure 1
Flowchart of the strategy for molecular species detection in meat and meat products.
Table 1
Table 1
PCR primer names and sequences. The target sequence and the resulting specificity is displayed in column 4. The gene, from which the primer is derived from, including the GenBank accession number, is given in column 5. The location of the primer binding sites is given in column 3.
Sequence (5´® 3´) | Localisation | Target Sequence and Degree of Specificity | Sequence Name and Accession Number | Reference | |
cytb1 | cca tcc aac atc tca gca tga tga aa | 15417-15436 | mitochondrial, universal primers | Sus scrofa mitochondrion, AF034253.1 | [3] |
cytb2 | gcc cct cag aat gat att tgt cct ca | 15769-14744 | |||
mel1 | cca tcc aac atc tcc gct tga tga aa | 14975-15000 | mitochondrial, adapted to poultry | Gallus gallus gallus mitochondrion, AP003322.1 | [19] |
mel2 | gcc cct cag aat gat att tgt ccc ca | 15333-15308 | |||
cerv1 | cca tca aat att tca tcm tga tga aa | 70-95 | mitochondrial, adapted to Cervidae | Cervus elaphus cyt b gene, AJ000021.1 | this work |
cerv2 | gct cct cag aat gat att tgt cct ca | 428-403 | |||
equus1 | ccc tca aac att tca tca tga tga aa | 14257-14282 | mitochondrial, adapted to Equidae | Equus caballus mitochondrion, X79547.1 | this work |
equus2 | gct cct caa aag gat att tgg cct ca | 14615-14590 | |||
MM1 | ctg ctc tgc ctg ccc tgg act | 1003-1023 (Bos taurus) 1311-1331 (Ovis aries) 681-701 (Capra hircus) | Chromosomal growth hormone gene, specific for Bovinae and Caprinae | Bovine growth hormone gene, M57764.1 Ovine growth hormone gene, M37310.1 Capra hircus growth hormone gene, partial, AF534522 | this work |
MM2 | ccc cgt ttc tgc tcc cct aac c | 1272-1251 | |||
MM3 | ggt cct cag ttc cct ccc att gt | 1528-1510 (Ovis aries) 899-875 (Capra hircus) |
Table 2
Table 2
Reaction conditions of the PCR systems. Temperature and time profiles are optimised for the ABI 9700 thermal cycler.
Step | cytb1/2 | MM1/2 and MM1/3 | mel1/2 | cerv1/2 and equus1/2 | |
Initial denaturation | 95°C 15 min | 95°C 15 min | 95°C 15 min | 95°C 15 min | |
40 cycles | denaturation | 94°C 30s | 94°C 30s | 94°C 30s | 94°C 30s |
primer annealing | 55 °C 1min | 71°C 1 min | 50°C 30s | 60°C 30s | |
primer extension | 72°C - 45 s | 72°C 45 s | 72°C 45 s | ||
Final extension | 72°C - 7 min | 72°C - 7 min | 72°C - 7 min | 72°C - 7 min | |
Table 3
Table 3
Restriction fragments in the published mitochondrial cytochrome b sequences according to GenBank. The 1st fragment is located at the 5´end of the PCR product including the primer cytb1, mel1, cerv1, or equus1, resp., depending on the detection system applied. The 2nd fragment is located at the 3´end including the primer cytb2, mel2, cerv2 or equus2. The following fragments are located in between the two primers. Location of primers is important when using fluorescence labelled primers and an automated fragment analysis detection system.
Enzym and recognition sequence | HaeIII gg/cc | HinfI g/antc | Pst I ctgca/g | Mbo I /gatc | AluI ag/ct | RsaI gt/ac | TaqI t/cga | Xba I t/ctaga | |||||||||||||||||
Equus caballus (horse) | 74 | 21 | 159 | 105 | 44 | 81 | 234 | 359 | 359 | 304 | 55 | 359 | 359 | 359 | |||||||||||
Ovis aries (sheep) | 74 | 126 | 159 | 63 | 296 | 359 | 244 | 115 | 359 | 359 | 359 | 359 | |||||||||||||
Capra hircus (goat) | 74 | 55 | 230 | 161 | 198 | 359 | 31 | 115 | 213 | 359 | 359 | 141 | 218 | 258 | 101 | ||||||||||
Bos taurus (cattle) | 74 | 285 | 44 | 198 | 117 | 359 | 359 | 190 | 169 | 359 | 359 | 258 | 101 | ||||||||||||
Sus scrofa (pig) | 74 | 132 | 153 | 359 | 359 | 244 | 115 | 115 | 244 | 359 | 141 | 218 | 359 | ||||||||||||
Capreolus capreolus capreolus (roe deer) | 74 | 126 | 159 | 44 | 315 | 359 | 244 | 115 | 359 | 359 | 359 | 258 | 101 | ||||||||||||
Dama dama dama (fallow deer) | 74 | 285 | 161 | 198 | 359 | 244 | 115 | 304 | 55 | 359 | 168 | 191 | 258 | 101 | |||||||||||
Cervus elaphus (red deer) | 74 | 126 | 159 | 44 | 315 | 359 | 244 | 115 | 359 | 238 | 121 | 141 | 191 | 27 | 258 | 101 | |||||||||
Alces alces (elk) | 359 | 161 | 198 | 359 | 247 | 78 | 37 | 359 | 359 | 170 | 189 | 261 | 98 | ||||||||||||
Equus asinus (donkey) | 74 | 126 | 159 | 44 | 198 | 117 | 359 | 359 | 359 | 359 | 359 | 359 | |||||||||||||
Oryctolagus cuniculus (rabbit) | 44 | 132 | 30 | 153 | 233 | 126 | 359 | 244 | 115 | 359 | 359 | 359 | 359 | ||||||||||||
Lepus europaeus (hare) | 227 | 132 | 161 | 72 | 126 | 359 | 62 | 297 | 109 | 250 | 359 | 359 | 359 | ||||||||||||
Gallus gallus domesticus (chicken) | 74 | 21 | 159 | 105 | 161 | 188 | 10 | 359 | 359 | 359 | 149 | 210 | 359 | 359 | |||||||||||
Meleagris gallopavo (turkey) | 74 | 285 | 161 | 198 | 102 | 257 | 359 | 359 | 149 | 109 | 101 | 359 | 359 | ||||||||||||
Anas platyrhynchos (duck) | 55 | 286 | 18 | 161 | 198 | 359 | 31 | 328 | 359 | 359 | 359 | 359 | |||||||||||||
Cairina moschata (moschus duck) | 74 | 285 | 161 | 198 | 359 | 359 | 359 | 154 | 205 | 141 | 131 | 87 | 359 | ||||||||||||
Anser domestica (goose) | 74 | 126 | 159 | 359 | 359 | 359 | 130 | 229 | 154 | 205 | 359 | 359 | |||||||||||||
Ursus arctos (brown bear) | 74 | 21 | 159 | 105 | 63 | 296 | 359 | 31 | 328 | 101 | 258 | 176 | 121 | 62 | 359 | 359 | |||||||||
Felis catus (cat) | 74 | 21 | 11 | 250 | 44 | 81 | 40 | 77 | 117 | 359 | 359 | 190 | 49 | 120 | 214 | 145 | 359 | 359 | |||||||
Canis familiaris (dog) | 233 | 136 | 54 | 296 | 9 | 359 | 31 | 115 | 213 | 85 | 244 | 30 | 284 | 33 | 42 | 359 | 359 | ||||||||
Rattus norvegicus (rat) | 359 | 359 | 359 | 244 | 81 | 34 | 359 | 59 | 33 | 267 | 359 | 359 | |||||||||||||
Mus musculus (mouse) | 359 | 44 | 315 | 359 | 244 | 115 | 359 | 281 | 78 | 141 | 218 | 359 | |||||||||||||
Homo sapiens (man) | 248 | 21 | 90 | 161 | 198 | 359 | 53 | 191 | 115 | 359 | 359 | 141 | 218 | 359 | |||||||||||
Struthio camelus (ostrich) | 227 | 132 | 233 | 126 | 359 | 31 | 328 | 359 | 149 | 205 | 5 | 359 | 359 | ||||||||||||
Table 4
Table 4
Restriction fragments of the cattle specific MM1/2 PCR and sheep/goat specific MM1/3 PCR. In this table fragments of closely related species are also listed. DNA of closely related species could be amplified when reaction conditions are not stringent enough. Generation of PCR products of non-listed species cannot be excluded because of the lack of data for many wildlife animals. RFLP is always necessary to verify the nature of the PCR product generated.
HinfI | HaeIII | AluI | |||||||||
MM1/2 (cattle specific) | cattle | 160 | 92 | 18 | 211 | 59 | 136 | 102 | 32 | ||
buffalo | 160 | 92 | 18 | 166 | 104 | 136 | 102 | 32 | |||
red deer | 160 | 91 | 18 | 184 | 43 | 26 | 16 | 135 | 102 | 32 | |
sheep | 160 | 90 | 18 | 209 | 59 | 134 | 133 | ||||
MM1/3 sheep/goat specific | sheep | 160 | 40 | 18 | 159 | 59 | 134 | 84 | |||
red deer | 160 | 40 | 18 | 133 | 43 | 26 | 16 | 102 | 32 | 84 | |
buffalo | 160 | 40 | 18 | 166 | 54 | 102 | 32 | 86 | |||
cattle | 160 | 43 | 18 | 162 | 59 | 102 | 32 | 87 | |||
Figure 2
Figure 2
Detection of horsemeat in pork using the primer system equus1/2 and REase HinfI. HinfI cuts the PCR product of horse in fragments of 234, 81, and 44 bp. Lane 1 and 9: 50 bp marker; lane 2: 100% pork; lane 3: 0,1 % horsemeat in pork; lanes 4: 1% horsemeat in pork; lane 5: 2% horsemeat in pork, lane 6: 5% horsemeat in pork, lane 7: 10% horsemeat in pork; lane 8: 100% horsemeat. Equine DNA is amplified superior to other species with the equus1/2 system. Because of the sequence homology in the conserved region of the cytochrome b gene where the primers are located, DNA from other animals is amplified if no equine DNA is present; so porcine DNA is amplified with the primers equus1/2 (lane 2). The resulting but the RFLP pattern is different, HinfI does not cut the porcine PCR product.
Figure 3
Figure 3
Detection of beef using the oligonucleotides MM1/2 and REase HaeIII. Lanes 1 and 8: 50 bp marker; lane 2: 1% beef in pork; lane 3: 2% beef in pork; lane 4: 5% beef in pork; lane 5: 100% beef; lane 6: 97% beef, 1% pork, 1% chicken, 1% turkey; lane 7: 85% beef, 5% pork, 5% chicken, 5% turkey
Table 5
Table 5
Result of proficiency studies for species detection in tinned sausage. Each year two samples were analysed; these samples were nominated A and B as demonstrated in the table. The composition of the samples varied from year to year and is given in column 2. The percentage of non-porcine material is given in brackets.
Year | Sample Consistence | Results |
2001 | A: pork, lard, pork liver, veal (1,4%), turkey (4%) | pig positive, cattle positive, sheep negative, chicken negative, turkey positive |
B: pork, lard, pork liver | pig positive, cattle negative, sheep negative, chicken negative, turkey negative | |
2002 | A: turkey | pig negative, cattle negative, sheep negative, chicken negative, turkey positive |
B: pork, lard, bacon rind, beef (0,8%), chicken (7,1%), mutton (5,3%) | pig positive, cattle positive, sheep positive, chicken positive, turkey negative | |
2003 | A: pork, lard, bacon rind, beef (0,7%), chicken (5%) | pig positive, cattle positive, sheep negative, chicken positive, turkey negative |
B: lard, bacon rind, beef (39%), turkey (5,9%) | pig positive, cattle positive, sheep negative, chicken negative, turkey positive | |
2004 | A: pork, lard, bacon rind, mutton (3%), turkey (5,9%) | pig positive, cattle negative, sheep positive, chicken negative, turkey positive |
B: pork, lard, bacon rind, beef (19%), chicken (9,5%), turkey (9,5%) | pig positive, cattle positive, sheep negative, chicken positive, turkey positive |
Figure 1
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http://edocs.tu-berlin.de/diss/2003/butschke_andreas.pdf