Biochemical Methods for Meat Species Identification : a Review

BIOCHEMICAL METHODS FOR MEAT SPECIES IDENTIFICATION : A REVIEW

LUMLEY I.D.

Laboratory of the Government Chemist, Teddington, UK

S-V.MP1

INTRODUCTION

The reliable identification of the species of origin of bulk meats, particularly after butchering, dicing or mincing, has been a problem faced by analysts for many years. Species identification is even more problematical in processed and heated products such as smoked and cooked sausages, canned and cooked meats. Identification of the major meat species present (by weight) has been possible but identification of low percentages of other meat species, which may be present in binary or ternary admixture, has been virtually impossible until recent years.

Traders or Customs and Health authorities may wish to confirm that consignments of meat and meat products being purchased or imported/exported are of the species declared, whilst enforcement authorities may wish to confirm that meats being purchased are of the nature, quality and substance demanded by the consumer and that label claims relating to composition are correct and not misleading. There have been various examples in the last decade of the substitution or dilution of an expensive meat species with meat from a cheaper species; horse and kangaroo substitutions make the headlines but substitution with 'acceptable' meats such as poultry and pork does occur depending upon price differentials at a particular time.

Gross substitution can occur where the analyst would be looking for high percentages (eg >10%) of non-declared meat species, whilst adventitious contamination may mean that the analyst is looking for low percentages of non- declared species, eg 1-10% (m/m); this could be in raw or cooked products. If the analyst reports the presence of one or more undeclared species in a product, the question invariably asked by the customer is "how much is present". Therefore when selecting analytical methods for meat species identification the analyst must consider:

is the method sufficiently specific and sensitive; can the method be applied to cooked or heated meats and meat products; can the method be used quantitatively.

Analytical Methods Available

Methods for species identification must detect or determine a natural component of meat which is a unique indicator of the species of origin, and lipids, proteins, and more recently DNA have provided the target for spéciation studies. In this paper the physical methods of lipid chromatography and electrophoresis of proteins are mentioned in overview only, the main methods under review being immunoassay and DNA analysis.

Lipid Analysis

Examination of the fatty acid isomer profile by capillary gas chromatography, determination of fatty acids at the 2- Position in triglycerides after enzymic hydrolysis and gas chromatography and examination of triglyceride profile hy gas chromatography and/or HPLC with mass detection can provide information which will allow tentative identification of a some meat species (eg horse, pork, kangaroo), but interpretation of data from raw meats in admixture is difficult. Interpretation of data from highly processed and cooked foods where more than one meat species is present is almost impossble.

Electrophoretic Separation of Characteristic Proteins

Many papiers have been published which describe the use of electrophoresis for the identification of meat spiecies.

1 Isoelectric focussing of soluble proteins has been used to characterise animal species, some in binary mixtures at the 2-5% level of addition, and electrophoretic separation of muscle lactate dehydrogenase, esterase isoenzymes and cratinine kinase have been used to identify animal species. Electrophoresis followed by immunoblotting can produce useful information and can unravel complex protein electrophoretograms.

There are several problems with the use of electrophoresis for meat speciation; it requires significant operator skill if reproducible results are to be obtained, not all laboratories have electrophoresis equipment, interpretation of results can be difficult for some species, particularly for meat products when two or more species are mixed, limited quantitative information is achievable, and this approach is not generally applicable to cooked or heated products. Despite these drawbacks electrophoresis can provide useful complimentary evidence of adulteration.

Immunoassay Procedures

Immunoassay procedures have been applied to problems of meat speciation for many years. In the 1890's they were used to identify horse proteins, in a 1942 text on food analysis (Nicholls) serological techniques were proposed as the best means of detection of horse meat in raw beef, and in the early 1950's the FDA in the USA used these techniques to detect beef adulteration with horse meat. Despite this long history of the application of immunoassay to meat speciation, robust methods have not been developed to meet all the needs of analysts.

Immunodiffusion Methods

Immunodiffusion methods are ideal for routine species identification of raw meats. They are simple to use, inexpensive, provide a relatively rapid result and some commercial kits are available which contain all the reagents required. Many papers have been published which give details of the production of species specific antisera by immunisation of various animals with a range of different and often unidentified antigens extracted from meats. Hayden (1978) used agar gel immunodiffusion (AGID) to detect serum proteins from pig, horse and rabbit flesh in extracts of ground beef which contained the flesh of these species. Antisera were raised in goats and rabbits by injection of whole species serum. Qualitative detection of each species in beef at the 1 -3% level was claimed to be possible. In a subsequent paper Hayden (1979) describes the production of antisera to ovine, porcine and equine myoglobin. This antigen was chosen because it is relatively thermostable and could perhaps yield antisera which could be used to identify the species of origin of heated meats. Extracted myoglobins were heated at 90°C in phosphate buffered saline (PBS) prior to immunisation and cross-reactivity of the resulting rabbit anti-lamb antiserum with beef was overcome by absorbing out with extracts of beef muscle. Use of the antisera in AGID allowed the detection of the flesh of the corresponding species in raw and mildly heated products but assay reliability decreased with increased heating of the sample. The detection limit was 3% raw pork in beef and 10% in the same product heated to 70°C. Horse meat in heated beef sausage could be detected at the 3% level. Hayden later published (1981) details of an AGID procedure which used rabbit antisera raised against thermostable antigens of horse, pig, lamb and poultry adrenals which could detect the flesh of the target species in sausages cooked to a core temperature of 71 °C. Thermostable antigens were extracted from species adrenals by blending with isotonic saline; the extract was then heated for 1 hour at 98 °C and autoclaved for 30 minutes prior to precipitation of the antigens and immunisation. Cross-reactivity was observed between beef and lamb, and chicken and turkey could not be differentiated. Detection levels were between 5% and 10% (m/m).

Kang'ethe et al (1986) were another group who worked on the development of AGID methods to identify meat species in heated products. Fresh meat antigens (FMA's) were extracted from meats by homogenisation with isotonic saline, cooked meat antigens (CMA's) were obtained by holding FMA's at 100° C for 1 hour and thermost able meat antigens (TMA's) were obtained by autoclaving CMA's for 30 minutes. TMA's were precipitated with ethanol and used to immunise goats and sheep. Resulting antisera were made species specific by adsorption with serum from cross-reacting species prior to AGID. Antisera to a range of species of bovidae significant in Kenya woe prepared and antisera to cattle TMA's could be used to detect the presence of beef in meat products that had been commercially sterilised. Applications did not extend to meats commonly consumed in Europe.

Schweiger et al (1983) developed an AGED procedure for the detection of chicken and turkey muscle in beef and pork products by raising antisera against the muscle protein troponin. Turkey muscle was washed, defatted, dried and powdered and muscle proteins extracted using a sodium carbonate-lithium chloride- di thioerythritol mixture. After various protein precipitation and washing stages extracted troponin T was purified using free-floW

2 electrophoresis and used as the immunogen in rabbits. Actin must be separated from troponin T because it was found to be similar in all animal species investigated, and therefore any antibodies recognising actin in samples would produce false positives. Results indicated that the presence of chicken or turkey could be detected in raw and products cooked to a temperature of 70°C, and that troponin could be extracted from cooked sausages using Tris-HCl or barbiturate buffers. This paper also reports the application of immunoelectrophoresis and counter- immuno electrophoresis. It is interesting that this is one of the few reports of an immunological procedure designed to detect muscle as opposed to plasma or blood proteins which can be significant when interpreting results of such examinations.

Swart and Wilks (1982) adopted the approach taken by many laboratories and used commercially available anti species antisera from Wellcome Research Laboratories (UK) in an AGIO assay format (they prepared anti- kangaroo antiserum by immunising rabbits with kangaroo blood as this was not commercially available). Detection limits for sheep, horse and kangaroo meats in beef ranged from 5 to 20% depending upon the species being detected and the effects of cross-reacting antibodies were nullified by addition of the relevant species proteins.

Mageau et al (1984) and Cutrufelli et al (1986,1987, 1988, 1989) published a series of papers which describe AGED procedures for the detection of bovine, avian, porcine and ovine meats using reagent impregnated filter paper discs to overcome the need for reagent handling (ie antisera and control species antigens), so that the kits could be used 'in the field'. The overnight Rapid Bovine Identification Test (ORBIT) used pre-prepared agar-gel immunodiffusion plates and paper discs impregnated with rabbit, sheep or goat anti-bovine antiserum. Bovine serum albumin was used as the immunogen and sheep and goat antisera were preferable as they lacked cross reactivity with sheep antigens, and larger volumes of serum could be collected. The discs which were impregnated with the anti-bovine antiserum and control bovine serum were stable for at least a year at 4°C. The Poultry Rapid Overnight Field Identification Test (PROFIT) used goat anti-poultry antisera raised against chicken and turkey serum albumin, the Porcine Rapid Identification Method (PRIME) used goat anti-porcine antisera and the Serological Ovine Field Test (SOFT) used calf anti-ovine antisera. ORBIT and PROFIT were exposed to a collaborative study in ten laboratories who analysed 30 raw meat samples (Cutruffelli et al, 1987) and shown to be reliable and capable of detecting adulteration at the 10% (m/m) level.

An early reference to a dip-stick assay was reported by Patterson and Spencer (1983). They developed a rapid dip-stick test for meat speciation in which the solid phase was coated with species specific antisera. Once coated, the sticks were placed in PBS-Tween extracts of meat samples, then placed in homologous horse-radish peroxidase (HRPO) conjugated antiserum and then placed in enzyme substrate. A positive reaction was indicated by a colour change and 1% (m/m) of meat in admixture could be detected.

Commercially Available AGID Kits

Various AGID kits for meat speciation have been produced commercially but many do not appear to survive long in the market-place. Rhone-Poulenc Diagnostics Ltd now sell the ORBIT, PROFIT, PRIME and SOFT test kits which can be conveniently used to identify meat species present in raw meat products. Kits to detect deer and horse are now being produced. The Binding Site (Birmingham, UK) also market pre-prepared AGID plates and anti species affinity purified antisera for meat species testing and anti-species antisera are also available from Sigma. If analysts purchase anti-species antisera for use in in-house AGID assays it is imperative that the species specificity of each batch purchased is evaluated against all possible animal species which may be present. It is often necessary to absorb out cross-reacting antibodies.

An alternative to AGID meat speciation is the FAST Immunostick Test Kit produced by Cortecs Diagnostics (UK), which can be used for beef, horse, pork, sheep/goat, poultry, rabbit and kangaroo detection. Immuno-reagents are immobilised on a dip-stick and identification of meat species via a colour change can be achieved in less than one hour. Detection limits for species in admixture are about 1% (m/m) for lean meat samples. FAST immunosticks for buffalo, goat, chicken, turkey and deer and under development.

Summary of AGID Methods

A range of applications of AGID for meat speciation have been published during the last 16 years but no great

3 advances have really been made. Raw meats are identified via blood or plasma proteins rather than via muscle protein, and few methods can be applied to cooked samples and antisera described for this application are not commercially available. The analyst may prefer to use commercially available kits as all reagents are supplied and good quality control is exercised, they show good species specificity, are relatively cheap, easy to use and are ideal for screening large numbers of samples. Perhaps the one disadvantage is that the analyst is reliant on one or two suppliers who may or may not last long in the market place. In the authors' laboratory we use commercial kits but also have validated in-house AGID methods which employ commercially available anti-species antisera. This allows simple and cost effective routine screening of raw meat samples using a suite of immunological reagents with no dependence on one particular commercial supplier. Commercially available AGID methods can only be applied to raw meats and they only detect blood or plasma proteins. There is a need for methods which can be applied to cooked samples and detect muscle proteins. Detection of muscle proteins implies that you are detecting 'meat' not perhaps just adventitious contamination of samples with blood from an undeclared species.

Enzyme Linked Immunosorbent Assay (ELISA)

ELISA methods have been used in biomedical research since the early 1970's but one of the first applications to food analysis was the determination of soya protein in meat products by Hitchcock et al{ 1981). Advantages of ELISA over AGED methods are that much less antisera is required, results can be obtained in several hours, large sample numbers can be processed and quantitative results may be obtained with some assays. Two of the first reported applications of ELISA to meat speciation were published by Whittaker et al (1982) and Kang'ethe et al (1982). Both groups of workers used anti-species antisera raised in rabbits against serum albumins from the target species (beef, sheep, horse, kangaroo, pig and camel), and purified the antisera by affinity chromatography against immobilised species serum albumins. Antigens were extracted from samples with buffer or saline and an indirect ELISA used which involved coating the microtitre plate with sample extracts, addition of the rabbit anti-species antiserum, and detection of the bound rabbit antibody by addition of staphylococcal protein A or anti-rabbit IgG both conjugated to HRPO and enzyme substrate. Detection limits of 3% (m/m) were reported by Kang’ethe (1982). Whittaker et al (1983) later published a more comprehensive report of their ELISA procedure. Patterson et al (1984) published details of a double sandwich ELISA procedure capable of detecting below 1% (m/m) of meat species in admixture. Species specific capture antibodies were raised in sheep or cattle and coated onto microtitre plates. Sample extracts were added and bound meat proteins were detected using a second species specific rabbit antisera and visualised using Protein A conjugated to HRPO and an enzyme substrate. The ELISA was capable of differentiating sheep and goat, and beef and buffalo using sheep anti-goat and sheep anti-buffalo as capture antisera and sheep anti-goat and rabbit anti-buffalo as detecting antisera. In a subsequent paper Patterson and Spencer (1985) differentiated phylogenically related species using monospecific antisera produced by immunisation in target related species; cattle were immunised with whole buffalo serum, sheep with whole goat serum and horses with whole donkey serum. Affinity purification and concentration by ultrafiltration were used to produce antisera which could detect 1% (m/m) or less of each target meat species using the previously described ELISA format.

In the same year Jones and Patterson (1985) published an ELISA procedure which used antisera directed towards porcine serum albumin (PSA) raised in rabbits and sheep to detect pig meat in meat mixtures. Rabbit anti-PSA was used as a capture antibody and sheep anti-PSA used for detection of immobilised sample PSA followed by visualisation with anti-sheep IgG HRPO conjugate and enzyme substrate. 0.5% pork in beef sausages could be detected. The same authors then reported a procedure (1986) in which commercially available anti-species antisera (anti-cow, anti-horse, anti-pig, anti-sheep/goat) could be used for meat speciation which incorporated integral assay inhibition of heterologous cross-reactivity by addition of heterologous serum albumins prior to ELISA.

Similar methods were published by Ayob et al (1989) who used crystalline porcine serum albumin (PSA) to produce anti-PSA antisera from rabbits. Cross-reactivity was overcome by affinity chromatography using immobilised species serum albumins. Two ELISA formats were used. An indirect competitive procedure involved coating the microtitre plate with PSA then addition of dilutions of the test solutions or standards. After incubation with the anti-PSA antiserum, goat anti-rabbit IgG peroxidase conjugate and enzyme substrate were used for detection. In the direct assay anti-PSA antiserum was coated onto microtitre plates and dilutions of test solutions or standards added. Detection was via addition of HRP-PSA conjugate and enzyme substrate. Antigens were extracted from samples using phosphate buffered saline (PBS) containing Tween (PBST). The direct assay was

4 more sensitive than the indirect competitive assay and 1% (m/m) pork in beef detected. Also the direct assay was simple and rapid. The amount of PSA in porcine muscle and offals was determined using the direct ELISA. The amount in muscle (3.8 mg/g) was considerably less than offal tissues (11.9-39.2 mg/g), a fact which would affect attempts at quantitation of pork meat in products containing offal. The authors did not evaluate the natural variation of PSA in porcine muscle within and between animals.

Griffiths and Billington (1984) used commercially available anti- species antisera for ELISA and evaluated the procedure for the estimation of the apparent beef content of beef joints and model mixtures via determination of beef serum proteins. A competitive assay was used in which microtitre plates were coated with beef serum, sample extracts (saline) were incubated with rabbit anti- beef antisera then loaded onto the microtitre plate, and after incubation excess antibody was detected using goat anti-rabbit IgG conjugated to alkaline phosphatase and enzyme substrate. ELISA results agreed well with the known beef content of model mixtures (but calibration was from same meats) whereas a wide variation in results was recorded for the fat free beef content of a range of beef joints which was attributed to differences in the amount of residual blood in the various joints (coefficient of variation of results was 29%). This is an observation which must be borne in mind when interpreting semi-quantitative data from this type of assay.

Following the Griffiths and Billington observation, Martin etal (1988a) comment on the fact that most anti-species antisera are raised against whole blood serum or serum albumins, and because of the variable amounts of residual blood in meats, these antisera cannot be used to quantify amounts of undeclared meat species. They extracted sarcoplasmic antigens from lean pig, horse, chicken and beef meat with isotonic saline and raised antibodies against the pig antigens in rabbits. The antisera were affinity purified against the sarcoplasmic extracts from the other meat species. Microtitre plates were coated with anti-pig sarcoplasmic protein (anti-PSP) antisera and residual sites blocked with PBST-BSA, aliquots of sample extracts were added and the bound sample antigens detected with anti- PSP-HRPO conjugate and enzyme substrate. The authors present evidence of the specificity of the anti-PSP antisera and state that 1 -50% (m/m) pig substitution in raw lean beef was possible. A second paper (Martin et al 1988b) discusses the preparation of anti-chicken sarcoplasmic protein (anti-CHSP) antisera. It is stated that the sarcoplasmic extracts contain haemoglobin and chicken serum albumins; it could be assumed that the pork sarcoplasmic extracts also contain the corresponding proteins. The authors state that this should not affect assay response but no experimental evidence is provided to substantiate this claim (what is the effect of blood and plasma?), neither is any data provided which shows the natural variation of sarcoplasmic proteins extracted from various muscles within and between animals. This is necessary if methods are to be used to estimate amounts of meat species present in admixture.

In a subsequent paper Martin et al (1991) report on the preparation of hybridoma cell lines for preparation of monoclonal antibodies to CHSP. Microtitre plates were coated with the purified capture monoclonal antibody and after blocking with BSA, extracts from meat mixtures were added, and after incubation and washing, rabbit anti- CHSP, goat anti-rabbit IgG-HRPO conjugate and enzyme substrate were added for detection of captured antigens. Calibration lines from 1% to 100% chicken are presented and quantification suggested. Martin et al (1992) later described a procedure for the imunoaffinity isolation of pig specific soluble muscle proteins which may be used for the development of monoclonal antibodies to detect and quantify pig meat in meat mixtures.

The Laboratory of the Government Chemist, in collaboration with Beeches Biotech, have developed an indirect ELISA which is designed to detect and quantify lamb muscle fibre proteins in the presence of pork and beef meat. The polyclonal anti-lamb muscle fibre antisera do not detect blood proteins from lamb, pork or beef and therefore the assay can be said to detect muscle. The ELISA procedure is currently being evaluated in the UK by inter laboratory study and results will be reported in the literature at the earliest opportunity.

Cooked Meats

All of the antisera used in the ELISA applications mentioned so far were raised against serum albumins, which are thermally labile, and therefore can only be applied to raw meat products. Kang ethe and Lindqvist (1987) described a procedure for the extraction of thermostable muscle antigens (TMAs) from buffalo tissue which is similar to that reported by Kang'ethe et al in 1986. Co-extracted gelatin from samples inhibited antigen absorption onto the microtitre plate but this was partially overcome by chromatographic fractionation of the antisera, but the ELISA format was found not to be suitable for speciation of cooked meats. The authors suggest a sandwich ELISA may

5 be the method to overcome the problems of gelatin..

Berger et al (1988) described a complex and lengthy procedure for the extraction of antigens from pork and chicken muscle which yielded potent species specific antisera which could be used to identify target antigens in cooked and canned products. From 1 kg of muscle tissue about 25 mg of antigen was obtained which was used to immunise rabbits, and the resulting antisera were purified by selective precipitation and chromatography. The purified IgG was coated onto microtitre plates, sample and control extracts were added, biotinylated IgG was then used to recognise the bound target antigen and visualisation achieved by the addition of streptavidin-peroxidase conjugate and enzyme substrate. The authors state that although the immunising antigens are heat resistant and species specific, neither heat resistance or specificity are absolute. The poultry ELISA showed virtually no cross reaction with other meat species (horse, beef, pork, sheep, deer, kangaroo) but showed a strong reaction with chicken and turkey even after samples had been heated to 120°C for 15 minutes. The pork ELISA performed similarly except that some cross-reactivity was shown with raw tissue extracts. The authors state that the method cannot be used quantitatively. Patterson and Jones (1989) also reported the development of an ELISA procedure for the detection of heated pig meat The paper describes two ELISA methods designed to detect heated lean meats, particularly pork, in a variety of meat products. The protocol for production of the antigen is not described in detail because it is stated that the procedure is being commercially developed; it is just stated that thermostable muscle components are prepared by a special autoclaving and extraction procedure and used to produce the anti-species muscle antisera. Sheep and goats were used as the principle host animals. The specificity of antisera were improved using a tailored blocking solution of heterologous serum albumins (see 1986 reference). Antigens were extracted from samples by autoclaving at 121 °C for a range of times, depending on the cooked state of the sample. Extracted sample antigens were coated directly onto the microtitre plate in an indirect assay, anti-goat IgG peroxidase conjugate and ABTS being used as the detection system. The indirect ELISA produced a linear response of optical density versus percentage of pig meat in lean meat mixtures with beef, lamb or chicken. The anti-pig antisera did recognise pork offals and fat to varying degrees although offals from other species were not recognised. The assay permits the detection of 5% pork lean meat mixed with other lean meats. A significant variation in the response to individual pig muscle meant that the assay could not be used to absolutely evaluate the lean meat content of products. The paper does not report applications to commercially canned and cooked products; model mixtures only were used.

Sawaya et ai (1990a,b) described the application of HPLC of triglycerides, GC of fatty acids, HPLC of ophidine dipeptides and ELISA for the detection of pork in processed meats. For ELISA, antigens were prepared from pork by extraction with saline followed by autoclaving at 120°C for 30 minutes. The filtered extract was used to immunise sheep; antisera raised in rabbits could not be made species specific. Mixtures of pork with beef and sheep meats and commercially canned meat products were homogenised with saline and autoclaved for 30 minutes to extract antigens. The sheep anti-porcine antisera was mixed with extracts from autoclaved beef, sheep and horse meats to block cross-reactions with heterologous species prior to a competitive ELISA. Microtitre plates were coated with porcine muscle extract then sample and standard mixture extracts added followed by the sheep anti- porcine antiserum. Goat IgG HRP conjugate and enzyme substrate were used as the detection system. The authors report pork could be detected at the 2% level in commercially canned products and in model meat mixtures which had been heated at up to 120°c for 30 minutes.

In one of the most recent papers on identification of heated meats Sherikar et al (1993) prepared thermostable antigens from adrenals and muscles by mincing, sonicating and heating (98 °C, 15 mins) in normal saline solution followed by centrifiigation and autoclaving of the supernatant for 30 minutes. Final products were precipitated using ethyl alcohol then dried and powdered. Buffalo, sheep, goat and pig were the species of interest. Antisera were raised in rabbits and specificity improved by absorption with antigens from other non-target species. The authors provide useful information on the electrophoretic study of the extracted thermostable antigens from the various species and identify them as troponin T. The antisera were used in AGIO, counter-immunoelectrophoresis (CIE), unlabelled antibody peroxidase antiperoxidase (PAP) and ELISA methods. The ELISA procedure of Kang’ethe (1982) was followed. Meat mixtures were 'cooked' at 100° C for 15 minutes. In mixtures containing cattle, buffalo, sheep and goat the AGID method had a limit of detection of 10% (m/m) whereas pork was detected at 5% (m/m) using AGID and CIE; ELISA and PAP could detect the presence of 1 % (m/m) of all species. Cooked products could be extracted with water for ELISA and PAP but the extraction protocol for thermostable antigens had to be used for AGID and CIEP.

6 Commercial ELISA Kits

Cortecs Diagnostics (UK) market ELISA kits designed to identify meat species in raw and cooked meats. The raw meat ELISA kit is capable of detecting cow, horse, pig, sheep, poultry and kangaroo meats in admixture while the cooked meat ELISA kit is capable of detecting beef, pork, poultry and sheep. These kits are ideal for screening meat products and about 1 -3% of each meat species in admixture can be detected. All reagents necessary for the assay are included in each kit. The kits cannot be used to estimate the amount of a particular meat species in a mixture.

Summary - ELISA A wide range of ELISA's have been applied to meat speciation since the early 1980's. Many are designed to detect blood plasma proteins and are suitable for the relatively rapid screening of raw meat samples but they cannot be applied to cooked products, and estimation of the amount of a meat in a mixture of meats is very difficult. One advantage is that some anti-species antisera are commercially available and allow the development and use of in- house ELISA's.

ELISA’s designed to identify the species of origin of meats in cooked products usually involve a complex extraction of thermostable antigens and antisera must then be purified to achieve species specificity. Thermostability is not absolute and assay response and specificity does diminish with prolonged cooking or heating and these assays cannot be used quantitatively. In addition, antisera are not available commercially.

Several ELISA's have been reported which are designed to detect muscle proteins, and with more investigation may be capable of identifying and determining the amount of a meat species in a mixture of meats. Applications of these assays to cooked products have not been reported, and antisera are not yet commercially available.

The majority of papers report the production of antisera which are not commercially available and so where do these facts leave the food analyst who does not have access to animal house facilities? The analyst is most likely to rely on the use of commercially available ELISA kits for raw and cooked meat speciation. In-house ELISA’s which use commercially available anti-species antisera to plasma proteins can be successfully used on raw meat products as long as antisera specificity is monitored with every assay and it is recognised that great care has to be exercised if the analyst attempts to obtain semi-quantitative information from such assays.

26 years ago colleagues at the Laboratory of the Government Chemist (Hubbard and PocUmgton, 1968) stated in a paper on meat speciation by lipid analysis that "the wider application of serological techniques is limited by the restricted range of antisera available". The situation has not significantly improved since that tune. Many interesting reports on the production of antisera for ELISA have been published but most are research papers and commercially available antisera do not often materialise.

DMA Methods for Meat Speciation From the previous review of immunological methods it is e t a that identification of meat species present in highly Processed and heated meat products is difficult and assay specificity a problem, particularly if we wish to differentiate between closely related species, eg. sheep » d goat Jieef and buffalo. DMA is a relatively stable molecule and specific recognition of unique sequences of DNA.from samples can provide the basis of a highly specific assay for species ident.fication. The principle of DNA based methods ,s quite straightforward. DNA ,s «traced from the meat product, partially purified ^ “ 8 ““ y '1?'" membrane. A species spLific segment of labelled DNA (a DNA probe) is foen allowed to hybntoe * h any complimentary sequences»! DNA which are immobilised on die membrane. If a species ^ f f i c labelledprobe i.L,. ; • wjH be bound to the membrane and visualisation of the probe label hjindises with its complimentary requeuedw,U £ ^ ^ „ ¿ lled DNA aWside— canon of the s p a te s ! T s S S y rfUrehybridisation conditions used. Imtially DNA probes were Probe for the species of interest and foe * ™ ^ ° '1A a^ niK8Cent labels can be used. There are far more user- radiolabelled but now colorimetric, fluorescent or ctiemiiunuu friendly for applications in food and enforcement laboratories.

7 The potential of DNA methods using simple dot- or slot-blot assay formats on nylon membranes have been investigated by Bauer et al (1987), Wintero et al (1990), Chikuni et al (1990) and Ebbehoj and Thomsen (1991). These workers used total genomic species DNA as the probe, but these probes contained highly repetitive base sequences which were homologous between closely related species. Cross-hybridisation between related species thus occurred and methods were not sufficiently species specific for general application. Pork and chicken were clearly identified but specific ruminant species identification was not possible. EbbehRj and Thomsen (1991b) attempted to overcome this problem by adding unlabelled DNA from the cross-hybridising species which allowed discrimination between some ruminants and some differentiation of sheep and goat.

Saikietal (1988) reported a procedure which used the polymerase chain reaction (PCR) for raw and cooked meat speciation using growth hormone gene specific primers. A modification of this technique using sheep satellite DNA primers for PCR amplification followed by Apa II restriction digestion of the product enabled differentiation of sheep and goat meats (Chikuni et al 1994). Bartlett and Davidson (1992) and Forrest and Carnegie (1994) have reported on the use of FINS, Forensically Informative Nucleotide Sequencing, for identifying the animal origin of biological specimens and gourmet meats respectively. The procedure involves extraction of DNA from raw and highly processed foods followed by PCR amplification of a specific segment of DNA, the mitochondrial cytochrome b gene, determination of the nucleotide sequence of the amplified DNA sequence, and phylogenic analysis of the nucleotide sequence using a database with subsequent matching of the most closely related species.

The PCR based techniques are relatively expensive, technically demanding and prone to contamination because of the extreme sensitivity of the reaction. This sensitivity is required for pathogen detection for example, but is not required for detection of meat species in foodstuffs which are present at percentage levels.

There is clearly a need for a relatively simple and robust DNA based procedure which can be used by food analysts to identify meat species present in raw and highly processed products. A simple slot-blot assay has been developed in the Laboratory of the Government Chemist which has successfully been applied to the specific identification of pork, beef, rabbit, sheep and goat in admixture (Parkes et al 1994). Species specific oligonucleotide probes have been developed which recognise relatively short sequences of DNA therefore a considerable amount of DNA degradation can be accommodated without influencing assay specificity. Total genomic DNA is extracted from authentic tissue and cooked and processed products with a buffer-SDS-EDTA mixture and after incubation wi ribonuclease A and proteinase K, DNA is extracted with phenol-chloroform and precipitated with ethanol. Sample DNA’s are loaded onto nylon membranes and digoxigenin labelled species specific probes added for the hybridisation stage. After removal of non-specifically bound probe by a series of stringency washes, bound species specific probes are visualised by chemiluminescent reaction and detection on X-ray film.

Species specific oligonucleotide probes were derived from satellite DNA sequences and oligonucleotides synthesised commercially. Goat and sheep can be differentiated using the assay procedure outlined and meat species in highly processed products such as canned frankfurters, pates, fermented sausages and canned pet food can be identified. Work to date also suggests that some semi-quantitative information can be obtained from these assays. A range of assay refinements are underway, including the adoption of a microtitre plate format.

DNA Methods - Summary

DNA based methods for meat species identification provide a way to identify the species of origin in raw and cooked meat products. Total genomic probes can be used to identify distantly related species but tailor oligonucleotide probes need to be designed for differentiation of closely related species. This is now possible using a variety of techniques. Non-radioactively labelled probes can be used in relatively simple assay formats which may mean that commercially available meat speciation kits based on DNA methods could soon be available for routine use by food analysts.REFERENCES

Ayob, M.K., Ragab, A.A., Allen, J.C., Faraj, R.S., Smith, C.J., (1989). An improved rapid ELISA technique for detection of pork in meat products. J. Sci. Food Agric., 49: 103-116.

8 Bartlett, S.E., Davidson, W.S., (1992). FINS (Forensically Informative Nucleotide Sequencing): A procedure for identifying the animal origin of biological specimens. Bio Techniques, 17(1): 408-411.

Bauer, C., Teifel-Greding, I, Liebhardt, E. (1987). Species identification of heat denaturized meat samples by DNA analysis. Archiv für Lebensmittelhygiene, 38: 149-176.

Berger, R.G., Mageau, R.P., Schwab, B., Johnston, R.W., (1988). Detection of poultry and pork in cooked and canned meat foods by enzyme-linked immunosorbent assays. J. Assoc. Off. Anal. Chem., 71(2): 406-409.

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