Nematodes

  Species identification

Molecular Methods

DNA based molecular techniques 

• DNA is a molecule of choice for species specifications due to its stability during heating and processing 
• DNA molecular-based species specification is possible in the foods obtained from identification rendered meat products, genetically modified foods, etc 

DNA hybridization technique 

• It is a qualitative or semi-quantitative technique of meat species speciation in which species-specific DNA sequence is detected
• Specie Specific probes are developed for this technique
• It detects admixtures that contribute as little as 5% or less to a product

Polymerase Chain Reaction (PCR)

• PCR is a rapid method
• has a high degree of selectivity and sensitivity.
• It is a qualitative test for meat species specification
• There are two main techniques for amplification of genetic marker, i.e., mono-locus-specific primers for amplification of a concrete DNA fragment and multi-locus amplification of non-targeted DNA
• By this technique closely related meat species can be identified with the discrimination between male and female raw meat.

PCR techniques using multi-locus primers

Random amplified polymorphic DNA fingerprinting (RAPD) 

• It is a modified PCR technique in which DNA fingerprints are generated in a very short period of time which can be visualized on gel electrophoresis 

Amplified fragment length polymorphism

• In this technique, about 500 primer combinations produce selected markers which are converted into a single nucleotide marker for highly specific genotyping of the meat species

PCR techniques using mono-locus primers 

Species-specific PCR: 

• In this technique, species-specific DNA in femtograms (fg) and picograms (pg) can be detected in both processed and unprocessed meat samples by using targeted amplification of rRNA genes
• By this technique, very old samples even of more than 100 million years can also be identified

 Multiplex PCR 

• In this technique, many targets are simultaneously amplified which helps in the detection of many species in a short period of time
• Organ meat can also be detected like the mixing of duck liver in goose liver can easily be identified
• For that purpose multiplex PCR with common forward and species-specific reverse primers are used.

Nested and Hemi nested PCR:

• This technique can also be used in the meat showing spoilage.
• But in such a case, two-stage PCR is generally used.
• First stage low stringency primer pair is used for amplification while in second nested PCR technique is applied
• By using polymorphism in the mitochondrial 16S rRNA gene, the meat of several wild animals can be detected at a time. 

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) 

• A modified form of PCR in which the conserved target gene is amplified
• THE resultant PCR product is digested with specific restriction endonuclease enzyme to get a restriction pattern or fingerprints
• This is also a suitable method for identification of even degraded DNA with apomorphic sites 
• A suitable method for the analysis of the very low amount of meat (1 mg)
• Faster and more sensitive method than DNA hybridization
• By this technique, two species of animals can be differentiated even after heating at 120°C. 
• Proper identification of males and females can be done by using PCR and genomic DNA extraction from raw muscles.

Advantage 

• PCR-RFLP analysis of the mitochondrial Cyt b gene to differentiate between beef, sheep, pork, chicken, donkey, and horse meats in meat products
• Sausages, frankfurters, hamburgers, hams, and cold cut meats
• A potential technique to rely on for authentication of halal (lawful or permitted) meat origin.

Drawbacks of PCR-RFLP

• It is not a hundred percent accurate methods
• because of the existence of some intraspecific variability within the species as in sheep, turkey, and chicken meat.

Forensically informative nucleotide sequencing (FINS) 

• In this technique, the conserved region of gene-specific to a species is amplified and the PCR product is cloned, sequenced and the sequence is analyzed
• For this purpose 12S rRNA, gene sequence analysis is the most preferred method for differentiation of beef, buffalo meat, mutton, and chevon.

Real-Time PCR (RT-PCR)

• In this technique, PCR products are detected and monitored during amplification in the same reaction vessel with the help of fluorescent compounds. This technique makes identification and defects of the product during its amplification
• Quantification of the meat species may be possible

Conclusions

• Although several methods are available for meat species specifications no single method can be applied in all cases
• As the physical, chemical, and anatomical methods are more suitable for raw meat while minced or comminuted meat requires sophisticated technique.