Molecular Identification Techniques And Applications Of Meat Products Originated From Livestock And Poultry

One of the most consumed agricultural products is meat,which is rich in protein,minerals and essential vitamins and plays a key role in the health of human food.Meat adulteration,mainly for the purpose of economic benefits,is widespread and results in serious risk for the public health,violations of religious custom and disruption of meat market.To ensure the safety of meat products in the market,it is necessary to establish some accurate and fast detection techniques for animal derived meat products.The present paper is aimed to construct specific PCR technique via bioinformatics method to detect those eleven animal-derived components,in which the targeted nuclear genes included:the nuclear pore associated protein 1（NPAP1）of pig,B and T lymphocyte associated（BTLA）of cattle,MHC class I heavy chain（MHCX1）of horse,hemoglobin subunit beta-C-like（HBBC）of goat,POTE ankyrin domain family member G like（Potegl）of mouse,scavenger receptor family member expressed on T-cells 1（Scart1）of rat,lactase phlorizin hydrolase（LPH）of rabbit,KLF transcription factor 18（KLF18）of dog,prolactin like（PRLL）of chicken,protein phosphatase Mg²⁺/Mn²⁺dependent 1H（PPM1H）of duck,and glucosamine（N-acetyl）-6-sulfatase（GNS）of goose.The main results were as follows.1.In the specific PCR technique to identify pork-derived components,the target fragment was amplified in length of 393 bp with the nucleotide sequence in 99%similarity to the target region.The minimum detection limit was 1.00 ng/μL of porcine genomic DNA and the spiking level was as low as 1%of pork.Random inspection of meat products in the market via the specific technique confirmed that beefsteak contained pork adulteration.2.As for bovine detection,both common PCR and quantitative PCR techniques were constructed,in which the amplificated fragment was 238 bp in length and the sequence similarity of 99%to the target gene.The specific PCR technique presented a minimum detection limit of1.00 ng/μL of bovine genomic DNA and as low as 1%of beef additive.The quantitative PCR technique detected a minimum adulteration amount of 0.1%beef,0.0064 ng/μL bovine genomic DNA and constructed a systematic error calibration curve for the adulteration amount.During the random sampling from the market,it was confirmed the beef products were substituted by pork ingredients.3.A PCR technique specific to the detection of equine-derived components was established,the target fragment was amplified to be 463 bp and the sequence showed 99%similarity to the target gene.The minimum detection limit was 1.00 ng/μL of equine genomic DNA and as low as 1%of horsemeat addition amount.Market meat products were randomly tested and not a single adulteration sample was checked.4.The 364 bp target fragment was amplified by specific PCR to detect goat meat,in which the fragment presented 99%similarity to the target sequence.The minimum detection limit was1.00 ng/μL of goat genomic DNA and the adulteration limit was 1%of goat meat.Total of 20%the market lamb rolls sampled were found to be duck replacements.5.Another PCR method to differentiate mouse meat from the others was constructed with target sequence of 400 bp.The amplificated sequence was 99%similar to the target region.The minimum detection limit was 1.00 ng/μL of mouse genomic DNA and as low as 1%of mouse meat.No mouse meat components was found in the random sampling of meat products from market.6.Regarding the detection of rat meat origin,specific PCR and quantitative PCR techniques were established,in which the amplified target fragment was 189 bp and the sequencing results showed 98%similarity to the target gene.The minimum detection limit for specific PCR was 1.00ng/μL of rat genomic DNA and as low as 1%rat meat spiking.The quantitative PCR technique was used to detect the minimum adulteration of 0.1%rat meat,0.032 ng/μL rat genomic DNA and constructed a systematic error calibration curve for the adulteration amount.Market meat products were tested and no rat-derived ingredients were measured.7.To identify the source of rabbit meat,specific PCR and quantitative PCR techniques were established,in which the target fragment was amplified at 171 bp and the nucleotide sequence gave 96%similarity to the target gene.The minimum detection limit of rabbit genomic DNA and rabbit meat addition amount is 1.00 ng/μL and 1%by specific PCR,0.032 ng/μL and 0.1%by quantitative PCR.The systematic error calibration curve for the adulteration of rabbit meat was constructed.Market steak,lamb rolls,beef rolls,salami,sausages and other meat products were tested and no rabbit meat components was detected.8.Specific PCR was used to detect the source of dog meat,the target fragment was amplified in length of 512 bp with the nucleotide sequence of 96%similarity to the target region.The minimum detection limit was 1.00 ng/μL of canine genomic DNA and as low as 1%of canine meat addition.Random sampling of meat products in the market did not reveal any source ingredients of dog meat.9.In the specific PCR technique to check chicken-derived components,the target fragment was amplified in length of 371 bp with the nucleotide sequence of 97%similarity to the target region.The technique indicated the detection limit of 1.00 ng/μL of chicken genomic DNA and as low as 1%of chicken spiked.Testing of meat products such as market steaks,lamb rolls and beef rolls did not find chicken-derived ingredients.10.As for duck meat source testing,both specific PCR and real-time quantitative PCR methods were performed.The target fragment was amplified at 182 bp and the sequence was 95%similar to the target location.Specific PCR technique had a minimum detection limit of 1.00ng/μL of duck genomic DNA,and as low as 1%of duck meat adulteration.The quantitative PCR technique detected a minimum adulteration level of 0.1%duck,0.032 ng/μL of duck genomic DNA and constructed an adulteration systematic error calibration curve.Duck meat substitution for beef and lamb detected from the market meat products.11.The specific PCR and quantitative PCR techniques for detecting goose origin,amplifying a target fragment of 153 bp and the sequence showed 95%similarity to the target gene.The minimum detection limit of specific PCR technique was 1.00 ng/μL of goose genomic DNA and as low as 1%of goose meat adulteration.The minimum detection limit of the quantitative PCR technique was 0.032 ng/μL of goose genomic DNA and a minimum adulteration of 0.1%of goose meat.A systematic error calibration curve of adulteration was constructed.Random sampling of meat products in the market did not reveal any goose component.Conclusion:This study constructed eleven detection technique for animal-derived meat products with the advantages of high sensitivity,accuracy,good repeatability,low cost and wide applicability.Those techniques would be helpful for the detection of prohibited and adulterated meat ingredients in the meat product market and maintain the safety of meat food market for the livestock,poultry and the people.