There are many PCR-based methods for animal species identification; however, their detection numbers are limited or could not identify unknown species. We set out to solve this problem by developing a universal primer PCR assay for simultaneous identification of eight animal species, including goat, sheep, deer, buffalo, cattle, yak, pig, and camel. In this assay, the variable lengths of mitochondrial DNA were amplified using a pair of universal primers. PCR amplifications yielded 760 bp, 737 bp, 537 bp, 486 bp, 481 bp, 464 bp, 429 bp, and 359 bp length fragments for goat, sheep, deer, buffalo, cattle, yak, pig, and camel, respectively. This primer pair had no cross-reaction with other common domestic animals and fish. The limit of detection varied from 0.01 to 0.05 ng of genomic DNA for eight animal species in a 20
The consumption of meat and meat products is increasing each year in the world. On the other hand, food authenticity issues in the form of adulteration and improper description have existed for as long as food has been offered for sale. In China, “hang a sheep head, sell vinegar” is a widely known proverb. However, meat adulteration affects food safety, quality, and many other respects, becoming a public focus recently. Since the 1980s, many immunological and molecular methods for species identification in food products have been developed [
With the development of Western China, more and more local meat products of mutton, beef, yak, deer, and camel have been sold to all parts of the country. There have been a few analytical methods for the identification of yak [
In recent years, universal primer PCRs have been used and further developed for animal species identification combination with other DNA analysis methods [
The samples of known species origins including goat
Genomic DNA was extracted from meat samples using an Animal Tissue DNA Extraction Kit (Takara) according to supplied instructions. Following DNA extraction, the purity and concentration of all the DNA samples were confirmed using a UV-Vis spectrophotometer (NanoDrop 2000, Thermo). DNA samples were diluted to a final concentration of 10 ng/
The mitochondrial DNA (mtDNA) sequences of all the above animals were retrieved from the NCBI database and were aligned using ClustalW sequence alignment tool to select the congenerous conserved regions. Two regions were selected as primer design areas, and a pair of universal primers was designed to amplify variable length mtDNA sequences from genomic DNA of yak, deer, goat, sheep, pig, camel, cattle, and buffalo but no other animals. There were several insertion-deletion polymorphisms in the amplified sequences, which, in theory, could yield different length fragments for each animal. The primer sequences are as follows: forward primer (5′-CCTCCCTAAGACTCAGGGAA-3′) and reverse primer (5′-AGCGGGTTGCTGGTTTCACG-3′). The designed primers were also screened for unique specificity to check cross-species binding with other animal or plant species using the online BLAST local alignment tool in the NCBI database.
PCR amplifications were carried out using a MJ-200 thermal cycler in a total of 20
To further discriminate between targeted animals, expected nucleotide sequences were restriction mapped with the Mapdraw program of DNASTAR (NY, USA). After testing, one restriction enzyme
To further validate this assay, each of the PCR products was purified and then sequenced in Hangzhou Qingke Biotechnology Company and then were analyzed using the BLAST local alignment tool. Fifteen commercial samples were screened using this developed assay, and each of the PCR products obtained was extracted and purified separately and then digested by the restriction enzyme
In the present study, all the genomic DNA samples were measured. The spectrophotometric assessment results showed that concentrations varied between 20 and 200 ng/
PCR amplification results for eight animal species. Lanes 1–8 represent goat, sheep, deer, buffalo, cattle, yak, pig, and camel, respectively. Lane M represents DNA marker; the lengths of different PCR products were noted above the band.
The specificity of primers was checked against the extracted DNA samples from common animals, which included dog, chicken, horse, rat, donkey, rabbit, duck, frog, and fish. The specificity test results showed that no cross amplification was detected. The sensitivity was tested using serially diluted DNA samples from goat, sheep, deer, buffalo, cattle, yak, pig, and camel, and until the PCR products could not be visualized on 2.0% agarose gel. DNA band patterns (Figure
Sensitivity test for pig DNA. Lanes 1–6 represent the PCR products with serial dilution of pig DNA template. The content of pig DNA was 30.0, 10.0, 1.0, 0.1, 0.01, and 0.001 ng. Lane N was negative control.
Each of the PCR products yielded different patterns for eight animal species after
The PCR-RFLP result for eight target animals. Lane M represents DNA marker; lanes 1–8 represent goat, sheep, deer, buffalo, cattle, yak, pig, and camel, respectively.
To confirm the developed method, each of the PCR products was purified and sequenced. The sequencing results indicated that the sizes and sequences of all the PCR products corresponded exactly to that of the expected amplicons. The similarity parameters were as high as 100% in accordance with the GenBank database. One exception was buffalo (98.2%). This difference was caused by two single nucleotide polymorphic loci detected in the referenced sequence of buffalo compared to GenBank Accession no. AY702618. The sequencing results were fully in accordance with the expected amplicons, which indicates that this developed assay had a higher specificity.
The fifteen commercial meat products were detected using this developed assay, two animal ingredients were found in two of the fifteen samples, while other samples had only one ingredient (Figure
Identification results of commercial meat samples. Samples in lanes 1–7 were labeled as deer, mutton, mutton, beef, pork, camel, and yak meat. Lane 8 was soybean DNA, and lane 9 was negative control. But, the samples in lanes 1 and 2 contained pork.
With the development of economy, meat products become a daily food for consumption. Furthermore, yak, camel, and deer meats are new popular meat products in the Chinese market, especially as leisure foods. The price of these meat products is higher than that of common meat products such as beef and pork. On the other hand, the rising price and decreasing availability of high-quality meat drive some meat producers to misrepresent and/or adulterate meat and meat products. In fact, food adulteration has been around for a long time. The most common economic fraudulence widely spread in meat industry is adulteration or substitution of costlier meat with cheaper or inferior meat. Preventing meat adulteration in foods is important for economic, religious, and health reasons. In addition, identification of the species origin in processed meat products is an important task in food hygiene, food codex, food control, and veterinary forensic medicine.
In order to protect consumers from fraud and adulteration, many meat and/or animal species identification methods have been developed to date. DNA assays have become common methods in practice because they have many advantages over other analyses. The stability of mtDNA is higher than that of genomic DNA and it is distributed in all the tissues. Therefore, mtDNA sequences are preferential for DNA barcoding in species identification [
In the past few years, PCR-RFLP assays had been used to identify food animal species targeting CytB gene [
In the present study, a modified PCR assay was developed using universal primers for simultaneous identification of goat, sheep, deer, buffalo, cattle, yak, pig, and camel. Also, this assay could be further developed into RFLP method; the fragments of different lengths produced from PCR products could be identifying the animal species. This method can detect any targeting individual species within a meat mixture, which had better discrimination and specificity than many other multiple PCR methods.
This developed PCR-RFLP assay was a simple and rapid technology for identifying multiple species. This method did not require expensive equipment, and the optimization procedure was simple in comparison with multiplex PCR, real-time PCR, and other technologies that are used for the detection of two or more animal species. Interestingly, this method could identify individual species within a meat mixture by separating different size PCR products. This method was developed based on conventional PCR platform and required a common PCR thermal cycler, so it could be carried out in most laboratories. Furthermore, the developed PCR had no cross-reaction with DNA samples from common meats, such as duck, chicken, rat, frog, fish, donkey, and horse, and the PCR system had a high sensitivity at a minimum level of 0.001 ng of DNA. In general, the detection sensitivity for animal species such as poultry, ruminant, and pig using common PCR and real-time PCR was up to 0.1–1% of the ingredients and was also able to detect common meat species down to 0.15–0.01 ng of DNA [
In conclusion, a pair of universal primers for amplifying variable length fragments in eight animal species was designed and a PCR assay was developed, which could be further developed into PCR-RFLP. This developed method can be used to identify eight animal species. This method was specific, sensitive, and reliable in the simultaneous identification of goat, sheep, deer, buffalo, cattle, yak, pig, and camel. This proposed method was relatively simple and rapid, does not require expensive equipment, and could be performed in most laboratories. It is a practical approach for routine analysis to determine fraudulent and/or mislabeled substitution in meat products.
The authors have declared that no conflicts of interest exist.
The authors would like to thank all the people for collecting the samples and they also gratefully acknowledge financial support from the National Natural Science Foundation of China (Nos. 31672394 and 31360540), the Science and Technology Department of Zhejiang for public welfare Technology Application Research (2017C32081), and the Major State Basic Research Development Program of China (2017YFD0501904).