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AssigneesLife Genetics Lab, LLC., Board of Supervisors of Louisiana State University and Agricultural and Mechanical College
InventorsSudhir K. Sinha, Jaiprakash G. Shewale, Jerilyn A. Walker, Mark A. Batzer
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A family of PCR assays is disclosed for determining, both qualitatively and quantitatively, presence of material from a predetermined species source and for quantifying the amount of such material. The assays are based respectively on SINEs uniquely characteristic of pig species, cow species, chicken species, and ruminant sub-order, and having a high copy number. The assays disclosed permit rapid, inexpensive evaluation of meat samples to facilitate elimination from their diet of pork or beef by persons desiring to avoid such food sources; as well as the assay of cattle feed to determine presence therein of ruminant-source proteins, which are a potential source of bovine spongiform encephalopathy (BSE), commonly referred to as “mad cow disease.” The assays amplify the predetermined unique SINEs and the resulting amplified mixture is then evaluated qualitatively by electrophoresis on gel containing ethidium bromide or quantitatively by SYBR Green-based detection or TaqMan chemistry. The invention also extends to kits, primers, and other products used in connection with the assays. The amplicons are selected to be from about 100 to 170 bp long.
A process for assaying a sample of unknown content to determine the presence of bovine-source material in the sample, said process comprising the steps of:
A process for assaying a sample of unknown content to determine the presence of chicken-source material in the sample, said process comprising the steps of:
Dependencies are inferred from the claim text.
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Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of…: with…
Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes: for detection or identification of organisms
Sugars
Measuring or testing processes involving enzymes, nucleic acids or microorganisms
Term ended 14 Sep 2025 (estimated)
1. Technical Field
The present invention concerns assays for determining presence, and for quantitation, of species sources of DNA. More specifically, the invention concerns assay methods for detecting and quantitating ruminant-source material in animal feed and pork material in foods. In addition, the invention extends to assay methods for detecting and quantitating beef and chicken material in foods of mixed or complex sources, and to products used in performing the foregoing methods. In addition, the invention extends to detecting and quantitating ruminant-source, pork-source, beef-source, and chicken-source material in cosmetics and other substances, which may be ingested by humans.
2. Related Art
FIG. 1 is a chromatography display for the assays of the invention, showing UV fluorescence visualization of results of gel chromatography of: (A) the bovine-specific assay, (B) the porcine-specific assay, (C) the chicken-specific assay, and (D) the ruminant species assay.
FIG. 2 shows the quantitation range for the four assays of the invention: (A) the bovine-specific assay, (B) the porcine-specific assay, (C) the chicken-specific assay, and (D) the ruminant species assay.
FIG. 3 shows cross amplification of DNA templates derived from 14 species for the four assays of the invention: (A) the bovine-specific assay, (B) the porcine-specific assay, (C) the chicken-specific assay, and (D) the ruminant species assay.
As previously indicated, the SINEs to be amplified by PCR are selected to uniquely identify the species, genus, etc. of interest, i.e., the subject of the assay. That is, each SINE of interest is selected from a genomic subset common to members of the particular type of animal that is the target of the given assay. But this SINE is one not found in genomes of other types of animal that are non-targets of the given assay.
Bovine SINE families such as Bov-tA, Bov-A, and Bov-B are common to all ruminant members of the order Artiodactyla such as shown when using the invention's Bov-tA2 ruminant assay (illustrated in FIG. 1D). See C. Jobse, J. B. Buntjer, N. Haagsma, H. J. Breukelman, J. J. Beintema, J. A. Lenstra, Evolution and recombination of the bovine DNA repeats, J. Mol. Evol. 41 (1995) 277-283. But these elements have also undergone recombination events throughout bovine evolution such that some sequence variants have formed satellites of the original SINE families. Some of these satellites, such as the 1.711B bovine repeat (Gen-Bank No. V00116) used in the bovine (beef) assay of the present invention, emerged after the radiation of the Bovidae approximately 5-15 million years ago and are absent from other ruminant species (see FIG. 1A). See Jobse et al. (1995). Therefore, such elements can advantageously be used to distinguish beef samples from other ruminant-derived samples. The 1.711B bovine repeat is thought to occupy 7.1% of the bovine genome. R. E. Streeck, A multicopy insertion sequence in the bovine genome with structural homology to the long terminal repeats of retroviruses, Nature 298 (1982) 767-768.
The porcine SINE PRE-1 used in the porcine assay of the invention (GenBank No. Y00104) is present in the common domestic pig, Sus scrofa, and other members of the Suidae family, but is absent from other genomes (see FIG. 1B). The PRE-1 SINE sequence reportedly diversified at least 43.2 million years ago and has about 100,000 copies per genome. See D. S. Singer, L. J. Parent, R. Ehrlich, Identification and DNA sequence of an interspersed repetitive DNA element in the genome of the miniature swine, Nucleic Acids Res. 15 (1987) 2780; H. Yasue, Y. Wada, A swine SINE (PRE-1 sequence) distribution in swine-related animal species and its phylogenetic analysis in swine genome, Anim. Genet. 27 (1996) 95-98. Although, technically speaking, this assay detects members of the Suidae family, rather than just the common domestic pig Sus scrofa, the distinction is immaterial since only the common domestic pig is used for commercial meat purposes and, also, any member of the Suidae family is an “unclean” animal proscribed by the Bible (because, although hoofed, they do not chew the cud).