WO2012059102A2 - Procédé de détection d'adn contaminant dans des échantillons biologiques - Google Patents

Procédé de détection d'adn contaminant dans des échantillons biologiques Download PDF

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WO2012059102A2
WO2012059102A2 PCT/DK2011/000127 DK2011000127W WO2012059102A2 WO 2012059102 A2 WO2012059102 A2 WO 2012059102A2 DK 2011000127 W DK2011000127 W DK 2011000127W WO 2012059102 A2 WO2012059102 A2 WO 2012059102A2
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seq
probe
sequence
ruminant
reverse primer
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PCT/DK2011/000127
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WO2012059102A3 (fr
Inventor
Jørgen FINNEMANN
Jesper Worm
Peter Hagedorn
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Leo Pharma A/S
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria

Definitions

  • the present invention relates to a real-time PCR-based method of detecting contaminant DNA in samples of biological material, as well as target polynucleotide sequences, oligonucleotide primers and probes and kits comprising said oligonucleotide primers and probes for use in the method.
  • BSE bovine spongiform encephalopathy
  • FDA Food and Drug Administration
  • manufacturers of food and feed products and other materials of biological origin to demonstrate that the products in question do not contain any material of ruminant, in particular bovine, origin.
  • methods have been developed that permit detection of undesired material of bovine or other ruminant origin.
  • the earliest reported methods involved either microscopic examination of animal feed to detect bovine proteins (Tartaglia et al., J. Food Prot. 61 (5), 1998, pp. 513-518) or an immunological assay (ELISA) which was also developed to detect bovine proteins in aminal feeds (Reveal®, Neogen Corp., Lansing, Michigan).
  • PCR-based methods were not very sensitive (Tartaglia et al, op. cit ) and therefore carried a significant risk of false negatives, i.e. contaminant bovine DNA was not detected in all instances such that the problem of detecting bovine contamination was not adequately solved.
  • improved PCR methods have been developed for detecting bovine material in animal feeds, primarily intended for cattle.
  • WO 2008/074522 discloses a method of amplifying ruminant DNA in a sample of animal feed or feed ingredient, wherein DNA extracted from the sample is treated with RNAse and then amplified using conventional PCR methods, including nested PCR.
  • PCR method 50-58 report on the development of a quantitative PCR method to characterize the animal origin of low molecular weight heparin.
  • the PCR method reported by Houiste et al. uses amplification of highly repeated regions (satellite DNA) of the genome, and the DNA detection limit is indicated to be as low as 1 ppm for bovine DNA and 10 ppm for ovine DNA.
  • the ppm is calculated on the basis of the weight of the DNA by weight of heparin and the crude porcine heparin is indicated to contain a median of 10 pg porcine DNA/mg heparin.
  • a content of 10 pg porcine DNA per mg of heparin equals 10,000 ppm (by weight).
  • ppm is calculated based on the ratio of ruminant to porcine mitochondrial DNA copies.
  • An object of the present invention is to provide a highly sensitive real-time PCR-based method of detecting DNA of ruminant origin in human foodstuffs or food ingredients, animal feeds or feed ingredients, therapeutic proteins and polysaccharides, and cosmetic or pharmaceutical ingredients of biological origin, especially derived from porcine material.
  • Another object of the invention is to provide target nucleotide sequences and
  • oligonucleotides to be used as primers and probes in the method the target nucleotide sequences being derived from the mitochondrial genome of ruminants and selected so as to be specific to a large number of ruminant species and particularly to ruminants of the family Bovidae, in particular the most common domesticated bovids, i.e. cattle, sheep and goats.
  • a further object of the invention is to provide a highly sensitive real-time PCR-based method of detecting DNA of porcine origin in human foodstuffs, animal feeds,
  • therapeutic proteins and polysaccharides and cosmetic or pharmaceutical ingredients of biological origin, especially derived from bovine material.
  • a still further object of the invention is to provide target nucleotide sequences and oligonucleotides to be used as primers and probes in the method, the target nucleotide sequences being derived from the porcine mitochondrial genome and selected so as to be specific to all breeds of the genus Suidae.
  • the present invention relates to an isolated, contiguous, ruminant-specific polynucleotide sequence of about 300 base pairs in length derived from the ruminant mitochondrial genome and comprising the sequence
  • TNAAANCCTC TTATTTCTAG AANTATAGGA ATNGAACCTA CTCCTAAGAA NCCAAAACTC TTCGTGCTCC CAATTACACC AAATTCTANT AGTAAGGTCA GCTAATTAAG CTATCGGGCC CATACC (SEQ ID NO : 5) ;
  • N is any nucleotide
  • the invention relates to ol igonucleotide sequences su itable for use as forwa rd or reverse primers in a method of amplifying any one of the ru mi nant-specific polynucleotide target sequences of SEQ ID NO : 1-7, as well as oligonucleotide sequences suitable for use as probes for hybridizing to a portion of any one of the polynucleotide ta rget sequences SEQ ID NO : 1-7.
  • the i nvention relates to an isolated, contig uous, porcine-specific polynucleotide sequence of about 300 base pa i rs in length derived from the porcine mitochondrial genome and comprising the sequence
  • TAAAGATCAA AAGGAGCAGG TATCAAGCAC ACCTATAACG GTAGCTCATA ACGCCTTGCT CAACCACACC CCCACGGGAA ACAGCAGTGA TAAAAATTAA GCCATGAA (SEQ ID NO : 872) ; CTAGGTTATA TGCAACTACG AAAAGGACCC AACGTTGTAG GCCCCTACGG CCTACTCCAA
  • the invention relates to oligonucleotide sequences suitable for use as forward or reverse primers in a method of amplifying any one of the porcine- specific polynucleotide target sequences of SEQ ID l ⁇ IO: 872-876, as well as
  • the invention relates to a method of detecting ruminant contaminant material in a biological sample, the method comprising the steps of
  • step (c) detecting the presence of the amplified ruminant-specific target sequence, wherein detection indicates the presence of ruminant contaminant material in the sample.
  • SEQ ID NO: l may be amplified using a forward primer of SEQ ID NO:9-42, a reverse primer of SEQ ID NO: 43-76, and a probe of SEQ ID NO: 77-110, as shown in Table 1 below;
  • SEQ ID NO: 2 may be amplified using a forward primer of SEQ ID NO: 111-159, a reverse primer of SEQ ID NO: 160-208, and a probe of SEQ ID NO: 209-257, as shown in Table 2 below;
  • SEQ ID NO: 3 may be amplified using a forward primer of SEQ ID NO: 258-286, a reverse primer of SEQ ID NO: 287-315, and a probe of SEQ ID NO: 316-344, as shown in Table 3 below;
  • SEQ ID NO:4 may be amplified
  • the invention relates to method of detecting porcine
  • SEQ ID NO:872 may be amplified using a forward primer of SEQ ID NO: 878-938, a reverse primer of SEQ ID NO: 939-999, and a probe of SEQ ID NO: 1000-1060, as shown in Table 8 below;
  • SEQ ID NO:873 may be amplified using a forward primer of SEQ ID NO: 1061-1186, a reverse primer of SEQ ID NO: 1187-1312, and a probe of SEQ ID NO: 1313-1438, as shown in Table 9 below;
  • SEQ ID NO:874 may be amplified using a forward primer of SEQ ID NO: 1439-1513, a reverse primer of SEQ ID NO: 1514-1588, and a probe of SEQ ID NO : 1589-1663, as shown in Table 10 below;
  • SEQ ID NO:875 may be amplified using a forward primer of SEQ ID NO: 1664-1951, a reverse primer of SEQ ID NO: 1952-2239, and a
  • NO:876 may be amplified using a forward primer of SEQ ID NO: 2528-2592, a reverse primer of SEQ ID NO: 2593-2657, and a probe of SEQ ID NO: 2658-2722, as shown in Table 12 below.
  • Fig. 1 is a schematic representation of the bovine, ovine and caprine mitochondrial genomes, wherein the positions of the consensus ruminant-specific target sequences SEQ ID NO: 1-7 are shown as pale grey regions 1-7 highlighted by arrows.
  • Fig. 2 is a schematic representation of the porcine mitochondrial genome, wherein the porcine-specific target sequences SEQ ID NO:872-876 are shown as pale grey regions 872-876 highlighted by arrows.
  • Fig. 3 is a graph showing the fluorescence detected at repeated cycles of PCR.
  • Fig. 4 is a schematic representation of a PCR plate layout for detecting the copy number of porcine mitochondrial DNA in a test sample.
  • Sus cal refers to wells containing a calibration sample comprising 100,000 copies of a porcine-specific target sequence per ⁇ .
  • Contr. sample refers to wells containing 1 copy of a ruminant-specific target sequence and 400,000 copies of a porcine-specific target sequence per pi to verify proper detection of porcine mitochondrial DNA in test samples.
  • NTC refers to a negative control containing buffer, but no ruminant- or porcine-specific target sequence, to verify the purity of the PCR reagents.
  • H refers to test samples containing DNA extracted from heparin. 0 refers to the first and 00 to the second extraction of an unknown sample.
  • Fig. 5 is a schematic representation of a PCR plate layout for detecting the copy number of ruminant mitochondrial DNA in a test sample.
  • J cal refers to wells containing a calibration sample comprising 8 copies of a ruminant-specific target sequence per ⁇ to determine the presence or absence of PCR inhibition in test samples.
  • Contr. sample refers to wells containing 1 copy of a ruminant-specific target sequence and 400,000 copies of a porcine-specific target sequence per ⁇ to verify proper detection of ruminant mitochondrial DNA in test samples.
  • NTC refers to a negative control containing buffer, but no ruminant- or porcine-specific target sequence, to verify the purity of the PCR reagents.
  • H refers to test samples containing DNA extracted from heparin.
  • 0 refers to the first and 00 to the second extraction of an unknown sample.
  • EC refers to an extraction control to verify the absence of contamination during DNA extraction. The shaded columns indicate wells that are spiked with 8 copies of a ruminant-specific target- sequence per ⁇ .
  • target sequence is intended to mean a single or double stranded polynucleotide sequence that is to be identified in the method of the invention.
  • the target sequence may be derived from the mitochondrial genome of porcine or ruminant species such that the ruminant-specific sequence is selected from portions of the mitochondrial genome with a high degree of sequence similarity (e.g. more than 96% sequence identity) among ruminant species, in particular of the family Bovidae comprising cattle, sheep and goats, but with a high degree of dissimilarity (e.g. less than 85% sequence identity) to the corresponding sequence of the porcine mitochondrial genome.
  • a high degree of sequence similarity e.g. more than 96% sequence identity
  • dissimilarity e.g. less than 85% sequence identity
  • the ruminant-specific target sequence is selected so as to be specific to a large number of ruminant species, including Gayal, Zebu, Bison, Kudu, Ibex, Chamois, Tahr, Chinese Antelope, Springbok, Gazelle, Blesbock, Gnu, Impala, Yellow- backed Duiker, Common Duiker, Oryx, Deer, Roedeer, Chinese Roedeer, Moose, Fallow Deer, Giraffe and Java Mouse-deer, and particularly to ruminants of the family Bovidae, in particular the most common domesticated bovids, i.e. cattle, sheep and goats.
  • porcine-specific sequence is selected from portions of the mitochodrial genome with a high degree of dissimilarity (less than 85% sequence identity) to the corresponding portions of the ruminant mitochondrial genome.
  • target sequences are selected from portions of the mitochondrial genome that have a high degree of sequence identity among a wide selection of breeds of ruminants and pigs, respectively.
  • an addtional . selection criterion was applied, i.e. the content of GC nucleotides which was set at between 45% and 55%.
  • percent sequence identity is intended to convey the degree of similarity between two sequences that are aligned for maximum correspondence by a local or global alignment algorithm such as the Smith-Waterman algorithm or the Needleman- Wunsch algorithm (Biological sequence analysis: probabilistic models of proteins and nucleic acids. R. Durbin, S. Eddy, A. Krogh, G. Mitchison. Cambridge University Press, 1998) or by computerized implementations of such algorithms as found in for example the Bioconductor (Gentleman et al. 2004 Genome Biology 5(10) : R80) package Biostrings in the statistical programming language R (www.r-project.org).
  • a local or global alignment algorithm such as the Smith-Waterman algorithm or the Needleman- Wunsch algorithm (Biological sequence analysis: probabilistic models of proteins and nucleic acids. R. Durbin, S. Eddy, A. Krogh, G. Mitchison. Cambridge University Press, 1998) or by computerized implementations of such algorithms as found in for example the Bioconductor (Gentleman et al. 2004 Genome Biology
  • nucleotide is intended to mean any naturally occurring nucleotide such as adenine (A), thymine (T), cytosine (C), guanine (G) or uracil (U) or a synthetic nucleotide such as inosine (I), 5-nitroindole or a pyrimidine or purine derivative capable of binding to any of the nucleotides A, T, C or G.
  • the nucleotide may be modified by methyl, ethyl, halogen, sulfur, carboxy or a sugar residue.
  • oligonucleotide is intended to mean a single-stranded nucleotide sequence composed of up to 50 nucleotides.
  • the sequence is preferably a DNA sequence, though the phosphate-deoxyribose backbone may be modified by phosphothioate, ribose, locked nucleic acids (LNA; Kumar et al., Bioorg. Med. Chem. Lett. 8(16), 1998, pp. 525-528), peptide nucleic acids (PNA; Nielsen et al, Anticancer Drug Des. 8(1), 1993, pp. 53-63), unlocked nucleic acids (UNA; Jensen et al., Nucleic Acids Symp. Ser. 52(1), 2008, pp. 133-134), or a derivative thereof.
  • oligonucleotides may be used as primers and probes in the PCR-based method of amplifying the ruminant or porcine target sequence.
  • primer is intended to mean an oligonucleotide sequence that is able to anneal to the 3' end of each complementary strand of the target sequence to be amplified. Primers are required for DNA polymerase extension of the separate strands of the target sequence such that the forward primer is used to extend one strand of double-stranded (ds) DNA and the reverse primer is used to extend the complementary strand of dsDNA. Primers that may be used to amplify target sequences according to the present method may have at least 90% sequence identity to the primers shown in Tables 1-12 below.
  • probe is intended to mean an oligonucleotide sequence capable of hybridizing to a single-stranded target sequence during PCR and usually labelled for detection of the presence of the target sequence.
  • polynucleotide sequence is intended to indicate a single- or double-stranded nucleotide sequence composed of from about 50 to about 300 nucleotides.
  • test sample is intended to indicate any sample of biological origin which is suspected of containing contaminant ruminant or porcine material detectable by means of the ruminant- or porcine-specific target sequence.
  • the test sample may be a food product, food ingredient, cosmetic ingredient, pharmaceutical ingredient, therapeutic protein or therapeutic polysaccharide.
  • the polynucleotide target sequences used in the present method are derived from mitochondrial DNA. This is advantageous because mitochondrial DNA is more abundant in mammalian cells than nuclear DNA as it is present in 100-1000 copies per cell for each copy of nuclear DNA. A higher amount of mitochondrial DNA is therefore likely to be present in the biological sample to be tested and is more likely to be detected by the present method reducing the risk of producing false negative results of the test. Furthermore, it has been found that certain portions of the mitochondrial genome are highly conserved in ruminant species, and it is therefore possible to identify several target sequences with at least 96% sequence identity between the bovine, ovine and caprine sequences (such sequences are termed "consensus sequences" in the following).
  • PCR amplification of these sequences upon extraction of DNA from a biological sample makes it possible to detect the presence of not only bovine, but also ovine and caprine DNA in the sa mple using just one set of pri mers a nd probe. Th is represents a significant si mplification of the PCR methods published so far. More specifically, the present ruminant target sequences represent consensus sequences of the bovine, ovine and caprine mitochondrial genome. The location of the bovine, ovine and caprine sou rce seq uences shown schematically i n Fig 1. is specifically found in the following portions of the bovine mitochondrial genome :
  • AACCTCACCA ATNCTTGCTA ATACAGTCTA TATACC ( SEQ ID NO : 2 is derived from positions 947 to 1042 which is within the region encoding 12S)
  • CATACC (SEQ ID IMO : 5 is derived from positions 4107 to 4232 which is within the region encoding tRNA) ANANCTTACA TCAATTGAAT GCAAATCAAC CACTTTAATT AAGCTAAATC CTNACTAGAN TGGTGGGCTC CACCCCC (SEQ ID NO : 6 is derived from positions 5392 to 5468 which is within the region encoding tRNA)
  • Position numbers refer to the bovine mitochondrial genome with GenBank ID : V00654.
  • One particular embodiment of a ruminant target sequence comprises the sequence
  • n is any nucleotide
  • This sequence is a partial sequence of SEQ ID NO : 5, and may be used as a target for amplification in the present method for detecting ruminant contamination in samples of crude porcine heparin, cf. Example 3 below.
  • oligonucleotide sequences shown (from 5' to 3') as forward primers, probes and reverse primers, respectively, in Table 1 below may be employed .
  • forward primer (F), probe (P) and reverse primer may (R) be selected as follows.
  • m be the maximum number of forward primers for a sequence region (e.g. 34 in Table 1).
  • Select a specific forward primer F Formula where / is an integer between 1 and m.
  • select a probe P j where j is an integer between / ' and m.
  • select a reverse primer R k where k is an integer between j and m.
  • the reverse primer may be selected from reverse primers where where k is between 14 and 34. This selection scheme ensures that the probe sequence is postioned downstream from the forward primer, and that the reverse primer is positioned downstream from the probe.
  • oligonucleotide sequences shown as forward primers, probes and reverse primers, respectively, in Table 2 below may be employed.
  • forward primer may be combined according to the selection criteria shown above.
  • the oligonucleotide sequences shown as forward primers, probes and reverse primers, respectively, in Table 3 below may be employed.
  • forward primer may be combined according to the selection criteria shown above.
  • oligonucleotide sequences shown as forward primers, probes and reverse primers, respectively, in Table 4 below may be employed.
  • GGTGACATGCCTAACG AGCCTGGTGATAGCTG TCATTTTCTGGACAAC
  • forward primer may be combined according to the selection criteria shown above.
  • AAACTCTTCGTGCTCC CAATTACACCAAATTC GCTGACCTTACTANTA
  • forward primer may be combined according to the selection criteria shown above.
  • the forward primer may comprise the sequence
  • the reverse primer may comprise the sequence
  • TTAATTAGCT GACCTTACTA NTAG (SEQ ID l ⁇ IO : 870, spanning SEQ ID Nos : 576-584 in Table 5)
  • IM is any nucleotide.
  • the probe may comprise the sequence
  • AAACTCTTCG TGCTCCCAAT TACA (SEQ ID NO : 871 spanning SEQ ID Nos:640-648 in
  • the oligonucleotide sequences shown as forward primers, probes and reverse primers, respectively, in Table 6 below may be employed.
  • CAATTGAATGCAAATC AACCACTTTAATTAAG TCTAGTNAGGATTTAG
  • forward primer may be combined according to the selection criteria shown above.
  • porcine-specific target sequences are shown schematically in Fig . 2 and may be found in the following portions of the porcine mitochondrial genome
  • AA (SEQ ID NO : 874 is derived from positions 7578 to 7699 which is within the region encoding COX1)
  • Position numbers refer to the porcine mitochondrial genome with GenBank ID :
  • porcine mitochondrial polynucleotide sequence comprises the sequence
  • oligonucleotide sequences shown (from 5' to 3') as forward primers, probes and primers, respectively, in Table 8 below may be employed .
  • forward primer may be combined according to the selection criteria shown above.
  • oligonucleotide sequences shown as forward primers, probes and reverse primers, respectively, in Table 9 below may be employed.
  • SEQ ID NO.-1077 SEQ ID NO: 1329) (SEQ ID NO:1203) ACGAAAAGGACCCAAC GTTGTAGGCCCCTACG ATGGGTTGGAGTAGGC
  • forward primer may be combined accord the selection criteria shown above.
  • oligonucleotide sequences shown as forward primers, probes and reverse primers, respectively, in Table 10 below may be employed.
  • GATTAGCTACCCTGCA CGGCGGCAATATTAAA GTATTGCGGGTGATCA
  • CTCTGGGCTTCATCTT CCTATTCACCGTAGGA CAATGCCCGTTAGACC
  • CTTCAICTTCCTATTC ACCGTAGGAGGTCTAA GCTAGTACAATGCCCG
  • forward primer may be combined according to the selection criteria shown above.
  • oligonucleotide sequences shown as forward primers, probes and reverse primers, respectively, in Table 11 below may be employed.
  • TCTTCACTGGATTCTT TTGAGCTTTCTACCAC TTGGTGCTAGGCTTGA

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Abstract

L'invention concerne un procédé de détection de matière contaminante de ruminant dans un échantillon biologique. Ce procédé consiste : (a) à extraire l'ADN génomique total de l'échantillon ; (b) à amplifier une séquence cible spécifique de ruminant présentant au moins 96% d'identité de séquence avec une des séquences SEQ ID NO: 1-7 susceptible d'être présente dans l'ADN extrait de l'échantillon, par la mise en contact de l'ADN extrait avec une amorce sens de SEQ ID NO:9-42, SEQ ID NO: ll l-159, SEQ ID NO: 258-286, SEQ ID NO : 345-377, SEQ ID NO:444-522, SEQ ID NO: 681-710 ou SEQ ID NO:771-803, une amorce anti-sens de SEQ ID NO :43-76, SEQ ID NO: 160-208, SEQ ID NO: 287-315, SEQ ID NO: 378-410, SEQ ID NO: 523-601, SEQ ID NO:711-740 ou SEQ ID NO: 804-836 et une sonde de SEQ ID NO:77-110, SEQ ID NO: 209-257, SEQ ID NO: 316-344, SEQ ID NO:411-443, SEQ ID NO: 602-680, SEQ ID NO: 741-770 ou SEQ ID NO:837-869, en présence d'un mélange de réaction contenant une ADN polymérase, un mélange de nucléotides et un tampon ; et (c) à détecter la présence de la séquence cible spécifique de ruminant amplifiée, la détection indiquant la présence de matière contaminante de ruminant dans l'échantillon.
PCT/DK2011/000127 2010-11-05 2011-11-04 Procédé de détection d'adn contaminant dans des échantillons biologiques WO2012059102A2 (fr)

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WO2024017382A1 (fr) * 2022-07-22 2024-01-25 中国食品药品检定研究院 Amorce pour la détection d'adn porcin et procédé de détection

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