U.S. patent application number 12/707828 was filed with the patent office on 2010-07-22 for oligonucleotide sequences that identify species of animal.
This patent application is currently assigned to NATIONAL INSTITUTE OF AGROBIOLOGICAL SCIENCES. Invention is credited to Koichi Kadowaki, Toyoko Kusama, Tetsuya Nomura.
Application Number | 20100184078 12/707828 |
Document ID | / |
Family ID | 35096706 |
Filed Date | 2010-07-22 |
United States Patent
Application |
20100184078 |
Kind Code |
A1 |
Kusama; Toyoko ; et
al. |
July 22, 2010 |
OLIGONUCLEOTIDE SEQUENCES THAT IDENTIFY SPECIES OF ANIMAL
Abstract
The present invention provides a method for identifying animal
species, said method comprises a step of amplifying a DNA fragment
by PCR using a DNA in a sample as a template and animal-specific
DNA sequences as a primer pair, wherein the animal-specific DNA
sequences are derived from a ATP synthase subunit 8 gene or a
region proximal thereto of a mitochondrial genome; and a step of
detecting the amplified DNA fragment.
Inventors: |
Kusama; Toyoko; (Chiba,
JP) ; Kadowaki; Koichi; (Ibaraki, JP) ;
Nomura; Tetsuya; (Saitama, JP) |
Correspondence
Address: |
TUROCY & WATSON, LLP
127 Public Square, 57th Floor, Key Tower
CLEVELAND
OH
44114
US
|
Assignee: |
NATIONAL INSTITUTE OF
AGROBIOLOGICAL SCIENCES
Tsukuba-shi
JP
INCORPORATED ADMINISTRATIVE AGENCY FOOD AND AGRICULTURAL
MATERIALS INSPECTION CENTER
Saitama-shi
JP
|
Family ID: |
35096706 |
Appl. No.: |
12/707828 |
Filed: |
February 18, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10826119 |
Apr 16, 2004 |
|
|
|
12707828 |
|
|
|
|
Current U.S.
Class: |
435/6.1 |
Current CPC
Class: |
C12Q 1/6888 20130101;
C12Q 1/6881 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of detecting animal-derived components in a sample,
comprising: providing a sample comprising DNA; adding a pair of DNA
primers and a DNA polymerase to the sample to form a PCR mixture,
the pair of DNA primers having a structure to prime a PCR reaction
if the sample comprises components derived from animals or a
specific grouping or species of animals; subjecting the PCR mixture
to conditions suitable for promoting PCR including bringing the PCR
mixture to a first temperature sufficient to denature the DNA in
the sample followed by a second temperature, less than the first
temperature, sufficient to anneal the pair of DNA primers to a
complementary DNA sequence; and determining the source of
components in the sample using one of the following criteria: 1)
observing a single PCR product when the sample comprises components
derived from animals or a specific grouping or species of animals;
and 2) observing a lack of a PCR product when the sample does not
comprise components derived from animals or a specific grouping or
species of animals, wherein the animals or the specific groups or
species of animals are selected from the group consisting of
mammal, ruminant, cattle, sheep, goat, deer, pig, horse, rabbit,
whale, chicken, and fish.
2. The method of claim 1, wherein the animals or the specific
grouping or species of animals are mammals, and wherein the pair of
DNA primers comprises a first primer having a DNA sequence of SEQ
ID NO: 1 and a second primer having a DNA sequence of SEQ ID NO:
2.
3. The method of claim 1, wherein the animals or the specific
grouping or species of animals are ruminants, and wherein the pair
of DNA primers comprises a first primer having a DNA sequence of
SEQ ID NO: 3 and a second primer having a DNA sequence of SEQ ID
NO: 4.
4. The method of claim 1, wherein the animals or the specific
grouping or species of animals are ruminants, and wherein the pair
of DNA primers comprises a first primer having a DNA sequence of
SEQ ID NO: 5 and a second primer having a DNA sequence of SEQ ID
NO: 6.
5. The method of claim 1, wherein the animals or the specific
grouping or species of animals are cattle, wherein the pair of DNA
primers comprises a combination of a first primer having a DNA
sequence and a second primer having a DNA sequence, and wherein the
combination of the first and the second primers is selected from
the group consisting of the following DNA sequence combinations:
SEQ ID NO: 9 and SEQ ID NO: 13; SEQ ID NO: 9 and SEQ ID NO: 12; SEQ
ID NO: 11 and SEQ ID NO: 13; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ
ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 8 and SEQ ID NO: 12; and
SEQ ID NO: 14 and SEQ ID NO: 15.
6. The method of claim 1, wherein the animals or the specific
grouping or species of animals are pigs, wherein the pair of DNA
primers comprises a combination of a first primer having a DNA
sequence and a second primer having a DNA sequence, and wherein the
combination of the first and the second primers is a combination of
a DNA sequence of SEQ ID NO: 17 and a DNA sequence of SEQ ID NO:
19; or a combination of a DNA sequence of SEQ ID NO: 18 and a DNA
sequence of SEQ ID NO: 22.
7. The method of claim 1, wherein the animals or the specific
grouping or species of animals are sheep, and wherein the pair of
DNA primers comprises a first primer having a DNA sequence
comprising SEQ ID NO: 23 and a second primer having a DNA sequence
comprising SEQ ID NO: 24.
8. The method of claim 1, wherein the animals or the specific
grouping or species of animals are goats, and wherein the pair of
DNA primers comprises a first primer having a DNA sequence
comprising SEQ ID NO: 25 and a second primer having a DNA sequence
comprising SEQ ID NO: 26.
9. The method of claim 1, wherein the animals or the specific
grouping or species of animals are chickens, and wherein the pair
of DNA primers comprises a first primer having a DNA sequence of
SEQ ID NO: 28 and a second primer having a DNA sequence of SEQ ID
NO: 30.
10. The method of claim 1, wherein the animals or the specific
grouping or species of animals are fish, and wherein the pair of
DNA primers comprises a first primer having a DNA sequence
comprising one selected from the group consisting of SEQ ID NOS:
32, 34, 38, and 39; and a second primer having a DNA sequence
comprising one selected from the group consisting of SEQ ID NOS:
33, 35, 36, 37, 40, and 41.
11. A method of detecting ruminant-derived components in a sample,
comprising: providing a sample comprising DNA; adding a pair of DNA
primers and a DNA polymerase to the sample to form a PCR mixture,
the pair of DNA primers having a structure to prime a PCR reaction
if the sample comprises components derived from ruminants or a
specific grouping or species of ruminants; subjecting the PCR
mixture to conditions suitable for promoting PCR including bringing
the PCR mixture to a first temperature sufficient to denature the
DNA in the sample followed by a second temperature, less than the
first temperature, sufficient to anneal the pair of DNA primers to
a complementary DNA sequence; and determining the source of
components in the sample using one of the following criteria: 1)
observing a single PCR product when the sample comprises components
derived from ruminants or a specific grouping or species of
ruminants; and 2) observing a lack of a PCR product when the sample
does not comprise components derived from ruminants or a specific
grouping or species of ruminants.
12. The method of claim 11, wherein the pair of DNA primers
comprises a first primer having a DNA sequence of SEQ ID NO: 3 and
a second primer having a DNA sequence of SEQ ID NO: 4.
13. The method of claim 11, wherein the pair of DNA primers
comprises a first primer having a DNA sequence of SEQ ID NO: 5 and
a second primer having a DNA sequence of SEQ ID NO: 6.
14. The method of claim 11, wherein the ruminants or the specific
grouping or species of ruminants are cattle.
15. The method of claim 14, wherein pair of DNA primers comprises a
combination of a first primer having a DNA sequence and a second
primer having a DNA sequence, and wherein the combination of the
first and the second primers is selected from the group consisting
of the following DNA sequence combinations: SEQ ID NO: 9 and SEQ ID
NO: 13; SEQ ID NO: 9 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID
NO: 13; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID
NO: 12; SEQ ID NO: 8 and SEQ ID NO: 12; and SEQ ID NO: 14 and SEQ
ID NO: 15.
16. The method of claim 11, wherein the ruminants or the specific
grouping or species of ruminants are sheep.
17. The method of claim 16, and wherein the pair of DNA primers
comprises a first primer having a DNA sequence comprising SEQ ID
NO: 23 and a second primer having a DNA sequence comprising SEQ ID
NO: 24.
18. The method of claim 11, wherein the ruminants or the specific
grouping or species of ruminants are goats.
19. The method of claim 18, wherein the pair of DNA primers
comprises a first primer having a DNA sequence comprising SEQ ID
NO: 25 and a second primer having a DNA sequence comprising SEQ ID
NO: 26.
20. The method of claim 11, wherein the pair of DNA primers
comprise a DNA sequence having a structure to anneal to a
mitochondrial ATP synthase subunit 8 gene or a region proximal
thereto of a mitochondrial genome.
21. The method of claim 11, wherein ruminants include one or more
selected from the group consisting of cattle, sheep, goats, and
deer.
22. The method of claim 11, further comprising preparing the sample
comprising DNA by extracting mitochondrial DNA from a sample
comprising one or more of animal and plant material.
23. The method of claim 22, wherein the sample is substantially
free from non-mitochondrial DNA.
24. A method of detecting ruminant-derived components in a sample,
comprising: providing a sample comprising DNA; adding a DNA primer
pair having a structure to anneal to a ruminant-derived DNA target
and prime a PCR reaction to a mitochondrial ATP synthase subunit 8
gene or a region proximal thereto of a mitochondrial genome of
ruminants and having a structure to not prime a PCR reaction of a
non-ruminant DNA target, to form a PCR mixture; subjecting the PCR
mixture to conditions and reagents suitable for promoting PCR
including bringing the PCR mixture to a first temperature
sufficient to denature the DNA in the sample followed by a second
temperature, less than the first temperature, sufficient to anneal
the pair of DNA primers to a complementary DNA sequence; and
determining the source of components in the sample using one of the
following criteria: 1) observing a single PCR product and assigning
the sample as containing ruminant-derived components; and 2)
observing a lack of a PCR product and assigning the sample as not
containing ruminant-derived components.
25. The method of claim 24, wherein the pair of DNA primers
comprises a first primer having a DNA sequence of SEQ ID NO: 3 and
a second primer having a DNA sequence of SEQ ID NO: 4.
26. The method of claim 24, wherein the pair of DNA primers
comprises a first primer having a DNA sequence of SEQ ID NO: 5 and
a second primer having a DNA sequence of SEQ ID NO: 6.
27. The method of claim 24, wherein the ruminant is cattle.
28. The method of claim 27, wherein pair of DNA primers comprises a
combination of a first primer having a DNA sequence and a second
primer having a DNA sequence, and wherein the combination of the
first and the second primers is selected from the group consisting
of the following DNA sequence combinations: SEQ ID NO: 9 and SEQ ID
NO: 13; SEQ ID NO: 9 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID
NO: 13; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID
NO: 12; SEQ ID NO: 8 and SEQ ID NO: 12; and SEQ ID NO: 14 and SEQ
ID NO: 15.
29. The method of claim 24, wherein the ruminants are sheep.
30. The method of claim 29, and wherein the pair of DNA primers
comprises a first primer having a DNA sequence comprising SEQ ID
NO: 23 and a second primer having a DNA sequence comprising SEQ ID
NO: 24.
31. The method of claim 24, wherein the ruminants are goats.
32. The method of claim 31, wherein the pair of DNA primers
comprises a first primer having a DNA sequence comprising SEQ ID
NO: 25 and a second primer having a DNA sequence comprising SEQ ID
NO: 26.
33. The method of claim 24, wherein ruminants include one or more
selected from the group consisting of cattle, sheep, goats, and
deer.
34. A method of detecting ruminant-derived components in a sample,
comprising: providing a sample comprising DNA; adding a pair of
ruminant-specific DNA primers consisting of a combination of a
first primer having a DNA sequence and a second primer having a DNA
sequence, and wherein the combination of the first and second
primers is a combination of a DNA sequence of SEQ ID NO. 3 and a
DNA sequence of SEQ ID NO. 4; and a combination of a DNA sequence
of SEQ ID NO. 5 and a DNA sequence of SEQ ID NO. 6; amplifying a
DNA sequence from the DNA in the sample by PCR, wherein the
amplified sequence comprises one or more sequences selected from
the group consisting of the sequence of a mitochondrial ATP
synthase subunit 8 gene and a region proximal thereto in a
mitochondrial genome of a ruminant; and observing a single PCR
product and assigning the sample as containing ruminant-derived
components.
35. The method of claim 34, wherein the sample comprises DNA
derived from one or more selected from the group consisting of
cattle, sheep, goats, and deer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of application Ser. No.
10/826,119 filed Apr. 16, 2004, the entire contents of which is
hereby incorporated by reference. Applicants request use of the
compliant computer readable "Sequence Listing" that is already on
file for application Ser. No. 10/826,119 filed Apr. 16, 2004 and
state that the paper or compact disc copy of the "Sequence Listing"
in this application is identical to the computer readable copy
filed for application Ser. No. 10/826,119 filed Apr. 16, 2004.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to methods for identifying
animal species and animal-derived DNA-specific primer pairs used
therein. More specifically, the present invention relates to
methods for identifying animal species that include a step of
amplifying an animal-specific DNA sequence derived from the ATP
synthase subunit 8 gene of a mitochondrial genome, and primer pairs
used in this amplification step.
[0004] 2. Description of the Related Art
[0005] Currently, there is a problem that cattle is infected with
bovine spongiform encephalopathy (BSE) by giving feed containing
meat and bonemeal derived from cattle infected with BSE. It has
been shown that BSE-like diseases may be present in various
livestock as well. Accordingly, since the emergence of BSE, there
has been a need to develop sensitive and effective method to
identify feed contaminated bone and bonemeal, and this has become a
particularly urgent matter for authorities.
[0006] Immunological methods and gene identification methods using
nuclear gene conventionally have been used as methods for
identifying animal species. Examples of immunological methods
include ELISA and immunoblotting. PCR is an example of a gene
identification method using nuclear gene. However, there are many
problems with methods for identifying the animal species that are
currently employed. For example, in meat and bonemeal that has been
heat-treated, there is a high likelihood that nucleic acids have
been fragmented. Furthermore, majority of feed in which meat and
bonemeal has been mixed is composed of plant-derived material.
Therefore, it is necessary to analyze trace amounts of
animal-derived components. There is a dire need for the development
of a detection method that is highly sensitive and effective and
that can be executed with respect to such heat-treated samples.
[0007] Accordingly, a method other than immunological methods and
gene identification methods employing nuclear gene that is for
detecting and identifying the animal species of animal-derived
components that are present in trace amounts is desirable. In
particular, it is crucial to identify the type of animal meat, meat
and bonemeal, or fishmeal used in feed given to livestock and pets.
Furthermore, it is desirable that the detection method is highly
sensitive and differentiate species of animal DNA present in trace
amounts from among large quantities of plant DNA or DNA of other
animal species.
SUMMARY OF THE INVENTION
[0008] The inventors of the present application focused on the
mitochondrial genome, which is inherited maternally and exists in a
greater number of copies than nuclear genome, and investigated the
use of mitochondrial gene as a target for identifying animal
species. Their research indicated that the homologous sequence with
ATP synthase subunit 8 gene (atp8 gene) from animal mitochondrial
genome is not present in plant (Oryza sativa) mitochondrial genome.
Thus, the inventors found that the DNA derived from the atp8 gene
can serve as a material for specific detection of trace amounts of
animal DNA among plant-based feed, in other word, plant atp8 gene
is very diverged from animal atp8 gene, and also that specific DNA
sequences of the animal mitochondrial atp8 gene can be used to
identify the animal species, thereby arriving at the present
invention.
[0009] The present invention provides a method for identifying
animal species, said method comprises:
[0010] amplifying a DNA sequence by PCR using a DNA in a sample as
a template and animal-specific DNA sequences as a primer pair,
wherein the animal-specific DNA sequences are derived from a ATP
synthase subunit 8 gene or a region proximal thereto of a
mitochondrial genome, and
[0011] detecting the amplified DNA sequence.
[0012] In a preferred embodiment, the animal is a mammal, and
further preferably, the primer pair is a combination of the DNA
sequence of SEQ ID NO: 1 and the DNA sequence of SEQ ID NO: 2.
[0013] In a further preferred embodiment, the mammal is selected
from the group consisting of cattle, sheep, goat, deer, pig, horse,
rabbit, and whale.
[0014] In a preferred embodiment, the animal is a ruminant, and
further preferably, the primer pair is a combination of the DNA
sequence of SEQ ID NO: 3 and the DNA sequence of SEQ ID NO: 4, or a
combination of the DNA sequence of SEQ ID NO: 5 and the DNA
sequence of SEQ ID NO: 6.
[0015] In a further preferred embodiment, the ruminant is selected
from the group consisting of cattle, sheep, goat, and deer.
[0016] In a preferred embodiment, the animal is a cattle, and
further preferably, the primer pair is a combination of DNA
sequences selected from the group consisting of the following DNA
sequence combinations: SEQ ID NO: 9 and SEQ ID NO: 13; SEQ ID NO: 9
and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 13; SEQ ID NO: 10
and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 8
and SEQ ID NO: 12; and SEQ ID NO: 14 and SEQ ID NO: 15.
[0017] In a preferred embodiment, the animal is a pig, and further
preferably, the primer pair is a combination of the DNA sequence of
SEQ ID NO: 17 and the DNA sequence of SEQ ID NO: 19, or a
combination of the DNA sequence of SEQ ID NO: 18 and the DNA
sequence of SEQ ID NO: 22.
[0018] In a preferred embodiment, the animal is a sheep, and
further preferably, the primer pair is a combination of the DNA
sequence of SEQ ID NO: 23 and the DNA sequence of SEQ ID NO:
24.
[0019] In a preferred embodiment, the animal is a goat, and further
preferably, the primer pair is a combination of the DNA sequence of
SEQ ID NO: 25 and the DNA sequence of SEQ ID NO: 26.
[0020] In a preferred embodiment, the animal is a chicken, and
further preferably, the primer pair is a combination of the DNA
sequence of SEQ ID NO: 28 and the DNA sequence of SEQ ID NO:
30.
[0021] In a preferred embodiment, the animal is a fish, and further
preferably, the primer pair is a combination of the DNA sequence
selected from the group consisting of SEQ ID NOS: 32, 34, 38 and 39
and the DNA sequence selected from the group consisting of SEQ ID
NOS: 33, 35, 36, 37, 40, and 41.
[0022] In a further preferred embodiment, the fish is selected from
the group consisting of sardine, flatfish, salmon, Alaska Pollack,
tuna, and lady crab.
[0023] In a further preferred embodiment, the sample is selected
from a group consisting of raw meat, raw fish, processed meat food
products, processed fish food products, food products containing
processed meat, food products containing processed fish, blood,
hair, body fluids, milk, milk processing products, meat and
bonemeal, bonemeal, fishmeal, fish soluble, and feed, fertilizer,
and feed additive containing them.
[0024] The present invention also provides a primer pair for
detection of a mammal-specific DNA, the primer pair being a
combination of the DNA sequence of SEQ ID NO: 1 and the DNA
sequence of SEQ ID NO: 2.
[0025] In a preferred embodiment, the mammal is selected from the
group consisting of cattle, sheep, goat, deer, pig, horse, rabbit,
and whale.
[0026] The present invention also provides a primer pair for
detection of a ruminant-specific DNA, the primer pair being a
combination of the DNA sequence of SEQ ID NO: 3 and the DNA
sequence of SEQ ID NO: 4, or a combination of the DNA sequence of
SEQ ID NO: 5 and the DNA sequence of SEQ ID NO: 6.
[0027] In a preferred embodiment, the ruminant is selected from the
group consisting of cattle, sheep, goat, and deer.
[0028] The present invention also provides a primer pair for
detection of a cattle-specific DNA, the primer pair being a
combination of DNA sequences selected from the group consisting of
the following DNA sequence combinations: SEQ ID NO: 9 and SEQ ID
NO: 13; SEQ ID NO: 9 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID
NO: 13; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID
NO: 12; SEQ ID NO: 8 and SEQ ID NO: 12; and SEQ ID NO: 14 and SEQ
ID NO: 15.
[0029] The present invention also provides a primer pair for
detection of a pig-specific DNA, the primer pair being a
combination of the DNA sequence of SEQ ID NO: 17 and the DNA
sequence of SEQ ID NO: 19, or a combination the DNA sequence of SEQ
ID NO: 18 and the DNA sequence of SEQ ID NO: 22.
[0030] The present invention also provides a primer pair for
detection of a sheep-specific DNA, the primer pair being a
combination of the DNA sequence of SEQ ID NO: 23 and the DNA
sequence of SEQ ID NO: 24.
[0031] The present invention also provides a primer pair for
detection of a goat-specific DNA, the primer pair being a
combination of the DNA sequence of SEQ ID NO: 25 and the DNA
sequence of SEQ ID NO: 26.
[0032] The present invention also provides a primer pair for
detection of a chicken-specific DNA, the primer pair being a
combination of the DNA sequence of SEQ ID NO: 28 and the DNA
sequence of SEQ ID NO: 30.
[0033] The present invention also provides a primer pair for
detection of a fish-specific DNA, the primer pair being a
combination of the DNA sequence selected from the group consisting
of SEQ ID NOS: 32, 34, 38 and 39 and the DNA sequence selected from
the group consisting of SEQ ID NOS: 33, 35, 36, 37, 40, and 41.
[0034] In a preferred embodiment, the fish is selected from the
group consisting of sardine, flatfish, salmon, Alaska Pollack,
tuna, and lady crab.
[0035] The present invention also provides a primer pair for
detection of a plant-specific DNA, the primer pair being a
combination of the DNA sequence of SEQ ID NO: 42 and the DNA
sequence of SEQ ID NO: 43.
[0036] Further, the present invention provides a method for
detecting animal-derived components present in mixed feed, said
method comprises:
[0037] amplifying a DNA sequence by PCR using a DNA in a sample as
a template and animal-specific DNA sequences as a primer pair,
wherein the animal-specific DNA sequences are derived from a ATP
synthase subunit 8 gene or a region proximal thereto of a
mitochondrial genome, and
[0038] detecting the amplified DNA sequence.
[0039] The present invention also provides a kit for detecting an
animal-derived component present in a sample, said kit comprises at
least one of primer pair specific for any animal described
above.
[0040] In a preferred embodiment, the kit further comprises a
primer pair for detection of a plant-specific DNA, said primer pair
is a combination of the DNA sequence of SEQ ID NO: 42 and the DNA
sequence of SEQ ID NO: 43.
[0041] Further, the present invention provides a method for
detecting plant-derived components present in sample, said method
comprises:
[0042] amplifying a DNA sequence by PCR using a DNA in a sample as
a template and plant-specific DNA sequences as a primer pair,
wherein the plant-specific DNA sequences are derived from a ATP
synthase subunit 8 gene or a region proximal thereto of a
mitochondrial genome, and
[0043] detecting the amplified DNA sequence.
[0044] Using the method of the present invention, it is possible to
detect, with high sensitivity, trace amounts of animal-derived DNA
in a sample. It is thus applicable to identify the animal species
of trace amounts of meat and bonemeal mixed into feed. More
particularly, detection is possible even if trace amounts of
cattle-derived meat and bonemeal are mixed into mixed feed for
livestock. Further, the primers for the detection of fish-specific
DNA according to the present invention are useful for detecting a
wide range of fishmeal derived from various fish species that has
been mixed into mixed feed or the like.
BRIEF DESCRIPTION OF DRAWINGS
[0045] FIG. 1 shows a sequence alignment of mitochondrial atp8
genes derived from various types of animals.
[0046] FIG. 2 shows a sequence alignment of chicken, cattle, and
pig mitochondrial atp8 genes.
[0047] FIG. 3 shows a sequence alignment of mitochondrial atp8
genes, and nearby regions, derived from various types of
animals.
[0048] FIG. 4 shows the positional relationship on the
mitochondrial atp8 gene of the primers used to specifically detect
cattle-derived DNA sequences.
[0049] FIG. 5 is a photograph showing the results of
electrophoresis after performing PCR using a primer pair (anicon5
and anicon3) for detecting mammal-specific DNA sequences and with
DNAs derived from various types of animals serving as
templates.
[0050] FIG. 6 is a photograph showing the results of
electrophoresis after performing PCR using a primer pair (rumicon5
and rumicon3) for detecting ruminant-specific DNA sequences and
with DNAs derived from various types of animals serving as
templates.
[0051] FIG. 7 is a photograph showing the results of
electrophoresis after performing PCR using a primer pair (Fpr-F and
Fpr-R) for detecting ruminant-specific DNA sequences and with DNAs
derived from various types of animals serving as templates.
[0052] FIG. 8 is a photograph showing the results of
electrophoresis after performing PCR using a primer pair (cow3 and
cow51) for detecting cattle-specific DNA sequences and with DNAs
derived from various types of animals serving as templates.
[0053] FIG. 9 is a photograph showing the results of
electrophoresis after performing PCR using various primer pairs for
detecting cattle-specific DNA sequences and with DNAs derived from
various types of animals serving as templates.
[0054] FIG. 10 is a photograph showing the results of
electrophoresis after performing PCR using various primer pairs for
detecting cattle-specific DNA sequences and with DNAs derived from
various types of animals serving as templates.
[0055] FIG. 11 is a photograph showing the results of
electrophoresis after performing PCR using a primer pair (Fpc-F and
Fpc-R) for detecting cattle-specific DNA sequences and with DNAs
derived from various types of animals serving as templates.
[0056] FIG. 12 is a photograph showing the results of
electrophoresis after performing PCR using a primer pair (pig51 and
pig3) for detecting pig-specific DNA sequences and with DNAs
derived from various types of animals serving as templates.
[0057] FIG. 13 is a photograph showing the results of
electrophoresis after performing PCR using a primer pair (pig5-3
and pig32-2) for detecting pig-specific DNA sequences and with DNAs
derived from various types of animals serving as templates.
[0058] FIG. 14 is a photograph showing the results of
electrophoresis after performing PCR using a primer pair (sheep5
and sheep3) for detecting sheep-specific DNA sequences and a primer
pair (goat5 and goat3) for detecting goat-specific DNA sequences,
and with DNAs derived from various types of animals serving as
templates.
[0059] FIG. 15 is a photograph showing the results of
electrophoresis after performing PCR using a primer pair for
detecting chicken-specific DNA sequences and with DNAs derived from
various types of animals serving as templates.
[0060] FIG. 16 is an electrophoresis photograph showing the results
of the detection of DNA, using mammal-specific primer pairs, using
various primer pairs, in mixed feed for livestock that contains
cattle meat and bonemeal.
[0061] FIG. 17 is an electrophoresis photograph showing the results
of the detection of DNA, using ruminant-specific primer pairs, in
mixed feed for livestock that includes cattle meat and
bonemeal.
[0062] FIG. 18 is an electrophoresis photograph showing the results
of the detection of DNA, using cattle-specific primer pairs, in
mixed feed for livestock that contains cattle meat and
bonemeal.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0063] The mitochondrial genome is essential for the biosynthesis
of the enzymes required for oxidative phosphorylation (electron
transport system), and mitochondrial DNA codes for ATP synthase and
cytochrome c oxidase, and the like. ATP synthase is composed of
several subunits, and as discussed above, the inventors found that
the ATP synthase subunit 8 gene (atp8 gene) is present in the
animal mitochondrial genome but homologous sequence with animal
atp8 gene is not present in the plant (Oryza sativa) mitochondrial
genome. It thus became possible to identify animal species even
when plant-derived DNA is present in large quantities in the
feed.
[0064] Several animal atp8 genes are known. For example, taking the
cattle atp8 DNA sequence (cattle) as a reference, the atp8 DNA
sequences of several animal species were aligned so that homology
with the cow sequence is achieved, and this is shown in FIG. 1.
FIG. 1 shows the DNA sequence of the atp8 gene of 1: cattle, 2:
alpaca, 3: cat, 4: dog, 5: goat (1), 6: goat (2), 7: horse, 8:
antelope, 9: mouse, 10: rabbit, 11: rat, 12: donkey, 13: sheep, 14:
deer, 15: sperm whale, and 16: razorback whale. It should be noted
that the arrow in the diagram indicates the start position of the
cattle atp8 reading frame.
[0065] FIG. 2 shows the atp8 genes of cattle, chicken, and pig
aligned with one another. Further, FIG. 3 shows the atp8 genes of
fish, chicken, cattle, sheep, and pig aligned with one another, and
the underlined portion indicates the position of the atp8 gene of a
pilchard.
[0066] As shown in FIGS. 1 to 3, there is diversity of DNA
sequences among the atp8 genes. This diversity can be used to
detect specific sequences in various types of animals, thereby
allowing various animal species to be identified.
[0067] To identify various types of animals, examples of methods
for detecting a DNA sequence specific to an animal species include
Southern Blotting and PCR (polymerase chain reaction). PCR is
preferably used in the present invention because it permits
detection even with very small DNA samples and it allows accuracy
to be improved. In the present invention, PCR is carried out using
a pair of primers including a DNA sequence specific to a target
animal species, and a DNA fragment that is amplified is
detected.
[0068] PCR is for example carried out as follows. First, two
regions are selected as primers from any of regions of the atp8
gene and regions proximal thereto having a DNA sequence specific to
a target animal species, and the selected primers are synthesized.
Using this pair of primers, the DNA fragment of the region
sandwiched between the two primers is amplified by PCR. A sample
containing the amplified DNA fragments is then subjected to
electrophoresis to determine whether that DNA fragment is
present.
[0069] A primer is a DNA sequence of nucleotides in any length, and
is suitably selected based on the sequence alignment of the atp8
genes of various types of animals in publicly available databases.
For example, in order to detect DNA specific to mammals, a region
having higher homology with mammals and low homology with
non-mammals and having appropriate nucleotide at the 3' end can be
chosen. In order to detect DNA specific to a certain animal, a
region having specificity to that animal and low specificity with
other animals can be chosen. Since primers are used in pairs, two
regions, that is, a region on the 5' side and a region on the 3'
side, are selected. If more than two regions can be selected as
primers, then it is possible to use various primer
combinations.
[0070] For example, FIG. 4 shows a DNA sequence of cattle
mitochondrial atp8 gene, and the markings in the diagram are the
regions (sequences) that can be used as primers for detecting
cattle-specific DNA. DNA sequences for specifically detecting
mammals (anicon5 [SEQ ID NO: 1] and anicon3 [SEQ ID NO: 2]) and DNA
sequences for specifically detecting ruminants (rumicon5 [SEQ ID
NO: 3] and rumicon3 [SEQ ID NO: 4]) are also shown in FIG. 4. A
more detailed explanation follows in the Examples. For example,
mammal specific DNA can be detected when anicon5 is used as the 5'
primer and anicon3 is used as the 3' primer. Likewise, ruminant
specific DNA can be detected when rumicon5 and rumicon3 are used as
primers. Alternatively, although not shown, it is also possible to
combine Fpr-F [SEQ ID NO: 5] and Fpr-R [SEQ ID NO: 6] to detect
ruminant specific DNA with greater accuracy. As the 5' primer, any
one of cow5 [SEQ ID NO: 7], cow51 [SEQ ID NO: 8], cow52 [SEQ ID NO:
9], cow53 [SEQ ID NO: 10], and cow54 [SEQ ID NO: 11] is used, and
as the 3' primer, either cow3 [SEQ ID NO: 12] or cow31 [SEQ ID NO:
13] is used. For example, the primer pairs of cow52 and cow31,
cow52 and cow3, cow54 and cow31, cow53 and cow3, cow54 and cow3,
and cow51 and cow3 are capable of detecting cattle-specific DNA.
Alternatively, although not shown, it is also possible to combine
Fpc-F [SEQ ID NO: 14] and Fpc-R [SEQ ID NO: 15] to detect
cattle-specific DNA with greater accuracy.
[0071] The regions surrounded by squares in FIGS. 1 to 3 indicate
examples of DNA sequences used in tests for specifically detecting
DNAs derived from the respective animal species.
[0072] The DNA sequences of the regions selected as primers are
synthesized by normally employed methods. Typically, nucleotides
are extended on a support medium using an automated DNA
synthesizer, then removed from protecting group and cleaved from
the support medium. Then, they can be purified using a normally
employed method (such as column chromatography) to obtain primers
of interest.
[0073] Samples that can be measured include raw meat, raw fish,
processed meat food products, processed fish food products, food
products containing processed meat, food products containing
processed fish, blood, hair, body fluids, milk, milk processing
products, meat and bonemeal, bonemeal, fishmeal, fish soluble, and
feed, fertilizer, and feed additive containing them. Extraction of
mitochondrial DNA from these samples is, for example, performed as
follows. Approximately 50 mg to 500 mg of a sample (for example, 50
mg in the case of raw meat, 100 mg to 500 mg in the case of a dried
powder sample) is suspended in about 10 times that amount of
buffered solution, ground using a bead grinding method, for
example, and then extracted using a commercially available tissue
cell mitochondrial DNA extraction kit (manufactured by Wako Pure
Chemical Industries, Ltd., for example). Such kits allow purer
mitochondrial DNA to be collected in that little genome DNA in the
tissue cells is contaminated. For example, to the sample ground is
added a reagent in the kit, and centrifuged, and the pellets are
collected to concentrate. It is thus possible to more efficiently
extract mitochondrial DNA with little contamination of genome DNA,
which is present in large quantities in the sample. The method for
extracting DNA is not limited to those described herein, and it is
apparent to those skilled in the art to employ other methods for
extracting DNA.
[0074] There are no particular limitations regarding the amount of
primer used, but generally is it preferable that approximately 0.4
.mu.M is used.
[0075] PCR is performed on the pretreated sample, that is, either
the mitochondrial DNA or total DNA extracted from the sample, using
the primer pair selected above, so as to amplify the DNA fragment
of the region sandwiched between the primers. PCR is executed under
conditions in which it is ordinarily performed, and conditions that
are appropriate for each primer pair are set. For example, DNA
fragment is, firstly, heat denatured at 95.degree. C. for 9
minutes; then subjected to the cycle of reactions of denaturing at
92.degree. C. for 30 seconds to one minute, annealing at 40 to
65.degree. C. for 30 seconds to two minutes, and extending at
72.degree. C. for 30 seconds to two minutes, which is repeated 30
to 50 times; and finally, allowed to react at 72.degree. C. for
five minutes to finish PCR. AmpliTaq GOLD polymerase or the like is
used as the DNA polymerase. The size of the PCR product (DNA
fragment) amplified by the primer pair can be approximately 100 by
to approximately 300 bp, although this varies depending on the
number of bases between the primer pair that has been selected. The
PCR product is then subjected to agarose or polyacrylamide gel
electrophoresis, for example, under conditions in which the above
DNA fragments can be separated.
[0076] The DNA fragments on the gel subjected to electrophoresis
can be detected by detection means normally employed by those
skilled in the art, such as ethidium bromide staining, silver
staining, fluorescence detection, and Southern hybridization, and
can be confirmed by DNA sequencing.
[0077] Thus, if mitochondrial DNA derived from a species of
interest is present in the sample, then, amplified DNA fragments
can be detected on the gel. Limits to detection may vary depending
on various factors, such as the type and combination of the primer
pair used, the amount of sample, the PCR conditions, and the
detection method. If appropriate conditions are selected, then the
presence of DNA can be detected with high sensitivity even in trace
samples. For example, if appropriate conditions are selected when
the sample is a mixed feed that contains cattle-derived meat and
bonemeal, then contamination of only 0.001 wt % cattle meat and
bonemeal in the feed can be detected using a specific
cattle-specific primer pair.
[0078] It should be noted that when a sample is a mixed feed, if a
plant DNA-specific primer pair (for example, the combination of
placon5 [SEQ ID NO: 42] and placon3 [SEQ ID NO: 43]) is used as a
control experiment, then it is possible to confirm whether or not
DNA has been appropriately extracted from the sample. Furthermore,
by using a plant DNA-specific primer pair, a plant-derived
component present in a sample can be detected.
[0079] The primer pairs of the present invention may be provided in
the form of a kit for detecting an animal-derived component present
in a sample. The kit comprises at least one of primer pair specific
for any animal described above. Preferably, the kit further
comprises the plant DNA-specific primer pair as a control.
[0080] It should be noted that the primer sequence has been
specified in the Examples discussed below, but there is no
intention to limit the present invention to the following Examples.
It is intended that the present invention includes in its scope
sequences that include those primer sequences, or those sequences
with one or more base sequence substitutions, deletions, or
insertions or addition of nucleotide sequences to the 5' end, and
sequences which by changing the hybridizing conditions can
hybridize with a DNA of interest and allow a DNA derived from a
specific animal species to be detected specifically.
EXAMPLES
Primer Synthesis
[0081] Primer regions to be used in the following Examples were
selected based on the sequence alignment of the atp8 gene and
regions proximal thereto of various animals shown in FIGS. 1 to 3.
The DNA sequences of the selected regions were synthesized using an
automated DNA synthesizer.
Example 1
Specific Detection of Mammal-Derived DNA Sequence
[0082] DNAs from 15 types of meat samples, these being cattle,
sheep, goat, deer, pig, horse, rabbit, whale, chicken, codfish,
salmon, sardine, crab, prawn, and clam, were prepared as follows.
Each DNA sample was diluted in an appropriate concentration in
buffer solution (10 mM Tris-HCl, pH 7.5, 20 mM EDTA, pH 7.5),
ground using a bead grinding method, and then DNA was extracted
using the mtDNA Extractor CT Kit (manufactured by Wako Pure
Chemical Industries, Ltd.), which is a commercially available
tissue cell mitochondrial DNA extraction kit.
[0083] With each of these DNA samples serving as a template, PCR
was performed using anicon5 [SEQ ID NO: 1] and anicon3 [SEQ ID NO:
2] as the 5' primer and the 3' primer, respectively. The PCR
conditions were as follows: reaction buffer solution (10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl.sub.2, 0.001% (w/v)
gelatin); heat denaturing at 95.degree. C. for 9 minutes, followed
by the cycle of reactions of denaturing at 92.degree. C. for one
minute, annealing at 55.degree. C. for two minutes, and extending
for two minutes at 72.degree. C. repeated 45 times, and lastly, the
product was allowed to react at 72.degree. C. for five minutes.
After PCR, the reaction mixture was subjected to agarose gel
electrophoresis, and the PCR product (DNA fragments) was detected
by ethidium bromide staining. The results are shown in FIG. 5. In
the diagram, M is the molecular weight marker.
[0084] As is clear from FIG. 5, by performing PCR using anicon5
[SEQ ID NO: 1] and anicon3 [SEQ ID NO: 2] as primers with each
animal-derived DNA serving as a template, the PCR products
(amplified DNA fragments) were observed only in mammals (176 by
band position) (lanes 1 to 8). That is, it was demonstrated that by
using anicon5 [SEQ ID NO: 1] and anicon3 [SEQ ID NO: 2] as the
primer pair, it is possible to specifically detect only
mammal-derived DNA from among various types of animal meat samples.
Consequently, the combination of anicon5 [SEQ ID NO: 1] and anicon3
[SEQ ID NO: 2] is a primer pair that specifically detects the DNA
sequence derived from animal species of mammalian origin. The
regions that can be used as primers for detecting DNAs derived from
mammals are shown in FIG. 4. It should be noted that this primer
pair was designed so as to fail to amplify human DNA.
Example 2
Specific Detection of Ruminant-Derived DNA Sequence-1
[0085] With the 15 types of DNA samples prepared in the same manner
as in Example 1 (except that a different type of cattle DNA sample
was used in place of deer) serving as templates, PCR was performed
using rumicon5 [SEQ ID NO: 3] and rumicon3 [SEQ ID NO: 4] as the 5'
primer and the 3' primer, respectively. The PCR conditions were as
follows: heat denaturing at 95.degree. C. for 9 minutes, followed
by the cycle of reactions of denaturing at 92.degree. C. for one
minute, annealing at 45.degree. C. for two minutes, and extending
at 72.degree. C. for two minutes repeated 45 times, and lastly, the
product was allowed to react at 72.degree. C. for five minutes.
After PCR, the reaction mixture was subjected to agarose gel
electrophoresis, and the PCR product (DNA fragments) was detected
by ethidium bromide staining. The results are shown in FIG. 6.
[0086] As shown in FIG. 6, by performing PCR using rumicon5 [SEQ ID
NO: 3] and rumicon3 [SEQ ID NO: 4] as primers with each
animal-derived DNA serving as a template, PCR products from DNAs
derived from beef cattle, dairy cattle, sheep, and goat, which are
ruminants, were observed (band position indicated by the arrow). On
the other hand, PCR products (DNA fragments) could not be detected
in the DNA samples of animals that were not ruminants. That is, it
was demonstrated that by using rumicon5 [SEQ ID NO: 3] and rumicon3
[SEQ ID NO: 4] as the primer pair, it is possible to specifically
detect only ruminant-derived DNA from various types of animal meat
samples. Consequently, the combination of rumicon5 [SEQ ID NO: 3]
and rumicon3 [SEQ ID NO: 4] is a primer pair that specifically
detects the DNA sequence derived from animal species of ruminant
origin. The regions that can be used as the primers for detecting
DNAs derived from ruminants are shown in FIG. 4.
[0087] It should be noted that in rumicon5 [SEQ ID NO: 3], "r" is
"g" or "a", and "k" is "g" or "t". That is, rumicon5 [SEQ ID NO: 3]
is a primer mixture, but it should be apparent to those skilled in
the art that each can be used independently.
Example 3
Specific Detection of Ruminant-Derived DNA Sequence-2
[0088] With the 15 types of DNA samples prepared in the same manner
as in Example 1 serving as templates, PCR was performed using Fpr-F
[SEQ ID NO: 5] and Fpr-R [SEQ ID NO: 6] as the 5' primer and the 3'
primer, respectively. The PCR conditions were as follows: heat
denaturing at 95.degree. C. for 9 minutes, followed by the cycle of
reactions of denaturing at 92.degree. C. for 30 seconds, annealing
at 50.degree. C. for 30 seconds, and extending at 72.degree. C. for
30 seconds repeated 45 times, and lastly, the product was allowed
to react at 72.degree. C. for five minutes. After PCR, the reaction
mixture was subjected to agarose gel electrophoresis, and the PCR
product (DNA fragments) was detected by ethidium bromide staining.
The results are shown in FIG. 7.
[0089] As shown in FIG. 7, by performing PCR using Fpr-F [SEQ ID
NO: 5] and Fpr-R [SEQ ID NO: 6] as primers with each animal-derived
DNA serving as a template, PCR products from DNAs derived from
cattle, sheep, goat, and deer which are ruminants, were more
clearly observed (104 by band position). On the other hand, PCR
products (DNA fragments) could not be detected in the DNA samples
of animals that were not ruminants. That is, it was demonstrated
that by using Fpr-F and Fpr-R as the primer pair, it is possible to
more specifically detect only ruminant-derived DNA from various
types of animal meat samples. Consequently, the combination of
Fpr-F [SEQ ID NO: 5] and Fpr-R [SEQ ID NO: 6] is a primer pair that
specifically detects DNAs derived from animal species of ruminant
origin.
[0090] It should be noted that in Fpr-F [SEQ ID NO: 5] and Fpr-R
[SEQ ID NO: 6], "r" is "g" or "a", and "k" is "g" or "t". That is,
these are primer mixtures, but it should be apparent to those
skilled in the art that each can be used independently.
Example 4
Specific Detection of Cattle-Derived DNA Sequence-1
[0091] Specific detection of a cattle-derived DNA sequence was
performed using the various primer pairs listed in Table 1. PCR was
performed under the same conditions as those of Example 1, except
that DNAs prepared from cattle, sheep, goat, pig, and chicken meat
were served as templates. The results are shown in FIGS. 8 to
10.
TABLE-US-00001 TABLE 1 Primer Anealing Result 5' cow51 SEQ ID NO: 8
55.degree. C. 2 min FIG. 8 3' cow3 SEQ ID NO: 12 5' cow52 SEQ ID
NO: 9 52.degree. C. 2 min FIG. 9 3' cow3 SEQ ID NO: 12 5' cow53 SEQ
ID NO: 10 52.degree. C. 2 min FIG. 9 3' cow3 SEQ ID NO: 12 5' cow54
SEQ ID NO: 11 52.degree. C. 2 min FIG. 9 3' cow3 SEQ ID NO: 12 5'
cow5 SEQ ID NO: 7 52.degree. C. 2 min FIG. 9 3' cow31 SEQ ID NO: 13
5' cow51 SEQ ID NO: 8 56.degree. C. 2 min FIG. 10 3' cow31 SEQ ID
NO: 13 5' cow52 SEQ ID NO: 9 52.degree. C. 2 min FIG. 10 3' cow31
SEQ ID NO: 13 5' cow53 SEQ ID NO: 10 56.degree. C. 2 min FIG. 10 3'
cow31 SEQ ID NO: 13 5' cow54 SEQ ID NO: 11 56.degree. C. 2 min FIG.
10 3' cow31 SEQ ID NO: 13 5' cow5 SEQ ID NO: 7 46.degree. C. 2 min
not shown 3' cow3 SEQ ID NO: 12
[0092] The results shown in FIG. 8 indicate that if cow51 [SEQ ID
NO: 8] and cow3 [SEQ ID NO: 12] are used as the primer pair, then
DNA fragments the same size as the PCR product (DNA fragments)
obtained from cattle-derived meat did not find when sheep, goat,
pig, or chicken DNA served as templates. It is clear that the
combination of cow52 and cow3 of FIG. 9 and the combination of
cow52 and cow31 of FIG. 10 are particularly suitable primer pairs
for cattle-specific DNA detection, because similar DNA fragments
were not detected in the samples of other animal species. On the
other hand, as shown in FIG. 9, when cow5 [SEQ ID NO: 7] and cow31
[SEQ ID NO: 13] are used as the primer pair, a band of the same
size as the DNA fragment obtained using a cattle DNA as a template
was slightly found in sheep. Likewise, in FIG. 10, when the primer
pair cow51 [SEQ ID NO: 8] and cow31 [SEQ ID NO: 13], and the primer
pair cow53 [SEQ ID NO: 10] and cow31 [SEQ ID NO: 13] are used, a
band the same size as the DNA fragment obtained using cattle DNA as
a template was observed.
Example 5
Specific Detection of Cattle-Derived DNA Sequence-2
[0093] With the 15 types of DNA samples prepared in the same manner
as in Example 1 serving as templates, PCR was performed using Fpc-F
[SEQ ID NO: 14] and Fpc-R [SEQ ID NO: 15] as the 5' primer and the
3' primer, respectively. The PCR conditions were as follows: heat
denaturing at 95.degree. C. for 9 minutes, followed by the cycle of
reactions of denaturing at 92.degree. C. for 30 seconds, annealing
at 55.degree. C. for 30 seconds, and extending at 72.degree. C. for
30 seconds repeated 45 times, and lastly, the product was allowed
to react at 72.degree. C. for five minutes. After PCR, the reaction
mixture was subjected to agarose gel electrophoresis, and the PCR
product (DNA fragments) was detected by ethidium bromide staining.
The results are shown in FIG. 11.
[0094] As shown in FIG. 11, by performing PCR using Fpc-F [SEQ ID
NO: 14] and Fpc-R [SEQ ID NO: 15] as primers with each
animal-derived DNA serving as a template, the PCR product from only
cattle-derived DNA was detected (126 by band position). That is, it
was demonstrated that by using Fpc-F and Fpc-R as the primer pair,
it is possible to detect with extreme specificity only
cattle-derived DNA from various types of animal meat samples.
Consequently, the combination of Fpc-F [SEQ ID NO: 14] and Fpc-R
[SEQ ID NO: 15] is a primer pair that specifically detects DNAs
derived from animal species of bovine origin.
Example 6
Specific Detection of Pig-Derived DNA Sequence-1
[0095] Specific detection of a pig-derived DNA sequence was
performed using the various primer pairs listed in Table 2. PCR was
performed under the same conditions as those of Example 1, except
that DNAs prepared from cattle, sheep, goat, pig, and chicken meat
were served as templates.
TABLE-US-00002 TABLE 2 Primer Anealing Result 5' pig5 SEQ ID NO: 16
46.degree. C. 2 min not shown 3' pig3 SEQ ID NO: 19 5' pig51 SEQ ID
NO: 17 58.degree. C. 2 min FIG. 12 3' pig3 SEQ ID NO: 19 5' pig5
SEQ ID NO: 16 46.degree. C. 2 min not shown 3' pig31 SEQ ID NO: 20
5' pig5 SEQ ID NO: 16 46.degree. C. 2 min not shown 3' pig32 SEQ ID
NO: 21 5' pig51 SEQ ID NO: 17 55.degree. C. 2 min not shown 3'
pig31 SEQ ID NO: 20 5' pig51 SEQ ID NO: 17 55.degree. C. 2 min not
shown 3' pig32 SEQ ID NO: 21
[0096] FIG. 12 shows the results obtained by using pig51 [SEQ ID
NO: 17] and pig3 [SEQ ID NO: 19] as the primer pair. As is clear
from FIG. 12, PCR products (DNA fragments) were detected only in
the sample derived from pig meat (band position indicated by the
arrow). Consequently, this primer pair is useful for specifically
detecting pig-derived DNA sequences.
Example 7
Specific Detection of Pig-Derived DNA Sequence-2
[0097] With the 15 types of DNA samples prepared in the same manner
as in Example 1 serving as templates, PCR was performed using
pig5-3 [SEQ ID NO: 18] and pig32-2 [SEQ ID NO: 22] as the 5' primer
and the 3' primer, respectively. The PCR conditions were as
follows: heat denaturing at 95.degree. C. for 9 minutes, followed
by the cycle of reactions of denaturing at 92.degree. C. for 30
seconds, annealing at 60.degree. C. for 1 minute, and extending at
72.degree. C. for 1 minute repeated 45 times, and lastly, the
product was allowed to react at 72.degree. C. for five minutes.
After PCR, the reaction mixture was subjected to agarose gel
electrophoresis, and the PCR product (DNA fragments) was detected
by ethidium bromide staining. The results are shown in FIG. 13.
[0098] As shown in FIG. 13, by performing PCR using pig5-3 [SEQ ID
NO: 18] and pig32-2 [SEQ ID NO: 22] as primers with each
animal-derived DNA serving as a template, the PCR product from only
DNA derived from pig was observed (band position indicated by the
arrow). On the other hand, PCR products could not be detected in
the DNA samples of other animals. That is, it was demonstrated that
by using pig5-3 and pig32-2 as the primer pair, it is possible to
detect with only pig-derived DNA among various types of animal meat
samples. Consequently, the combination of pig5-3 [SEQ ID NO: 18]
and pig32-2 [SEQ ID NO: 22] is a primer pair that very specifically
detects DNAs derived from animal species of pig origin.
Example 8
Specific Detection of Sheep-Derived DNA Sequence
[0099] Specific detection of a sheep-derived DNA sequence was
performed using sheep5 [SEQ ID NO: 23] and sheep3 [SEQ ID NO: 24]
as the 5' primer and the 3' primer, respectively. PCR was performed
under the same conditions as those of Example 1, except that DNAs
prepared from cattle, sheep, goat, pig, and chicken meat were
served as templates and that the annealing conditions were set to
two minutes at 46.degree. C. The results are shown in FIG. 14.
[0100] These results demonstrate that specific detection of a
sheep-derived DNA sequence is possible using sheep5 [SEQ ID NO: 23]
and sheep3 [SEQ ID NO: 24] as the primer pair.
Example 9
Specific Detection of Goat-Derived DNA Sequence
[0101] Specific detection of a goat-derived DNA sequence was
performed using goat5 [SEQ ID NO: 25] and goat3 [SEQ ID NO: 26] as
the 5' primer and the 3' primer, respectively. PCR was performed
under the same conditions as those of Example 1, except that DNAs
prepared from cattle, sheep, goat, pig, and chicken meat were
served as templates and that the annealing conditions were set to
two minutes at 46.degree. C. The results are shown in FIG. 14.
[0102] These results demonstrate that specific detection of a
goat-derived DNA sequence is possible using goat5 [SEQ ID NO: 25]
and goat3 [SEQ ID NO: 26] as the primer pair.
Example 10
Specific Detection of Chicken-Derived DNA Sequence
[0103] Specific detection of a chicken-derived DNA sequence was
performed using the various primer pairs listed in Table 3 as the
5' and the 3' primers. PCR was performed under the same conditions
as those of Example 1, except that DNAs prepared from cattle,
sheep, goat, pig, and chicken meat were served as templates. The
results obtained by using chick5 [SEQ ID NO: 28] and chick3 [SEQ ID
NO: 30] as the primers are shown in FIG. 15.
TABLE-US-00003 TABLE 3 Primer Anealing Result 5' chick5 SEQ ID NO:
28 46.degree. C. 2 min FIG. 15 3' chick3 SEQ ID NO: 30 5' chick51
SEQ ID NO: 29 50.degree. C. 2 min not shown 3' chick3 SEQ ID NO: 30
5' chick5 SEQ ID NO: 28 50.degree. C. 2 min not shown 3' chick31
SEQ ID NO: 31 5' chick51 SEQ ID NO: 29 60.degree. C. 2 min not
shown 3' chick31 SEQ ID NO: 31
[0104] As shown in FIG. 15, specific detection of a chicken-derived
DNA sequence is possible using chick5 [SEQ ID NO: 28] and chick3
[SEQ ID NO: 30] as the primer pair (band position indicated by the
arrow).
Example 11
Detection of Cattle-Derived Component in Mixed Feed-1
[0105] Meat and bonemeal derived from cattle (Australian bred) was
mixed into mixed feed for livestock (main components: corn, milo,
gluten feed, bran, rice bran, soy oil cake, rapeseed oil cake) at a
predetermined ratio. Then, as in Example 1, 100 mg of the feed
obtained was suspended in ten times that amount of buffer solution,
and ground by a bead grinding method. Then, DNA was extracted using
a commercially available tissue cell mitochondrial DNA extraction
kit (manufactured by Wako Pure Chemical Industries, Ltd., Japan).
Using this DNA as a template, PCR was perfollued under the same
conditions as in Example 1 using anicon5 [SEQ ID NO: 1] and anicon3
[SEQ ID NO: 2], which are the primer pair for specifically
detecting mammalian-derived DNA DNA sequences used in Example 1,
Fpr-F [SEQ ID NO: 5] and Fpr-R [SEQ ID NO: 6], which are the primer
pair for specifically detecting ruminant-derived DNA sequences used
in Example 3, and Fpc-F [SEQ ID NO: 14] and Fpc-R [SEQ ID NO: 15],
which are the primer pair for specifically detecting cattle-derived
DNA sequences used in Example 5. The results are shown in FIG. 16.
In all cases where these primer pairs were used, it was possible to
detect cattle meat and bonemeal contained in the feed at 0.01 wt
%.
Example 12
Detection of Cattle-Derived Component in Mixed Feed-2
[0106] PCR was performed under the same conditions as in Example 2,
except that DNA extracted from mixed feed in the same manner as in
Example 11 was served as a template and that the primer pair
rumicon5 [SEQ ID NO: 3] and rumicon3 [SEQ ID NO: 4] for
specifically detecting ruminant-derived DNA sequences that is used
in Example 2 was used. The results are shown in FIG. 17. Detection
was possible even in a case where the feed contained 0.1 to 1 wt %
meat and bonemeal.
Example 13
Detection of Cattle-Derived Component in Mixed Feed-3
[0107] PCR was performed under the same conditions as in Example 3,
except that DNA extracted from mixed feed in the same manner as in
Example 11 was served as a template and that the cattlederived
DNA-specific primer pair of cow52 [SEQ ID NO: 9] and cow31 [SEQ ID
NO: 13] that was used in Example 3 was used. The results are shown
in FIG. 18. Detection was possible even in a case where meat and
bonemeal was only contained in the feed at a mere 0.001 wt %.
Example 14
Specific Detection of Fish-Derived DNA Sequence
[0108] Fishmeal is often a mixture of various types of fish. Thus,
several primer regions were selected based on the alignment of the
DNA sequences of the atp8 gene and proximal regions shown in FIG. 3
in order to detect a wide range of components derived from various
types of fish. The underlined portion in FIG. 3 is the atp8 gene of
a pilchard, and the asterisk bellowing the alignment indicates the
portion where bases are conserved among the DNA sequences.
[0109] A commercially available tissue cell mitochondrial DNA
extraction kit (manufactured by Wako Pure Chemical Industries,
Ltd., Japan) was used in the same manner as in Example 1 to prepare
DNA from the 26 types of plants and animals shown in Table 4 below.
PCR was carried out using this DNA solution as a template and using
the various combinations of 5' and 3' primers selected based on the
sequence alignment of FIG. 3. As for the primers used, the 5' side
primers used were fish5 [SEQ ID NO: 32], fish51 [SEQ ID NO: 34],
fish5-1 [SEQ ID NO: 38], and fish5-2 [SEQ ID NO: 39], and the 3'
side primers used were fish3-1 [SEQ ID NO: 33], fish3 [SEQ ID NO:
35], fish31 [SEQ ID NO: 36], fish31-1 [SEQ ID NO: 37], fish3-2 [SEQ
ID NO: 40], and fish3-3 [SEQ ID NO: 41]. The PCR conditions were as
follows: reaction buffer solution (10 mM Tris-HCl, pH 8.3, 50 mM
KCl, 1.5 mM MgCl.sub.2, 0.001% (w/v) gelatin); heat denaturing at
95.degree. C. for 9 minutes, followed by the cycle of reactions of
denaturing at 92.degree. C. for 30 seconds, annealing at 55.degree.
C. for 30 seconds, and extending at 72.degree. C. for 30 seconds
repeated 45 times, and lastly, the product was allowed to react at
72.degree. C. for five minutes. After PCR, the reaction mixture was
subjected to agarose gel electrophoresis, and the PCR product (DNA
fragments) was detected by ethidium bromide staining. In the
positive control, PCR was performed with a solution of
sardine-derived DNA obtained in the same manner as in Example 1
from crushed sardine meat serving as the template. In the negative
control, PCR was performed with a blank in which the template DNA
was not added to the PCR reaction mixture.
[0110] Table 4 shows the results obtained with the combination of
fish5 [SEQ ID NO: 32] and fish3-1 [SEQ ID NO: 33]. It should be
noted that in the assessment column of Table 4, the cases where a
band was not detected in the negative control, but a band of a
target size was detected in the positive control, and a band of the
same size as that detected in the positive control was detected in
the sample, were regarded as positive.
TABLE-US-00004 TABLE 4 Animal Assesment * Cattle(Beef cattle) -
Cattle(Holstein) - Cattle(Japanese black) - Cattle(Red hair) -
Cattle(F2) - Sheep - Goat - Deer - Pig - Horse - Rabbit - Whale -
Chicken - Human - Scad - Sardine + Flatfish .+-. Salmon + Alaska
pollack + Tuna + Crab - Lady crab + Prawn - Squid - Clam - Corn - *
+; Detection of band at 284 bp, .+-.; Detection of unclear band at
284 bp, -; Under detection limit of band at 284 bp Detection was
performed with agarose gel electrophoresis and ethidium bromide
staining.
[0111] As shown in the assessment column of Table 4, a band was
detected at the position of 284 by in DNA samples taken from
sardine, flatfish, salmon, Alaska Pollack, tuna, and lady crab. On
the other hand, a band was under detectable in mammalian, chicken,
and corn DNA samples. Consequently, it is clear that the primer
pair of fish5 [SEQ ID NO: 32] and fish3-1 [SEQ ID NO: 33] allows
the major fish types often used to produce fishmeal to be
detected.
[0112] Apart from this, about 100 kinds of fishmeal was examined by
extracting DNA in the same manner as above. The results showed that
a band can be detected at the position of interest in about 90
percent of the fishmeal. These results also indicated that wide
ranging detection is possible for DNA derived from various types of
fishes.
Example 15
Fish-Derived DNA Sequence Addition Testing and Detection Limit
[0113] Whether or not it is possible to detect fishmeal mixed into
commercially available mixed feed for raising cattle or chickenmeal
was examined. The raw material of the fishmeal was mackerel and
saury, and the fishmeal used was passed through a 0.5 mm mesh wire
sieve. As regards the mixed feed, commercially available mixed feed
for cattle was crushed with a crusher and a triturator, and was
passed through a 0.5 mm mesh wire sieve. It should be noted that
the mixture proportions of the mixed feed are as shown below in
Table 5. The chickenmeal was commercially available chickenmeal,
and had been passed through a 0.5 mm mesh wire sieve.
TABLE-US-00005 TABLE 5 Feed Type of material Ratio(%) Raw materials
Mixed feed for Chaff and bran 37 Corn gluten feed, Rice bran, Bran,
Soybean hull raising dairy cattle Cereals 36 Corn, Rye, Wheat
flour, Milo Vegetable oil cake 19 Rapeseed oil cake, Soy oil cake
Others 8 Syrup, Calcium carbonate, Salt Mixed feed for Cereals 66
Corn, Barley, Wheat raising beef cattle Chaff and bran 28 Bran,
Corn gluten feed Vegetable oil cake 4 Soy oil cake Others 2 Alfalfa
meal
[0114] Samples containing 1 wt % fishmeal in the above mixed feed
and chickenmeal were prepared. As regards these samples and the
samples without fishmeal, DNAs were extracted as in Example 1, and
PCR was performed using the primer pair of fish5 [SEQ ID NO: 32]
and fish3-1 [SEQ ID NO: 33].
[0115] Fish-derived DNA was detected in the mixed feed and
chickenmeal containing 1 wt % fishmeal. On the other hand, DNA was
not detected from the samples to which fishmeal was not added.
Consequently, it was clear that the fishmeal used in this example
can be detected even when contained at 1 wt %.
[0116] Next, the detection limit of fishmeal contaminated in mixed
feed was examined. Fishmeal was added at 10, 1, 0.1, and 0.01 wt %
to the mixed feed for raising dairy cattle of Table 5, and using
the same procedure as that of Example 14, DNA was extracted in
duplicate for each sample, and for each extracted sample PCR was
performed in duplicate. When both of the two DNA extracts resulted
in positive, an assessment of positive was reached. The detection
limit for the fishmeal when using the primer pair of fish5 [SEQ ID
NO: 32] and fish3-1 [SEQ ID NO: 33] was about 1 wt %.
[0117] The invention may be embodied in other forms without
departing from the spirit or essential characteristics thereof. The
embodiments disclosed in this application are to be considered in
all respects as illustrative and not limiting. The scope of the
invention is indicated by the appended claims rather than by the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are intended to be
embraced therein.
Sequence CWU 1
1
43120DNAArtificial Sequenceprimer 1aactagacac gtcaacatga
20220DNAArtificial Sequenceprimer 2aggtaaataa attttcgttc
20318DNAArtificial Sequenceprimer 3acrtcaacrt gactkaca
18419DNAArtificial Sequenceprimer 4tctggrttgt grtaraagt
19521DNAArtificial Sequenceprimer 5gacacrtcaa crtgactkac a
21622DNAArtificial Sequenceprimer 6arttctggrt tgtgrtaraa gt
22719DNAArtificial Sequenceprimer 7gtcaacatga ctgacaatg
19819DNAArtificial Sequenceprimer 8agacacgtca acatgactg
19921DNAArtificial Sequenceprimer 9atgatcttat caatattctt g
211021DNAArtificial Sequenceprimer 10gacatgccgc aactagacac g
211120DNAArtificial Sequenceprimer 11ctctccttgg tgacatgccg
201222DNAArtificial Sequenceprimer 12ttttaatatt tttgttggtg tc
221321DNAArtificial Sequenceprimer 13tcaaggggtg ttttgtttta a
211424DNAArtificial Sequenceprimer 14acaatgatct tatcaatatt cttg
241524DNAArtificial Sequenceprimer 15ccttcaaggg gtgttttgtt ttaa
241618DNAArtificial Sequenceprimer 16atgattcatt acaattac
181720DNAArtificial Sequenceprimer 17tgccacaact agatacatct
201824DNAArtificial Sequenceprimer 18atctacatga ttcattacaa ttac
241918DNAArtificial Sequenceprimer 19ttatttctca aggggtgc
182021DNAArtificial Sequenceprimer 20tgagttcaat tgattctggg c
212121DNAArtificial Sequenceprimer 21tgtttttgag ttttgagttc a
212224DNAArtificial Sequenceprimer 22ctatgttttt gagttttgag ttca
242321DNAArtificial Sequenceprimer 23aattctatca atatttttag t
212420DNAArtificial Sequenceprimer 24ctcaaggagt attttgtttc
202519DNAArtificial Sequenceprimer 25gccacaacta gacacatcr
192618DNAArtificial Sequenceprimer 26tctcaagggg tgttatgc
182721DNAArtificial Sequenceprimer 27ttctgggttg tggtagaagt c
212818DNAArtificial Sequenceprimer 28catcatactc ctaacttg
182921DNAArtificial Sequenceprimer 29cctaacttga ttcaccttct c
213018DNAArtificial Sequenceprimer 30tttaggttca tggtcagg
183121DNAArtificial Sequenceprimer 31gttcatggtc aggttcaggg g
213220DNAArtificial Sequenceprimer 32atcctcactc atacttgaag
203320DNAArtificial Sequenceprimer 33actggtcaaa gaagcttagt
203420DNAArtificial Sequenceprimer 34atcctcwctm atamttgaag
203520DNAArtificial Sequenceprimer 35agtatcatgg tcaggttcag
203620DNAArtificial Sequenceprimer 36agtrtcatgg tcagkttcag
203720DNAArtificial Sequenceprimer 37aytgrtcaaa gargcttagt
203820DNAArtificial Sequenceprimer 38atgcctcaat taaaccccgc
203920DNAArtificial Sequenceprimer 39gcccacaact tcaacaacga
204020DNAArtificial Sequenceprimer 40gccccctggt ttgcaattct
204120DNAArtificial Sequenceprimer 41acctctagtg acatgcctca
204220DNAArtificial Sequenceprimer 42atgttagaag gagctaaatc
204320DNAArtificial Sequenceprimer 43ccgaatgatt gtttagccaa 20
* * * * *