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Oligonucleotide sequences that identify species of animal

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Oligonucleotide sequences that identify species of animal


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.
Related Terms: Dna Sequences

Browse recent National Institute Of Agrobiological Sciences patents - Tsukuba-shi, JP
Inventors: Toyoko Kusama, Koichi Kadowaki, Tetsuya Nomura
USPTO Applicaton #: #20120270223 - Class: 435 612 (USPTO) - 10/25/12 - Class 435 


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The Patent Description & Claims data below is from USPTO Patent Application 20120270223, Oligonucleotide sequences that identify species of animal.

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CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Division of application Serial No. 12/707,828 filed Feb. 18, 2010, which is a Continuation of application Ser. No. 10/826,119 filed Apr. 16, 2004,the entire contents of both 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

1. Field of the Invention

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.

2. Description of the Related Art

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.

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.

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

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.

The present invention provides a method for identifying animal species, said method comprises:

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

detecting the amplified DNA sequence.

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.

In a further preferred embodiment, the mammal is selected from the group consisting of cattle, sheep, goat, deer, pig, horse, rabbit, and whale.

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.

In a further preferred embodiment, the ruminant is selected from the group consisting of cattle, sheep, goat, and deer.

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.

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.

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.

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.

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.

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.

In a further preferred embodiment, the fish is selected from the group consisting of sardine, flatfish, salmon, Alaska Pollack, tuna, and lady crab.

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.



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stats Patent Info
Application #
US 20120270223 A1
Publish Date
10/25/2012
Document #
File Date
04/15/2014
USPTO Class
Other USPTO Classes
International Class
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Dna Sequences


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