WO2014011023A1

WO2014011023A1 - A method for identifying epinephelus lanceolatus

Info

Publication number: WO2014011023A1
Application number: PCT/MY2012/000200
Authority: WO
Inventors: Kenneth Francis RODRIGUES; Shigeharu SENOO; Mabel Manjaji B. MATSUMOTO; Ahmad Zaidi TANI; Christopher Lok Yung VOO
Original assignee: Universiti Malaysia Sabah
Priority date: 2012-07-09
Filing date: 2012-07-09
Publication date: 2014-01-16

Abstract

The present invention relates to a method for identifying Epinephelus lanceolatus,characterized by the steps of extracting deoxyribonucleic acid from a biological sample; subjecting the deoxyribonucleic acid of the biological sample for amplification of a marker region in the presence of a pair of amplification primer, wherein the pair of amplification primer is independently selected from a group consisting of a first pair of amplification primer, a second pair of amplification primer, a third pair of amplification primer, a fourth pair of amplification primer, a fifth pair of amplification primer, a sixth pair of amplification primer, a seventh pair of amplification primer, and an eighth pair of amplification primer; and identifying the Epinephelus lanceolatus by detecting the presence of the amplified marker region.

Description

i

A METHOD FOR IDENTIFYING EPINEPHELUS LANCEOLATUS

Background of the Invention

Field of the Invention

This invention relates to a method for identifying fish species, and more particularly to a method for identifying Epinephelus lanceolatus in a biological sample.

Description of Related Arts

Generally, commercial fish products in Europe come from all parts of the world. Accurate species identification is not always an easy task, for example expensive fish can be labelled as a less valuable species and this has become a growing problem in fish production and distribution chain. Grouper from genera Epinephelus and Mycteroperca is highly appreciated among fish lovers and is cacategorised as an expensive group of fishes from the Family Serranidae. The high demand and popularity for the grouper fish have led to the substitution of grouper fillets with those of closely related species. In market, grouper fish is frequently misidentified as Nile perch (Lates niloticus) or the wreckfish (Polyprion americanus).

There are several species identification method known in the prior arts, such as U.S. Patent Application Publication Number 2002/0090663 A1 which disclosed an assay comprises billfish-specific antibody for identifying the presence of a particular species of billfish in a test sample. A lateral flow immunoassay device for identifying the presence of tissue from a particular species of billfish in a test sample includes a substrate onto which a billfish specific antigen-containing sample has been immobilized. The substrate has a first end having thereon the immobilized billfish specific antigen-containing sample and a second end adapted to receive a solution including an antibody that specifically binds the billfish specific antigen. However, the immunological methods may be compromised by cross-reactions among proteins from closely related species. Moreover, this identification method is inappropriate for frequent high-throughput sample analysis as it is time consuming, expensive, and complex to perform. U.S. Patent Number 6,949,508 disclosed a method for marking animals for subsequent identification by placing foreign proteins into the blood of an animal so that they can be recovered at a later time and used for identification. Proteins are administered to the animal from a water bath via the animal's gill (where appropriate), gut and, through the skin. The foreign protein is detectable in small amounts using immune assays which magnify the available signals or tags in an assay. However, the drawback of this patent includes an additional step of placing molecular tag into the blood of an animal. This step is time consuming and the protein may be denatured or degraded when placing the foreign protein into the blood.

Another example, such as U.S. Patent Number 4,392,236 disclosed an identification of migratory animals, such as adult fish and the like, by means of implanted coded tags, with tag coding involving one or more higher atomic numbered chemical elements in stable and solid form (elements with atomic numbers 40-42, 44-53, 55-60 and 62-83). The elements are identified in the live animal by selective X-ray irradiation of the implanted tag and spectral analysis of the fluorescent X-ray radiation emitted by the tag. Rapid analysis of the fluorescent X-ray radiation to identify the coding elements with a high level of confidence is obtained by using high intensity irradiation and controlled masking for essentially confining the irradiation to the tag and animal tissue surrounding the tag. It thereby improves the signal-to-noise ratio of the fluorescent X-ray radiation emitted by the coding elements. However, chemical marking method typically has disadvantages in attenuation as the fish grows. The chemical mark in animals' tissues may disappear after few years. Therefore, there is a need of present invention to overcome the aforesaid deficiencies of the prior art. Moreover, the present invention can identify the groupers with the molecular marker without sacrificing the animal to conserve the valuable biodiversity.

Summary of Invention

It is an objective of the present invention to provide a method for distinguishing Epinephelus lanceolatus from other species of groupers such as Epinephelus fuscoguttatus, Epinephelus coioides, and Epinephelus corallicola.

It is also an objective of the present invention to provide a method for identifying Epinephelus lanceolatus with high accuracy and specificity.

It is yet another objective of the present invention to provide a method for identifying Epinephelus lanceolatus without sacrificing the animal so as to conserve valuable biodiversity.

It is a further objective of the present invention to provide a method for identifying Epinephelus lanceolatus based on polymerase chain reaction.

Accordingly, these objectives may be achieved by following the teachings of the present invention. The present invention relates to a method for identifying Epinephelus lanceolatus, characterized by the steps of extracting deoxyribonucleic acid from a biological sample; subjecting the deoxyribonucleic acid of the biological sample for amplification of a marker region in the presence of a pair of amplification primer, wherein the pair of amplification primer is independently selected from a group consisting of a first pair of amplification primer comprising a forward primer 5'-ACTGCTACCCGACTCGTGAC-3' (SEQ ID NO: 1 ) and a reverse primer 5'-GCAAGGAAAGTGGAGAGAGC-3' (SEQ ID NO: 2), a second pair of amplification primer comprising a forward primer 5'-AGTTTGAGGGGGAAAAGCAT-3' (SEQ ID NO: 3) and a reverse primer 5'-CTGGCATTGACGAGAGCATA-3' (SEQ ID NO: 4), a third pair of amplification primer comprising a forward primer 5'-CCCATGTTAAAATGCCCAAC-3' (SEQ ID NO: 5) and a reverse primer 5'-GGATCGGTTGCGTAAGTTGT-3' (SEQ ID NO: 6), - a fourth pair of amplification primer comprising a forward primer 5'-GGGAGGCATTTGGTCAGATA-3' (SEQ ID NO: 7) and a reverse primer 5 -ACACACAGGCTGCTGACAAG-3' (SEQ ID NO: 8), a fifth pair of amplification primer comprising a forward primer 5'-CTGCCATGTTTTGGGTTTTT-3' (SEQ ID NO: 9) and a reverse primer 5'-GTGTAGGGGGAGGTCTGTGA-3' (SEQ ID NO: 10), a sixth pair of amplification primer comprising a forward primer 5'-ACGTCAGGGAGAAATTGTGC-3' (SEQ ID NO: 11 ) and a reverse primer 5'-CCAGACAGTGTGCTCCATTG-3' (SEQ ID NO: 12), a seventh pair of amplification primer comprising a forward primer 5'-TCCTGTGTGAAGCTGAATGC-3' (SEQ ID NO: 13) and a reverse primer 5'-TC ACAC G G GAC ATG AAC ACT-3' (SEQ ID NO: 14), and an eighth pair of amplification primer comprising a forward primer 5'-GAGGGCAGGAACACTGAGAA-3' (SEQ ID NO: 15) and a reverse primer 5'-ACTCTGCAGGTCCCAGCTT-3' (SEQ ID NO: 16); identifying the Epinephelus lanceolatus by detecting the presence of the amplified marker region.

Brief Description of the Drawings

The features of the invention will be more readily understood and appreciated from the following detailed description when read in conjunction with the accompanying drawings of the preferred embodiment of the present invention, in which:

Sequence Listing (according to PCT standard ST. 25) of a nucleotide sequence of a first pair of amplification primer comprising a forward primer 5 -ACTGCTACCCGACTCGTGAC-3' (SEQ ID NO: 1 ) and a reverse primer 5 -GCAAGGAAAGTGGAGAGAGC-3' (SEQ ID NO: 2); a second pair of amplification primer comprising a forward primer 5'-AGTTTGAGGGGGAAAAGCAT-3' (SEQ ID NO: 3) and a reverse primer 5'-CTGGCATTGACGAGAGCATA-3' (SEQ ID NO: 4); a third pair of amplification primer comprising a forward primer 5'-CCCATGTTAAAATGCCCAAC-3' (SEQ ID NO: 5) and a reverse primer 5'-GGATCGGTTGCGTAAGTTGT-3' (SEQ ID NO: 6); a fourth pair of amplification primer comprising a forward primer 5 -GGGAGGCATTTGGTCAGATA-3' (SEQ ID NO: 7) and a reverse primer 5'-ACACACAGGCTGCTGACAAG-3' (SEQ ID NO: 8); a fifth pair of amplification primer comprising a forward primer

5'-CTGCCATGTTTTGGGTTTTT-3' (SEQ ID NO: 9) and a reverse primer 5'-GTGTAGGGGGAGGTCTGTGA-3' (SEQ ID NO: 10); a sixth pair of amplification primer comprising a forward primer 5 -ACGTCAGGGAGAAATTGTGC-3' (SEQ ID NO: 1 1 ) and a reverse primer 5'-CCAGACAGTGTGCTCCATTG-3' (SEQ ID NO: 12);

,a seventh pair of amplification primer comprising a forward primer

5'-TCCTGTGTGAAGCTGAATGC-3' (SEQ ID NO: 13) and a reverse primer 5'-TCACACGGGACATGAACACT-3' (SEQ ID NO: 14); and an eighth pair of amplification primer comprising a forward primer 5'-GAGGGCAGGAACACTGAGAA-3' (SEQ ID NO: 15) and a reverse primer 5'-ACTCTGCAGGTCCCAGCTT-3' (SEQ ID NO: 16);

Detailed Description of the Invention

As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting but merely as a basis for claims. It should be understood that the

SUBSTITUTE SHEET RULE 26 drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modification, equivalents and alternatives falling within the scope of the present invention as defined by the appended claims. As used throughout this application, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Similarly, the words "include," "including," and "includes" mean including, but not limited to. Further, the words "a" or "an" mean "at least one" and the word "plurality" means one or more, unless otherwise mentioned. Where the abbreviations or technical terms are used, these indicate the commonly accepted meanings as known in the technical field. For ease of reference, common reference numerals will be used throughout the figures when referring to the same or similar features common to the figures. The present invention relates to a method for identifying Epinephelus lanceolatus, characterized by the steps of:

extracting deoxyribonucleic acid from a biological sample;

subjecting the deoxyribonucleic acid of the biological sample for amplification of a marker region in the presence of a pair of amplification primer, wherein the pair of amplification primer is independently selected from a group consisting of a first pair of amplification primer comprising a forward primer 5'-ACTGCTACCCGACTCGTGAC-3' (SEQ ID NO: 1 ) and a reverse primer 5'-GCAAGGAAAGTGGAGAGAGC-3' (SEQ ID NO: 2), a second pair of amplification primer comprising a forward primer 5 -AGTTTGAGGGGGAAAAGCAT-3' (SEQ ID NO: 3) and a reverse primer 5'-CTGGCATTGACGAGAGCATA-3' (SEQ ID NO: 4), a third pair of amplification primer comprising a forward primer 5'-CCCATGTTAAAATGCCCAAC-3' (SEQ ID NO: 5) and a reverse primer 5'-GGATCGGTTGCGTAAGTTGT-3' (SEQ ID NO: 6), a fourth pair of amplification primer comprising a forward primer 5'-GGGAGGCATTTGGTCAGATA-3' (SEQ ID NO: 7) and a reverse primer 5'-ACACACAGGCTGCTGACAAG-3' (SEQ ID NO: 8), a fifth pair of amplification primer comprising a forward primer 5'-CTGCCATGTTTTGGGTTTTT-3' (SEQ ID NO: 9) and a reverse primer 5 -GTGTAGGGGGAGGTCTGTGA-3' (SEQ ID NO: 10), a sixth pair of amplification primer comprising a forward primer 5'-ACGTCAGGGAGAAATTGTGC-3' (SEQ ID NO: 11 ) and a reverse primer 5'-CCAGACAGTGTGCTCCATTG-3' (SEQ ID NO: 12), a seventh pair of amplification primer comprising a forward primer 5'-TCCTGTGTGAAGCTGAATGC-3' (SEQ ID NO: 13) and a reverse primer 5'-TCACACGGGACATGAACACT-3' (SEQ ID NO: 14), and an eighth pair of amplification primer comprising a forward primer 5'-GAGGGCAGGAACACTGAGAA-3' (SEQ ID NO: 15) and a reverse primer 5'-ACTCTGCAGGTCCCAGCTT-3' (SEQ ID NO: 16);

identifying the Epinephelus lanceolatus by detecting the presence of the amplified marker region. In a preferred embodiment of the method for identifying Epinephelus lanceolatus, the biological sample is from the genus of Epinephelus.

In a preferred embodiment of the method for identifying Epinephelus lanceolatus, the amplified marker region is 252 to 474 nucleotides in length.

Below is an example of a method for identifying Epinephelus lanceolatus from which the advantages of the present invention may be more readily understood. It is to be understood that the following example is for illustrative purpose only and should not be construed to limit the present invention in any way.

Examples

A genomic library of the Epinephelus lanceolatus is constructed. In a preferred embodiment, the deoxyribonucleic acid is extracted from Epinephelus lanceolatus. The deoxyribonucleic acid and pUC19 vector are digested with the use of restriction endonuclease enzyme and electrophoretically resolved on 1.5% agarose gels, wherein the restriction endonuclease enzyme is a combination of BamHI (the first enzyme from Bacillus amyloliquifaciens strain H) and Hindlll (the third enzyme from Haemophilus influenza strain d). The digested pUC19 vector is excised from the gel and purified using QIAquick^® gel extraction kit (Qiagen). A plurality of deoxyribonucleic acid fragments and a set of digested pUC19 vectors are formed after the digestion reaction. Ligation is set up with the plurality of deoxyribonucleic acid fragments and the set of pUC19 vectors by using T4 deoxyribonucleic acid ligase (Fermentas) and ligation buffer. Ligation reaction is carried out at room temperature for 16 hours and transformed into chemically competent E. coli and screened on Lysogeny Agar containing Ampicilin and X-Gal. White colonies are chosen and screened using Colony Polymerase Chain Reaction and sequenced using an ABI Big Dye terminator cycle sequencing kit with M13 (-20) as forward and reverse primers.

With the availability of Epinephelus lanceolatus genomic library sequence, a group of amplification primers may be designed for identification of Epinephelus lanceolatus. In a preferred embodiment, the deoxyribonucleic acid is extracted from the biological sample, wherein the biological sample is from the genus of Epinephelus. The deoxyribonucleic acid of the biological sample is subjected to polymerase chain reaction (PCR) amplification of the marker region in the presence of a pair of amplification primer, wherein the pair of amplification primer is independently selected from the group consisting of the first pair of amplification primer comprising the forward primer 5'-ACTGCTACCCGACTCGTGAC-3' (SEQ ID NO: 1 ) and the reverse primer 5'-GCAAGGAAAGTGGAGAGAGC-3' (SEQ ID NO: 2), the second pair of amplification primer comprising the forward primer 5'-AGTTTGAGGGGGAAAAGCAT-3' (SEQ ID NO: 3) and the reverse primer 5'-CTGGCATTGACGAGAGCATA-3' (SEQ ID NO: 4), the third pair of amplification primer comprising the forward primer 5'-CCCATGTTAAAATGCCCAAC-3' (SEQ ID NO: 5) and the reverse primer 5'-GGATCGGTTGCGTAAGTTGT-3' (SEQ ID NO: 6), the fourth pair of amplification primer comprising the forward primer 5'-GGGAGGCATTTGGTCAGATA-3' (SEQ ID NO: 7) and the reverse primer 5'-ACACACAGGCTGCTGACAAG-3' (SEQ ID NO: 8), the fifth pair of amplification primer comprising the forward primer 5'-CTGCCATGTTTTGGGTTTTT-3' (SEQ ID NO: 9) and the reverse primer 5'-GTGTAGGGGGAGGTCTGTGA-3' (SEQ ID NO: 10), the sixth pair of amplification primer comprising the forward primer 5'-ACGTCAGGGAGAAATTGTGC-3' (SEQ ID NO: 11 ) and the reverse primer 5'-CCAGACAGTGTGCTCCATTG-3' (SEQ ID NO: 12), the seventh pair of amplification primer comprising the forward primer 5'-TCCTGTGTGAAGCTGAATGC-3' (SEQ ID NO: 13) and the reverse primer 5 '-TC AC AC G G G AC ATG AAC ACT-3 ' (SEQ ID NO: 14), and the eighth pair of amplification primer comprising the forward primer 5'-GAGGGCAGGAACACTGAGAA-3' (SEQ ID NO: 15) and the reverse primer 5'-ACTCTGCAGGTCCCAGCTT-3'(SEQ ID NO: 16).

In order to identify Epinephelus lanceolatus, the group of amplification primers is tested on the deoxyribonucleic acid of different species of biological sample such as Epinenephelus lanceolatus, Epinephelus fuscoguttatus, Epinephelus coioides, and Epinephelus corallicola, whereby the group of amplification primers only shows the amplification bands on Epinephelus lanceolatus and not on the deoxyribonucleic acid of different species of biological sample as shown in Table 1. From the Table 1 , "1 " or "2" indicates amplification band, whereas "0" indicates no band or smear. The Epinephelus lanceolatus is identified by detecting the presence of the amplified marker region, wherein the amplified marker region is preferably 252 to 474 nucleotides in length.

Table 1 : PCR amplification datasheet

^Discrimination based on polymorphism

'Although the present invention has been described with reference to specific embodiments, also shown in the appended figures, it will be apparent for those skilled in the art that many variations and modifications can be done within the scope of the invention as described in the specification and defined in the following claims.

Claims

Claims I/We claim:

1 . A method for identifying Epinepheius lanceolatus, characterized by the steps of:

extracting deoxyribonucleic acid from a biological sample;

subjecting the deoxyribonucleic acid of the biological sample for amplification of a marker region in the presence of a pair of amplification primer, wherein the pair of amplification primer is independently selected from a group consisting of a first pair of amplification primer comprising a forward primer 5'-ACTGCTACCCGACTCGTGAC-3' (SEQ ID NO: 1 ) and a reverse primer 5'-GCAAGGAAAGTGGAGAGAGC-3' (SEQ ID NO: 2), a second pair of amplification primer comprising a forward primer 5'-AGTTTGAGGGGGAAAAGCAT-3' (SEQ ID NO: 3) and a reverse primer 5'-CTGGCATTGACGAGAGCATA-3' (SEQ ID NO: 4), a third pair of amplification primer comprising a forward primer 5'-CCCATGTTAAAATGCCCAAC-3' (SEQ ID NO: 5) and a reverse primer 5'-GGATCGGTTGCGTAAGTTGT-3' (SEQ ID NO: 6), a fourth pair of amplification primer comprising a forward primer 5'-GGGAGGCATTTGGTCAGATA-3' (SEQ ID NO: 7) and a reverse primer 5'-ACACACAGGCTGCTGACAAG-3' (SEQ ID NO: 8), a fifth pair of amplification primer comprising a forward primer 5'-CTGCCATGTTTTGGGTTTTT-3' (SEQ ID NO: 9) and a reverse primer 5'-GTGTAGGGGGAGGTCTGTGA-3' (SEQ ID NO: 10), a sixth pair of amplification primer comprising a forward primer 5'-ACGTCAGGGAGAAATTGTGC-3' (SEQ ID NO: 1 1 ) and a reverse primer 5'-CCAGACAGTGTGCTCCATTG-3' (SEQ ID NO: 12), a seventh pair of amplification primer comprising a forward primer 5'-TCCTGTGTGAAGCTGAATGC-3' (SEQ ID NO: 13) and a reverse primer 5'-TCACACGGGACATGAACACT-3' (SEQ ID NO: 14), and an eighth pair of amplification primer comprising a forward primer 5'-GAGGGCAGGAACACTGAGAA-3' (SEQ ID NO: 15) and a reverse primer 5'-ACTCTGCAGGTCCCAGCTT-3' (SEQ ID NO: 16);

identifying the Epinephelus lanceolatus by detecting the presence of the amplified marker region.

A method for identifying Epinephelus lanceolatus, wherein the biological sample is from the genus of Epinephelus.

A method for identifying Epinephelus lanceolatus, wherein the amplified marker region is 252 to 474 nucleotides in length.