KR101789182B1 - Primer set for diagnosing infection of fish and method for diagnosing infection of fish using the same - Google Patents

Abstract

The present invention relates to a composition for the diagnosis of bacterial infection of fishes comprising a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2, and a method for diagnosing bacterial infection of fish using the composition. Since the primer set of the present invention can specifically amplify a reverse transcriptase derived from LINE, it is possible to diagnose bacterial infection of a fish, and it is economical because it can save time and cost for diagnosis of bacterial infection, It is possible to prevent the damage caused by the bacterial infection.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a primer set for detecting a bacterial infection in a fish, and a method for diagnosing a bacterial infection in a fish using the primer set.

The present invention relates to a composition for the diagnosis of bacterial infection of fishes comprising a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2, and a method for diagnosing bacterial infection of fish using the composition.

In vertebrate genomes, retro elements are widely distributed which cause insertion mutations and deletion mutations and provide poly (A) signals, palindromic miRNA sequences, and alternative promoters do. Retro elements are distinguished by LINE (long interspersed nuclear element), ERV (endogenous retrovirus) and SINE (short interspersed nuclear element). Autonomy LINE voluntarily entered the new genome area during evolution. LINE has a length of about 6 kb. LINE encodes two ORFs called ORF1 and ORF2. The reverse transcriptase is encoded by ORF2 and the transcription product derived from the LINE can be made to have retrotransformation activity. On the other hand, the pathogenic bacteria of fish include Streptococcus iniae causing Streptococcus disease, Streptococcus parauberis , Edwardsiella causing Ed ward disease ( Edwardsiella et al . Vibrio that causes tarda), Vibrio illness anggwil Leeum (Vibrio anguillarum), Vibrio Tangier Bay (Vibrio harveyi = V. carchariae = V. trachuri , Vibrio ordalii , Vibrio causing hypertrophic disease, Vibrio ichthyoenteri ), tenacibaculum causing a bacterial disease of the slide ( Tenacibaculum maritimum ) (syn, Flexibacter maritimus), Pasteurella piscicida ), and Nocardia kampachi (causing Nocardia disease ).

Among these pathogenic bacteria, most of them are caused by Streptococcal disease, Edwad disease, Vibrio disease, Slide bacterial disease, etc. According to the statistics of the Aquatic Species of Cheju Island, 5,760 tons in 2011, 26.07% of the total amount of Jeju Island (22,093 tons) And the amount is about 57.6 billion won. However, it is actually more than 70 billion.

These pathogenic bacteria are derived from the feces of land animals, and they are inhabited in wetlands such as a puddle of land, and it is presumed that they infect fishes by flowing into rivers and oceans due to heavy rainfall. However, there is still no definite epidemiological investigation.

For this reason, early diagnosis of bacterial diseases occurring in fish is very important to prevent the spread of diseases and treatments. However, it is required to improve the diagnostic method because the conventional method requires more than 2 days to diagnose by the method of bacterial segregation.

Therefore, the present inventors confirmed that the reverse transcriptase derived from the flounder's LINE increases the expression level in response to the bacterial infection, and the primer set capable of specifically amplifying the reverse transcriptase has an excellent diagnostic effect on the bacterial infection of the fish And completed the present invention.

An object of the present invention is to provide a composition and kit for the diagnosis of bacterial infection of fish, which comprises a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2.

Still another object of the present invention is to provide a method for detecting RNA, comprising: (a) extracting RNA from a sample; (b) carrying out RT-PCR using the primers set forth in SEQ ID NO: 1 and SEQ ID NO: 2, using the RNA of step (a) as a template; And (c) identifying the level of expression of the reverse transcriptase from the LINE in the RT-PCR product.

In order to achieve the above object, the present invention provides a composition for the diagnosis of bacterial infection of fish, comprising a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2.

In addition, the present invention provides a kit for the diagnosis of bacterial infection of fish, comprising a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2.

(A) extracting RNA from a sample; (b) carrying out RT-PCR using the primers set forth in SEQ ID NO: 1 and SEQ ID NO: 2, using the RNA of step (a) as a template; And (c) identifying the level of expression of the reverse transcriptase from the LINE in the RT-PCR product.

Since the primer set of the present invention can specifically amplify a reverse transcriptase derived from LINE, it is possible to diagnose bacterial infection of a fish, and it is economical because it can save time and cost for diagnosis of bacterial infection, It is possible to prevent the damage caused by the bacterial infection.

Figure 1 shows the alignment of reverse transcriptase sequences derived from three mature genetic elements LINE located within the flounder genome.
FIG. 2 is a diagram showing the result of comparing the reverse transcriptase sequence derived from the mating genetic element LINE identified in the flounder genome with the reverse transcriptase sequence present in the other fish genome. FIG.
FIG. 3 is a graph showing the results of comparing the expression levels of reverse transcriptase from the mobility gene LINE of the present invention in liver, spleen, gills and muscles before and after anthrax infection by Streptococcus parauberis (L: liver, S: spleen , G: gill, M: muscle).

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for the diagnosis of bacterial infection of fish, comprising a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2.

The primer set is composed of primers necessary for specifically amplifying a reverse transcriptase derived from a long interspersed nuclear element (LINE) of a flounder ( Paralichthys olivaceus ) for the diagnosis of a bacterial infection of a fish.

In order to identify genetic markers capable of diagnosing bacterial infections in fishes from flounder, the present inventors found three new LINE sequences in the flounder and sequenced them with known LINE sequences to obtain seven of the reverse transcriptase derived from the LINE The conserved domains were identified and primers containing them were prepared. The primer of the present invention is advantageous in that it is excellent in sensitivity to reverse transcriptase derived from the LINE of the flounder.

In the present invention, the term "primer" means a nucleic acid sequence having a short free 3 'hydroxyl group and capable of forming a base pair with a template of a complementary nucleic acid, Quot; means a short nucleic acid sequence which functions as a starting point. The primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

The primers can be prepared by a method of synthesizing nucleotides known to those skilled in the art.

The primer set of the present invention may include a modified sequence in which mutation occurs in a part of the individual base sequences constituting the primer set.

The modified sequence refers to a result obtained by performing PCR using a mutated primer and a result of performing PCR using a primer consisting of an unmodified sequence in substantially the same range.

The composition for diagnosing bacterial infection of fish of the present invention may further comprise a reagent, a thermostable DNA polymerase, a dNTP and a divalent metal ion necessary for carrying out an RT-PCR amplification reaction.

In the present invention, the fish may be at least one species selected from the group consisting of flounder (turmeric), turbot, dolphin, red sea bream, sandalfish, pufferfish, rockfish (rockfish), defense and eel.

Bacterial infections in the present invention is Streptococcus am ah (Streptococcus iniae), streptococcus para Ube is less (Streptococcus parauberis), Era tar ed and served (Edwardsiella tarda , Vibrio anguillarum , Vibrio harveyi = V. carchariae = V. trachuri), otherwise Vibrio climb (Vibrio ordalii), Vibrio yihi thio yen Terry (Vibrio ichthyoenteri , Tenacibaculum maritimum (syn, Flexibacter maritimus), Pasteurella piscicida , Nocardia kampachi , but are not limited thereto.

In the present invention, the diseases that can be caused by bacterial infection of fish include, but are not limited to, streptococcal disease, Edward's disease, Vibrio disease, intestinal opacity, sliding bacterial disease, pancreas disease, nocardia disease and the like .

In addition, the present invention provides a kit for the diagnosis of bacterial infection of fish, comprising a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2.

The kit includes a primer set of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention, and a reagent for performing a reverse reaction and an amplification reaction. The reagent for carrying out the amplification reaction in the kit may include reverse transcriptase, DNA polymerase, dNTPs, buffer and the like. In addition, components necessary for conducting electrophoresis to confirm whether the RT-PCR product is amplified can be further included in the kit of the present invention. Furthermore, it may contain an RNase inhibitor, DEPC-water, sterilized water and the like, if necessary, and may further include known components required for real-time RT-PCR.

The kit of the present invention can be prepared by putting the above composition into a plastic tube in lyophilized form, and the kit may further comprise, in addition to the composition, appropriate reagents and tools used for analysis, Suitable carriers, labels capable of producing a detectable signal, solubilizers, detergents, and the like may be included.

Such suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester , Polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

(A) extracting RNA from a sample; (b) carrying out RT-PCR using the primers set forth in SEQ ID NO: 1 and SEQ ID NO: 2, using the RNA of step (a) as a template; And (c) identifying the level of expression of the reverse transcriptase from the LINE in the RT-PCR product.

The sample may be a tissue of fish associated with the immune response, such as liver, spleen, gill, muscle, and preferably spleen or muscle tissue.

The RNA extraction in the step (a) may be performed according to a method commonly used in the art, and may be carried out using a commercially available RNA extraction kit.

The RNA isolated from the RT-PCR in the step (b) may be used as a template, and the cDNA may be synthesized through a reverse transcription reaction using a reverse transcriptase and then performed using a PCR reaction mixture. The PCR reaction may be performed using a PCR reaction mixture containing various components known in the art necessary for the PCR reaction. In addition to the cDNA synthesized by the reverse transcription reaction and the primer set provided in the present invention, the PCR reaction mixture includes an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and water. The PCR buffer solution may include Tris-HCl, MgCl ₂ , KCl, and the like. In this case, MgCl ₂ concentration greatly affects the specificity and yield of amplification. In general, when Mg ^{2 +} is excessive, nonspecific PCR amplification product is increased, and when Mg ^{2 +} is insufficient, the yield of PCR product is decreased . The PCR buffer solution may further contain an appropriate amount of Triton X-100 (Triton X-100).

The temperature, time, and number of cycles of each step of the PCR can be determined according to the conditions commonly practiced in the art.

In step (c), the level of expression can be confirmed by isolating the DNA of the PCR product according to a method well known in the art. Preferably by agarose gel or polyacrylamide gel electrophoresis or fluorescence analysis apparatus. At this time, in order to use the fluorescence analyzer, PCR is performed in the step (b) using a primer set with a fluorescent substance known in the art. When PCR products are identified by agarose gel electrophoresis, electrophoresis can be followed by electrophoresis with ethidium bromide staining. Methods for performing general reverse transcription, PCR and analysis of the results are well known in the art.

In addition, the expression level of the reverse transcriptase derived from the LINE can be confirmed by real-time RT-PCR without depending on the size of the DNA of the PCR product as described above.

The method for diagnosing a bacterial infection of a fish according to the present invention comprises the steps of: (d) detecting the level of expression of a reverse transcriptase from the LINE measured in the step (c) as compared with the expression level of a reverse transcriptase derived from the LINE measured in the control sample And determining that the fish is infected with bacteria or an immune response has occurred in the fish.

The "control" refers to a healthy fish not infected with bacteria.

Terms not otherwise defined herein have meanings as commonly used in the art to which the present invention belongs.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1: Sequence comparison of reverse transcriptase from three lines located in flounder dielectrics

Reverse transcriptase from three lines was identified through analysis of transposable elements in flounder, and the sequences corresponding to the known LINE sequence (GenBank Accession number AY136821) were compared. The results are shown in Fig.

As shown in Figure 1, the reverse transcriptase sequence is located in ORF2 of the LINE and has seven conserved reverse transcriptase domains (RTI to RTVII), with 89-99% sequence homology between the reverse transcriptase from the three LINEs Respectively.

Example 2: Phylogenetic analysis of reverse transcriptase from three lines located in flounder

The MEGA6 program was used to systematically analyze the reverse transcriptase from three LINEs located in the flounder genome through the neighbor-joining method. The phylogenetic tree of the reverse transcriptase from the three LINEs of Example 1 was obtained and the results are shown in Fig. Eight species of fish used for phylogenetic analysis were three-spined stickleback ( Gasterosteus aculeatus ; AC182753.1), European seabass ( Dicentrarchus labrax ; FQ310506.3), coral grouper ( Epinephelus corallicola ), Turquoise killifish ( Nothobranchius furzeri ; FQ310506.3), Cichlid endemic to Lake Victoria ( Haplochromis chilotes ), African cichlids ( Haplochromis burtoni ), Japanese rice fish ( Oryzias latipes ; AB054296.2), and Takifugu rubripes ( Takifugu rubripes ).

As shown in FIG. 2, it was confirmed that reverse transcriptase sequences derived from the previously known LINE sequence (GenBank Accession number AY136821) and the three LINEs of Example 1 were classified together.

Example 3: Design of a primer specific to a gene encoding a reverse transcriptase derived from LINE

Among the seven conserved reverse transcriptase domains identified in Example 1 above, the domains 4 to 5 were found to be relatively well conserved in the reverse transcriptase of other mobility factors, and as shown in Table 1, (RTIV to RTV) were designed.

Name of the primer order SEQ ID NO: OF-LINE-RT-Chr3-1-F 5'-CAAGTTAATCATGGAGTTCCACA-3 ' One OF-LINE-RT-Chr3-1-R 5'-TTCATCATGCTTCATGGAAAT-3 ' 2

The following experiments were conducted to compare the amount of reverse transcriptase expressed by tissues using the primer sets designed as described above.

Example  4: Streptococcus parauberis of  In the liver, spleen, gills, and muscle before and after anthrax infection Reverse-transcriptase  Identify expression levels

Flounder ( Paralichthys olivaceus ) from Korean fisheries. Cultured flounder provided only industrially produced food. For anthropogenic infection, healthy flounder was kept in a 22 ° C tank and infected with a sub-lethal dose of S. parauberis suspended in PBS. Seven days after infection, the tissues were isolated from liver, spleen, gill and muscle of the flounder, and all tissues before RNA extraction were frozen at -80 ° C.

To extract total RNA from liver, spleen, gill and muscle, RNA was extracted from the tissue as described above according to the manufacturer's protocol using Trizol reagent (Invitrogen, Carlsbad, Calif., USA). After extraction, total RNA concentration was measured using NanoDrop® ND-1000 UVvis Spectrophotometer.

Reverse transcription was performed using PrimeScript® RT Reagent Kit and gDNA Eraser (Takara, Japan) in a reaction mixture of 20 μl containing 500 ng of total RNA from each sample.

(5'-CAAGTTAATCATGGAGTTCCACA-3 'and 5'-TTCATCATGCTTCATGGAAAT-3') and 18s rRNA gene (accession No. EF126037) specific to the LINE designed as in Example 3 were used for the transcripts obtained by the reverse transcription reaction, (5'-GGTCTGTGATGCCCTTAGATGTC-3 'and 5'-AGTGGGGTTCAGCGGGTTAC-3') were used to perform quantitative real-time RT-PCR under the conditions shown in Table 2 below.

Name of the primer order mer Tm G.C% target OF-LINE-RT-Chr3-1-F 5'-CAAGTTAATCATGGAGTTCCACA-3 ' 23 57.6 39 OF-LINE-RT-Chr3-1 OF-LINE-RT-Chr3-1-R 5'-TTCATCATGCTTCATGGAAAT-3 ' 21 56.5 33 18s rRNA-F 5'-GGTCTGTGATGCCCTTAGATGTC-3 ' 23 60.7 52 18s rRNA gene
(EF126037) 18s rRNA-R 5'-AGTGGGGTTCAGCGGGTTAC-3 ' 20 62.0 60

LINE expression level was standardized using 18s rRNA gene, and the result is shown in FIG.

In Fig. 3, the white bar represents the control group, which is the flounder before infection, and the black bar represents the test group, which is flounder after infection. As shown in FIG. 3, as a result of the anthrax infection by Streptococcus parauberis , the reverse transcriptase derived from LINE showed the most prevalent expression level in the spleen and was also significantly overexpressed in the muscle.

Therefore, it is possible to deduce that the reverse transcriptase derived from the LINE is involved in the immune response in the spleen and muscle, and by discovering a reverse transcriptase derived from the LINE that expresses specifically in the spleen known as the organ associated with the immune response of the fish It has been confirmed that the primer set of the present invention can be used for rapid and accurate diagnosis when a fish is infected or when an immune response occurs in a fish.

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

<110> Pusan National University Industry-University Cooperation Foundation <120> Primer set for diagnosing infection of fish and method for          diagnosing infection of fish using the same <130> 1.292P <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for LINE <400> 1 caagttaatc atggagttcc aca 23 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for LINE <400> 2 ttcatcatgc ttcatggaaa t 21

Claims (5)

A composition for diagnosing Streptococcus parauberis infection of a flounder, comprising a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2, wherein the primer set is for amplification of a spleen sample.
2. The composition according to claim 1, wherein the primer sets represented by SEQ ID NO: 1 and SEQ ID NO: 2 specifically amplify a reverse transcriptase derived from a long interspersed nuclear element (LINE) of a flounder ( Paralichthys olivaceus ).
A kit for the diagnosis of Streptococcus parauberis infection of flounder, comprising a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2, wherein the primer set is amplified spleen sample.
(a) extracting RNA from a spleen sample;
(b) carrying out RT-PCR using the primers set forth in SEQ ID NO: 1 and SEQ ID NO: 2, using the RNA of step (a) as a template; And
(c) identifying the level of expression of the reverse transcriptase from the LINE in the RT-PCR product. &lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; delete

Images