KR101747684B1 - Method for detection of Heterocapsa triquetra by sandwich hybridization integrated with nuclease protection assay and kit therefor - Google Patents

Method for detection of Heterocapsa triquetra by sandwich hybridization integrated with nuclease protection assay and kit therefor Download PDF

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KR101747684B1
KR101747684B1 KR1020150086522A KR20150086522A KR101747684B1 KR 101747684 B1 KR101747684 B1 KR 101747684B1 KR 1020150086522 A KR1020150086522 A KR 1020150086522A KR 20150086522 A KR20150086522 A KR 20150086522A KR 101747684 B1 KR101747684 B1 KR 101747684B1
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probe
oligonucleotide
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nucleotide sequence
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이택견
서승석
황진익
박미례
장만
이주연
정승원
손민식
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한국해양과학기술원
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
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    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/301—Endonuclease
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    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/125—Sandwich assay format
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/50—Detection characterised by immobilisation to a surface

Abstract

The present invention relates to a detection method and a kit for detection of Hercapacastriquestra using nuclease protection assay integrated sandwich hybridization, and more particularly to a nuclease protection assay probe, a capture probe, And a method for detecting Hercapacetriquatra contained in a sample by performing a sandwich hybridization integrated with a nuclease protection assay (NPA-SH) using a signal probe, Kit.
According to the present invention, it is possible to detect hetercapsatriquatra so as to be distinguished from other microcavities very accurately, and quantitative detection is also possible. Therefore, it is expected that the occurrence of Hercapacetriquotra can be predicted, and it can contribute to the maintenance of a stable marine ecosystem as well as to reduce unexpected damage of fisheries and aquaculture.

Description

[0001] The present invention relates to a method and a kit for detection of Hercapacastriquute using sandwich hybridization,

The present invention relates to a detection method and a kit for detection of Hercapacastriquestra using nuclease protection assay integrated sandwich hybridization, and more particularly to a nuclease protection assay probe, a capture probe, And a method for detecting Hercapacetriquatra contained in a sample by performing a sandwich hybridization integrated with a nuclease protection assay (NPA-SH) using a signal probe, Kit.

Microalgae can grow rapidly when environmental factors such as sunlight, temperature, trace elements, nutrients, and phosphorus are sufficient. However, rapid proliferation of some marine microalgae can cause serious harm to marine ecosystems. Especially, the occurrence of these harmful algae causes the death of many marine life by releasing toxic substances by seawater. About 300 out of 5,000 marine microalgae known to date produce about 80 species of toxin, which adversely affect fish, shellfish and humans.

Among these harmful birds, Heterocapsa triquetra is a biplane algae that caused great outbreaks in coastal areas since its first identification in 1883, and it does not release toxicity, but has a serious impact on aquaculture in Asia, . Since the observations were reported in Helsinki in 1976, major outbreaks have been reported in Finland, the United States, and Hong Kong, and have caused massive destruction of fish and shellfish. For this reason, it is very important to accurately detect microalgae causing harmful algae. Conventional detection methods and quantitative analysis methods for microalgae causing large outbreaks are methods for observing morphological characteristics, but these methods are time consuming, accurate and require a lot of experience. In addition, the various shapes and sizes of microalgae vary depending on the environmental factors, and also vary depending on the stage of growth. For this reason, for the past 20 years, microfabrication through molecular biology such as RFLP (Restriction Fragment Length Polymorphism), Real-time PCR, Fluorescent In Situ Hybridization (FISH), Flow CAM (Flow Cytometry And Microscopy) and NASBA (Nucleic Acid- Algae detection techniques have been developed.

Of these methods, methods based on oligonucleotide probes have been the most widely used for microalgae detection. The rRNA-targeted sandwich hybridization assay (SHA) has been used for the qualitative detection of several microalgae including Psuedonitzschia pungens , Heterosigma akashiwo , Fibrocapsa japonica and Alexandrium fundyense . Nonetheless, it was difficult to reach as much specificity and reproducibility as desired due to the instability of RNA molecules and the non-sophistication of hybridization.

Recently, sandwich hybridization (SH) integrated with nuclease protection assay (NPA) has been developed based on SHA. The technology uses three different probes, capture probes, NPA probes, and signal probes, and also uses S1 nuclease, which degrades single-chain nucleic acids. This nuclease protection assay has been successfully applied to the detection of several microalgae including Prorocentrum micans , Skeletonema costatum and Phaeocystis globosa .

The present inventors tried to apply the NPA-SH technique to heretofore not applied Heterocapsa triqueltra. Heterocapsa triquera, which is a harmful alga, can be accurately detected from a sample and clearly distinguished from other microalgae And to develop a method to minimize errors.

As described above, various techniques for identifying species or detecting specific cells have been developed. However, due to various variables such as the characteristics of an organism or a cell, the state of a sample, the distribution of a pseudocell, and the characteristics of a genome, Finding a way is very tricky. The present inventors have been able to develop a sandwich hybrid detection method integrated with neclease protection analysis that is efficient in the detection of Heterocapsa tincture based on the experience gained through many years of research.

Cai, Q.S., Li, R.X., Zhen, Y., Mi, T.Z., Yu, Z.G., 2006. Detection of two Prorocentrum species using sandwich hybridization integrated with nuclease protection assay. Harmful Algae 5 (3), 300-309.

Therefore, a main object of the present invention is to provide a method for accurately detecting Hercapacetriquatra from a sample.

It is another object of the present invention to provide a kit for easily detecting the above-mentioned hetercapacetriquater.

According to one aspect of the present invention, the present invention provides a method for hybridization comprising hybridizing an oligonucleotide probe comprising the nucleotide sequence of SEQ ID NO: 1 with a nucleic acid in a sample; A single-stranded naturally-degrading step of treating a single stranded nuclease to decompose the unmarried oligonucleotide probe; A denaturing step of denaturing the hybridized oligonucleotide probe to a single strand; A capture probe hybridization step of hybridizing a capture probe specifically binding to the oligonucleotide probe and immobilized on a support with a modified oligonucleotide probe; A cleaning step for removing the unfavorite material from the trapping probe; A signal probe hybridization step of hybridizing a signal probe specifically bound to the oligonucleotide probe and labeled with a labeling substance with an oligonucleotide probe hybridized to the capture probe; A washing step for removing the unfavorable substance from the oligonucleotide hybridized to the trapping probe; And a labeling substance detection step of detecting a labeling substance of the signal probe, wherein the capture probe comprises an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 2, wherein the signal probe comprises a nucleotide sequence of SEQ ID NO: 3 The present invention provides a method for detecting Heterocapsa triquetra comprising the steps of:
According to another aspect of the present invention, there is provided an oligonucleotide probe for detection of Hercapacetriquotra comprising the nucleotide sequence of SEQ ID NO: 1; A capture probe that specifically binds to the oligonucleotide probe and is fixed to the support; And a signal probe that specifically binds to the oligonucleotide probe and is labeled with a labeling substance, the capture probe comprising an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 2, And a kit for detecting Heterocapsa triquetra comprising an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 3.

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According to the present invention, it is possible to detect hetercapsatriquatra so as to be distinguished from other microcavities very accurately, and quantitative detection is also possible. Therefore, it is expected that the occurrence of Hercapacetriquotra can be predicted, and it can contribute to the maintenance of a stable marine ecosystem as well as to reduce unexpected damage of fisheries and aquaculture.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the rRNA gene sequence of a heterokapatriquotra compared with a region corresponding to a nuclease protection assay (NPA) probe sequence of the present invention and rRNA gene sequences of other microalgae.
FIG. 2 is a schematic representation of the binding of the rRNA of Heterocarpa triquestra with the NPA probe of the present invention and the combination of the NPA probe with the capture probe and the signal probe.
Fig. 3 shows the results of testing the specificity of the various microalgae by carrying out the detection method of the present invention.
FIG. 4 shows the result (A) of the sensitivity test conducted by the detection method of the present invention at various cell concentrations and the regression curve (B) determined based on the result.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

<Examples>

1. Microalgae culture

Heterocapsa triquetra and Chattonella marina were purchased from South Sea Institute of Korea Ocean Research & Development Institute, Cochlodinium polykrikoides were purchased from Korea Ocean Research & Development Institute, Oceanic Sampling Library, Heterosigma akashiwo , Prorocentrum minimum and Scrippsiella trochoidea were distributed from Ansan, Korea Ocean Research & .

Each of the microalgae was inoculated into sterilized 30 psu of f / 2 medium and cultured at 20 ° C for 12 hours and 12 hours for dark reaction.

2. Determination of rRNA gene sequence of each microalgae prepared

Trizol was used to extract the RNA of each microalgae and cDNA was synthesized using reverse transcription system kit (Promega, USA). The amplified PCR product was cloned into a pGEM-T-easy vector (Promega, USA) after preparing the PCR product using the prepared cDNA as a template and a PCR primer (Table 1) for large subunit RNA and small subunit RNA.

The cloned gene was transformed into Escherichia coli DH-5 and submitted to Bioneer (Daejeon, Korea) for sequencing.

Based on the results of the nucleotide sequence analysis, NCBI blast search was performed to confirm the sequence of each species.

PCR primers for rRNA gene amplification Target gene PCR primer Large subunit RNA Forward 5'-CGGAGGAAAAGAAACTAAC-3 ' Reverse 5'-AGCTACTAGATGGTTCGAT-3 ' Small subunit RNA Forward 5'-ACCTGGTTGATCCTGCCAGT-3 ' Reverse 5'-TCACCTACGGAAACCTTGT-3 '

3. Probe design

11 species of microalgae ( C. polykrikoides , C. fulvescens , P. minimum , H. akashiwo , S. trochoidea , C. marina , H. triquetra , C. curvisetus , S. marinoi , T. nordenskioeldii , L. danicus ) Of the large subunit rRNA gene was selected by Mega 5.05 program. The most nuclease protection assay (NPA) probe of 60 nucleotides was designed and compared with H. triquetra SEQ ID NO: 1: CCACGCTTGCGCTGAAGCAGCAGGCAATCACATTAGCACGCACCAATCTTGCCAAGAAGC) (see Fig. 1).

A capture probe (biotin-GCTTCTTGGCAAGATTGGTGCGTGC) of 25 nucleotides conjugated with biotin was designed from the 3-terminal sequence of the NPA probe. From the 5-terminal sequence, 25 nucleotides linked with fluorescein (fluorescein) A signal probe (GCCTGCTGCTTCAGCGCAAGCGTGG-fluorescein) was designed (see Fig. 2).

4. nuclease protection assay sandwich hybridization integrated with nuclease protection assay (NPA-SH)

The NPA-SH was carried out in 2006 with reference to the paper by Cai et al.

4-1. Capture probe fixed

A 50 nM trapped probe dissolved in PBS was added to a microplate coated with streptavidin, reacted at 37 ° C for 2 hours, washed with PBST (0.5% tween-20) Respectively.

4-2. Cell lysis

(50% duty cycle and 450W output) (Ultrasonic Cell) were added to 0.5 ml of yeast tRNA containing lysis buffer (80% formamide, 450 mM NaCl, 5 mM Na 2 EDTA, 1% SDS, pH 6.4) Disrupter, model JY-92 II, Ningbokesheng Inc., Zhejiang, China) and then centrifuged at 13,000 rpm for 1 min at 4 ° C to remove cell debris.

4-3. Hybridization of NPA probe and S1 nuclease reaction

The NPA probe and the dissolved microalgae were mixed well and denaturation was carried out at 97 ° C for 15 minutes, followed by hybridization at 42 ° C for 2 hours. Subsequently, S1 nuclease was added, reacted at 42 ° C for 1 hour, and the reaction was terminated with a nuclease stop solution (62.5 mM NaOH, 30 mM Na 2 EDTA, 0.5 M PBS, pH 7.2).

4-4. Hybridization of capture probes

The S1 nuclease-terminated solution was denatured at 97 ° C for 15 minutes, then cooled at room temperature and injected into a microplate containing a capture probe, followed by hybridization at 50 ° C for one hour in a shaking incubator at 130 rpm.

4-5. Hybridization and detection of signal probe

Then, a 50 nM signal probe was added and reacted in a shaking incubator at 130 rpm for 30 minutes at 50 ° C. Then, anti-fluorescein-POD (1: 6,000), 3,3 ', 5,5'- tetramethylbenzidine solution The reaction was finally terminated with 2M H 2 SO 4 , and absorbance was measured at 450 nm and 630 nm using FLUOstar.

5. Specificity test

After incubating 1 ml of each microalgae culture solution for 1 minute with Rugol solution, the number of H. triasseri 10 4 cells was measured by repeating the experiment 100 times by microscope three times, and H. akashiwo , C. polykrikoides , C. marina , P. minimum , And S. trochoide were collected in 10 5 cells each in an e-tube.

Then, sonication (50% duty cycle and 450W output) was performed for 10 seconds with 0.5 mg of yeast tRNA containing lysis buffer (80% formamide, 450 mM NaCl, 5 mM Na 2 EDTA and 1% SDS, pH 6.4) NPA-SH was performed in the same manner as in Example 1. [

As a result, as shown in FIG. 3, the detection method of the present invention is capable of specifically detecting only H. triquetra by distinguishing it from other microalgae such as H. akashiwo , C. polykrikoides , C. marina , P. minimum and S. trochoide .

6. Sensitivity check

H. triquetra culture medium was measured 1㎖ repeated three times with a microscope populations were fixed 1 min at rugol solution, based on the average value f / 2 serial dilution in culture medium (serial dilution) in a manner that each of, or increase the volume 10 0 ~ 10 6 cells were collected in an e-tube.

Then, sonication (50% duty cycle and 450W output) was performed for 10 seconds with 0.5 mg of yeast tRNA containing lysis buffer (80% formamide, 450 mM NaCl, 5 mM Na 2 EDTA and 1% SDS, pH 6.4) And NPA-SH were repeated three times each in the same manner as in Example 1.

As a result, as shown in FIG. 4, the detection intensity was changed depending on the number of cells in the sample. Based on this result, a regression curve like FIG. 4B was derived.

Therefore, it was confirmed that the number of cells of H. triquetra in the sample can be accurately determined based on the absorbance value obtained by performing NPA-SH using the same method as in this embodiment.

<110> Korea Institute of Ocean Science & Technology <120> Method for detection of Heterocapsa triquetra by sandwich          hybridization integrated with nuclease protection assay and kit          therefor <130> PA-D15144 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Nuclease protection assay probe for Heterocapsa triquetra <400> 1 ccacgcttgc gctgaagcag caggcaatca cattagcacg caccaatctt gccaagaagc 60                                                                           60 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Capture probe for Heterocapsa triquetra <400> 2 gcttcttggc aagattggtg cgtgc 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Signal probe for Heterocapsa triquetra <400> 3 gcctgctgct tcagcgcaag cgtgg 25

Claims (14)

A hybridization step of hybridizing an oligonucleotide probe comprising the nucleotide sequence of SEQ ID NO: 1 with a nucleic acid in the sample;
A single-stranded naturally-degrading step of treating a single stranded nuclease to decompose the unmarried oligonucleotide probe;
A denaturing step of denaturing the hybridized oligonucleotide probe to a single strand;
A capture probe hybridization step of hybridizing a capture probe specifically binding to the oligonucleotide probe and immobilized on a support with a modified oligonucleotide probe;
A cleaning step for removing the unfavorite material from the trapping probe;
A signal probe hybridization step of hybridizing a signal probe specifically bound to the oligonucleotide probe and labeled with a labeling substance with an oligonucleotide probe hybridized to the capture probe;
A washing step for removing the unfavorable substance from the oligonucleotide hybridized to the trapping probe; And
And a labeling substance detecting step of detecting labeling substance of the signal probe,
Wherein the capture probe comprises an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 2,
The signal probe is heteroaryl kapsa tree Quebec trad (Heterocapsa triquetra) detection method which comprises the oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 3 comprises a nucleotide.
delete delete delete delete delete delete An oligonucleotide probe for detecting Heterocapsa triquetra comprising the nucleotide sequence of SEQ ID NO: 1;
A capture probe that specifically binds to the oligonucleotide probe and is fixed to the support; And
A signal probe that specifically binds to the oligonucleotide probe and is labeled with a labeling substance,
Wherein the capture probe comprises an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 2,
Wherein the signal probe comprises an oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 3. 2. A kit for detecting Heterocapsa triquetra , comprising:
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KR1020150086522A 2015-06-18 2015-06-18 Method for detection of Heterocapsa triquetra by sandwich hybridization integrated with nuclease protection assay and kit therefor KR101747684B1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5818195B2 (en) 2010-03-04 2015-11-18 三菱レイヨン株式会社 Probes and microarrays for detecting harmful or toxic dinoflagellates

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5818195B2 (en) 2010-03-04 2015-11-18 三菱レイヨン株式会社 Probes and microarrays for detecting harmful or toxic dinoflagellates

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Cai et al., Harmful Algae, Vol.5, pp.300-309, (2006)*
NCBI GenBank accession No : HQ902267.1 (2011. 3. 15.)*

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