JP2015146774A - Primer sets for detecting red tide, and methods for detecting red tide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide primer sets which are excellent in specificity, sensitivity and amplification efficiency, and can be easily used on-site, though there are detection methods drawing attention, which can promptly and correctly identify red tide plankton by nucleic acid amplification tests, and to solve the problem that there has been increased harm caused by red tides in recent years and accordingly methods for detecting red tide in the earliest stage have been in great demand.SOLUTION: Provided are: primer sets capable of detecting red tide by specifically amplifying a region of ITS1-5.8S ribosomal RNA-ITS2 or a region of Mitochondrial Cytochrome C Oxidase Subunit I in the red tide plankton genomes; and methods for detecting red tide by using these primer sets.

Description

The present invention is a primer set for detecting red tide and a red tide detection method.

Red tide is a phenomenon in which the water of the sea, lakes, and rivers is colored by the large proliferation of a specific type of plankton. In areas where aquaculture is thriving, the occurrence of red tide causes suffocation due to clogged or damaged plankton in the aquaculture of fish, toxins produced by plankton, decreased dissolved oxygen concentration due to decomposition of plankton carcasses, etc. Damage caused by drowning of large amounts of cultured fish is a problem.

Among planktons that cause red tides, there are Heterosigma akashiwo, Rafido algae such as Chattonella, and dinoflagellates such as Alexandrium, Karenia, and Cochlodinium. These red tide plankton are examined by observing the collected seawater with a microscope. A technology has also been developed to detect the occurrence of red tide plankton with high sensitivity.

Conventional red tide detection techniques include the method of detecting the occurrence of red tide by measuring the peak height of the output waveform using a phytoplankton sensor (see Patent Document 1), and the detection of shellfish belonging to bivalves in contact with the sample. A method for detecting harmful floating organisms such as red tide that measures changes in muscle action potential (see Patent Document 2), and phytoplankton that releases superoxide and / or hydrogen peroxide by the amount of chemiluminescence generated by luminol reagents. Examples thereof include a method for quantifying the concentration (see Patent Document 3), a fluorescence detector that can be detected with high sensitivity by measuring the emission wavelength of a red tide phosphor in water (see Patent Document 4), and the like.

Attempts have also been made to detect plankton that causes red tide with high sensitivity specifically by the nucleic acid amplification method (see Non-Patent Document 1 and Non-Patent Document 2).

JP 2002-045074 A JP 2000-093038 A JP-A-11-075893 JP 08-261934 A

Ryoma Kamikawa, Yoshihiko Sako et al, “Application of a Real-time PCR Assay to a Comprehensive Method of Monitoring Harmful Algae”, MicrobesEnviron. Vol.21, 2006 Masao Adachi, “Analysis of Alexandrium (Dinophyceae) Species UsingSequences of the 5.8s Ribosomal DNA and Internal Transcribed Spacer Regions”, J. Phycol. Vol.32, 1996

In recent years, damage caused by the red tide is increasing, and a method capable of detecting the occurrence of the red tide at an early stage is required. In view of this, a detection method using a nucleic acid amplification method, which is quick and accurate and can identify species of red tide plankton, has attracted attention. In the nucleic acid amplification method, it is only necessary to have a general nucleic acid amplification device or reagent, and no special equipment is required other than designing a primer set, so that it can be introduced at low cost.

However, the primer set described in the above-mentioned non-patent literature has a problem in specificity, such as amplification of plankton nucleic acids other than red tide plankton. Moreover, since the size of the amplification product was large, the amplification efficiency was low and could not be detected quickly. Therefore, there has been a demand for a primer set for nucleic acid amplification that is excellent in specificity, sensitivity, and amplification efficiency and can be easily introduced in the field.

In order to solve the above-mentioned problems, the inventors have developed the ITS1-5.8S ribosomal RNA-ITS2 region of the red tide plankton genome, or Mitochondrial.
A primer set for specifically amplifying and detecting a part of the Cytochromoe C Oxidase Subunit I region was designed, and further, a red tide detection method using these primer sets was invented.

That is, the present invention provides a red tide detection primer set consisting of one or more of the following (a) to (e).

(A) Primer set consisting of oligonucleotide shown in SEQ ID NO: 1 and oligonucleotide shown in SEQ ID NO: 2 (b) Primer set consisting of oligonucleotide shown in SEQ ID NO: 3 and oligonucleotide shown in SEQ ID NO: 4 (C) Primer set consisting of the oligonucleotide shown in SEQ ID NO: 5 and the oligonucleotide shown in SEQ ID NO: 6 (d) Primer set consisting of the oligonucleotide shown in SEQ ID NO: 7 and the oligonucleotide shown in SEQ ID NO: 8

Furthermore, the present invention provides a primer set for detecting a red tide comprising one or a plurality of primer sets described in (e) to (h) below, which is a primer set including a probe used in a quantitative nucleic acid amplification method.

(E) Primer set (f) comprising a probe composed of the oligonucleotide shown in SEQ ID NO: 9 in addition to the primer set consisting of the oligonucleotide shown in SEQ ID NO: 1 and the oligonucleotide shown in SEQ ID NO: 2 In addition to the primer set consisting of the oligonucleotide shown in SEQ ID NO: 3 and the oligonucleotide shown in SEQ ID NO: 4, in addition to the primer set comprising the oligonucleotide shown in SEQ ID NO: 10, a primer set (g) SEQ ID NO: In addition to the primer set consisting of the oligonucleotide shown in SEQ ID NO: 6 and the primer set consisting of the oligonucleotide shown in SEQ ID NO: 6, the primer set (h) shown in SEQ ID NO: 7 comprises a probe composed of the oligonucleotide shown in SEQ ID NO: 11 Oligonucleotide , And in addition to the primer set consisting of an oligonucleotide of SEQ ID NO: 8 includes a probe composed of an oligonucleotide depicted in SEQ ID NO: 12, primer set

Furthermore, the present invention provides a primer set designed to specifically amplify a nucleic acid by the LAMP method, wherein the primer set for detecting red tide is composed of one or more of the following (i) to (l): provide.

(I) a primer set consisting of the oligonucleotide shown in SEQ ID NO: 13, the oligonucleotide shown in SEQ ID NO: 14, the oligonucleotide shown in SEQ ID NO: 15 and the oligonucleotide shown in SEQ ID NO: 16 (j) in SEQ ID NO: 17 A primer set comprising the oligonucleotide shown, the oligonucleotide shown in SEQ ID NO: 18, the oligonucleotide shown in SEQ ID NO: 19, and the oligonucleotide shown in SEQ ID NO: 20 (k) the oligonucleotide shown in SEQ ID NO: 21, SEQ ID NO: A primer set consisting of the oligonucleotide shown in SEQ ID NO: 23, and the oligonucleotide set shown in SEQ ID NO: 25, the primer set consisting of the oligonucleotide shown in SEQ ID NO: 24 That oligonucleotides, oligonucleotide shown in SEQ ID NO: 27, and a primer set consisting of an oligonucleotide depicted in SEQ ID NO: 28

The present invention also includes an oligonucleotide in which one or a plurality of bases are deleted, added, or substituted in the oligonucleotide constituting the primer set of any one of the above primer set (a) to primer set (l). Provided is a red tide detection primer set that can function as a red tide detection primer set.

The present invention also provides a method for detecting red tide including a step of specifically amplifying a red tide plankton nucleic acid using the above-described primer set.

Since the nucleic acid amplification method using the primer set of the present invention has high species specificity, the occurrence of false positives caused by contamination with nucleic acids of other plankton can be suppressed. Further, the primer set of the present invention is designed so that the size of the amplification product is about 100 bases, and the nucleic acid amplification efficiency is high, and rapid and accurate detection is possible.

It is a figure which shows the sensitivity of the red tide detection by a quantitative nucleic acid amplification method. It is a figure which shows the species specificity of the red tide detection by the quantitative nucleic acid amplification method. It is a figure which shows the sensitivity of the red tide detection by LAMP method. It is a figure which shows the species specificity of the red tide detection by LAMP method. It is a figure which shows the detection of Heterosigma akashiwo in Ainan-cho waters. It is a figure which shows the detection of Cochlodinium polykrikoides in the sea area of Ainan town. It is a figure which shows the detection of Karenia mikimotoi in Ainan town sea area. It is a figure which shows the detection of Chattonella sp. In the sea area of Ainan-cho.

The present invention provides a primer set capable of detecting red tide plankton quickly and accurately, and a red tide detection method using the primer set.

By using the primer set of the present invention, Heterosigma akashiwo, Cochlodinium known as red tide plankton
polykrikoides, Karenia mikimotoi, Chattonella marina, Chattonella antiqua, and Chattonella ovata can be detected. More specifically, the primer sets (a), (e) and (i) are Heterosigma akashiwo, the primer sets (b), (f) and (j) are Cochlodinium polykrikoides, (c), (g ) And (k) primer sets can specifically detect Karenia mikimotoi, and (d), (h) and (l) primer sets can specifically detect Chattonella marina, Chattonella antiqua, and Chattonella ovata, respectively.

Heterosigma akashiwo, Cochlodinium of the present invention
The primer sets that detect polykrikoides and Karenia mikimotoi target the ITS1-5.8 S ribosomal RNA-ITS2 region of each red tide plankton genome. That is, it can hybridize under stringent conditions with a part of the nucleic acid having the base sequence of the ITS1-5.8 S ribosomal RNA-ITS2 region of red tide plankton and / or a part of the nucleic acid complementary to the base sequence. Since the 5.8 S ribosomal RNA region has high species specificity, red tide detection with excellent specificity is possible.

On the other hand, the primer sets of (d), (h) and (l) are Mitochondrial of the genomes of Chattonella marina, Chattonella antiqua, and Chattonella ovata.
Targets the Cytochromoe C Oxidase Subunit I region. In the genus Chattonella, the above-mentioned three species are known as red tide plankton. However, since the risk does not change greatly, it is preferable to detect without distinguishing the species. With the primer set of the present invention, it is possible to detect the above three types together.

That is, the primer set for detecting the genus Chattonella of the present invention comprises a part of a nucleic acid having a base sequence of the Mitochondrial Cytochromoe C Oxidase Subunit I region of the genome of the genus Chattonella and / or a part of a nucleic acid complementary to the base sequence. It can hybridize under stringent conditions.

The stringent conditions include, for example, conditions in which the temperature of the reaction solution used in a general nucleic acid amplification method is about 50 ° C. to about 70 ° C., preferably about 55 ° C. to about 65 ° C. .

Among the primer sets of the present invention, (a) to (d) are primer sets designed for general nucleic acid amplification methods, and (e) to (h) are quantitative nucleic acid amplification methods, particularly real-time nucleic acid amplification. A primer set including a probe used in the method (Real-time PCR), and (i) to (l) are primer sets designed for the LAMP method.

The present invention performs determination of the presence / absence of red tide plankton contained in a sample, quantification of the number of red tide plankton contained in a sample, and risk determination from the quantified red tide plankton by a nucleic acid amplification method. Can do. When quantifying the number of red tide plankton contained in a sample, a quantitative nucleic acid amplification method is preferably used.

Examples of the quantitative nucleic acid amplification method include a real-time nucleic acid amplification method. More specifically, in the real-time nucleic acid amplification method, TaqMan (registered trademark) Probe method (Roche), QProbe (registered trademark) method (Nippon Steel & Sumikin Environment Co., Ltd.), universal QProbe (registered trademark) method, intercalator method, etc. Is mentioned. Among these, TaqMan (registered trademark) Probe method can be preferably used.

When using the TaqMan (registered trademark) Probe method, a target-specific probe is designed by modifying the 5 'end with a fluorescent dye and the 3' end with a quencher substance. By adding this to the nucleic acid amplification reaction system simultaneously with the primer set, the probe specifically hybridizes to the template DNA. Since the probe is degraded by the 5 '→ 3' exonuclease activity of DNA polymerase, the fluorescent substance is released and emits fluorescence. Therefore, the amount of amplification product can be known in real time by measuring the fluorescence intensity in the reaction solution.

The probe of the present invention is specifically composed of the oligonucleotide shown in SEQ ID NO: 9, and the probe modified with a fluorescent dye and a quencher substance is composed of the oligonucleotide shown in SEQ ID NO: 10 for detection of Heterosigma akashiwo. The probe modified with the fluorescent dye and the quencher substance is composed of the oligonucleotide shown in SEQ ID NO: 11 for detection of Cochlodinium polykrikoides, and the probe modified with the fluorescent dye and the quencher substance is used for detection of Karenia mikimotoi. A probe composed of the oligonucleotide shown in FIG. 12 and modified with a fluorescent dye and a quencher substance can be used for the detection of Chattonella marina, Chattonella antiqua, and Chattonella ovata, respectively. However, the base sequence of the oligonucleotide constituting the probe is not limited to these as long as quantitative detection is possible.

The size of the amplification product when nucleic acid amplification was performed using each primer set was 127 bp for the primer set (a), 100 bp for the primer set (b), 99 bp for the primer set (c), ( In the primer set of d), it is 143 bp. In either case, since the size is around 100 bp, it is considered that an amplified product can be efficiently obtained and rapid and accurate detection can be achieved.

The LAMP (Loop-Mediated Isothermal Amplification) method (Patent No. 3313358) is a nucleic acid amplification method using a strand displacement reaction, and four types of primers are used. Starting from amplification products having loop structures at both ends, amplification products of a plurality of sizes having repetitive sequences are finally obtained. Quantitative detection is possible by electrophoresis or turbidity of the reaction solution.

In nucleic acid amplification by the LAMP method, four types of primers, F3, B3, FIP (Forward Inner Primer), and BIP (Backward Inner Primer) are used. In the present invention, specifically, in the detection of Heterosigma akashiwo, F3 is an oligonucleotide represented by SEQ ID NO: 13, B3 is an oligonucleotide represented by SEQ ID NO: 14, and FIP is an oligonucleotide represented by SEQ ID NO: 15. The oligonucleotide shown in SEQ ID NO: 16 is used for BIP. Cochlodinium
In the detection of polykrikoides, F3 has the oligonucleotide shown in SEQ ID NO: 17, B3 has the oligonucleotide shown in SEQ ID NO: 18, FIP has the oligonucleotide shown in SEQ ID NO: 19, and BIP has the sequence shown in SEQ ID NO: 20. Oligonucleotides are used.

For detection of Karenia mikimotoi, F3 has the oligonucleotide shown in SEQ ID NO: 21, B3 has the oligonucleotide shown in SEQ ID NO: 22, FIP has the oligonucleotide shown in SEQ ID NO: 23, BIP has the sequence shown in SEQ ID NO: 24 The oligonucleotides shown are used. In addition, Chattonella
marina, Chattonella antiqua, and Chattonella
In the detection of ovata, F3 has the oligonucleotide shown in SEQ ID NO: 25, B3 has the oligonucleotide shown in SEQ ID NO: 26, FIP has the oligonucleotide shown in SEQ ID NO: 27, and BIP has the sequence shown in SEQ ID NO: 28 Oligonucleotides are used. However, as long as each red tide plankton can be detected, the base sequence of the oligonucleotide of the primer is not limited thereto.

In addition to the oligonucleotides shown in SEQ ID NO: 1 to SEQ ID NO: 28, including oligonucleotides in which one or more bases of the oligonucleotide shown in SEQ ID NO: 1 to SEQ ID NO: 28 are deleted, substituted, or added, A primer set that can function as a red tide detection primer set by generating an equivalent amplification product and a red tide detection method using the primer set are included in the present invention.

  Furthermore, although it demonstrates in detail using an Example, this invention is not limited to them.

Example 1. Verification of quantitative nucleic acid amplification method using red tide plankton cultures Red tide plankton was detected by quantitative nucleic acid amplification method using red tide plankton cultures. Table 1 shows the primer sets and TaqMan® probe sequences for each red tide plankton. As the reaction conditions, the first heat denaturation was performed at 98 ° C. for 1 minute, and the temperature change at 96 ° C. for 1 second and 58 ° C. for 30 seconds was performed for 40 cycles. The total reaction time was 50 minutes.

The detection sensitivity of the obtained results is shown in FIG. 1 and the specificity is shown in FIG. Heterosigma akashiwo, Cochlodinium
Polykrikoides and Chattonella genera were detectable up to 0.001 cells / reaction solution, and Karenia mikimotoi was detectable up to 0.0001 cells / reaction solution. Heterosigma akashiwo, Cochlodinium
It was confirmed that polykrikoides and Karenia mikimotoi were detected in a species-specific manner, and that three species of the genus Chattonella were detected with the same primer set.

Example 2 Verification of LAMP method using red tide plankton cultures. Red tide plankton was detected by LAMP method using red tide plankton cultures. The primer set sequence for each red tide plankton is shown in Table 1. The reaction conditions were 65 ° C. and 1 hour.

The detection sensitivity of the obtained results is shown in FIG. 3, and the specificity is shown in FIG. Heterosigma akashiwo and Cochlodinium
It was possible to detect polykrikoides up to 0.01 cells / reaction solution, and Karenia mikimotoi and Chattonella genera up to 0.001 cells / reaction solution. Heterosigma
It was confirmed that akashiwo, Cochlodinium polykrikoides and Karenia mikimotoi were detected in a species-specific manner, and that three species of the genus Chattonella were detected with the same primer set.

Example 3 FIG. Demonstration of red tide detection in the waters of Ainan Town, Ehime Prefecture
From January 2012 to September 2012, red tides were detected by quantitative nucleic acid amplification in the sea area of Ainan, Ehime Prefecture. DNA is extracted from the sediment obtained by centrifuging 50 mL of seawater collected at five locations: Fukaura, Kura, Funakoshi, Fukuura, and Misso, and quantitative nucleic acid amplification is performed using a primer set that includes a TaqMan (registered trademark) probe. It was. Oligonucleotides having the sequences shown in Table 1 were used as primers and probes. As the reaction conditions, the first heat denaturation was performed at 98 ° C. for 1 minute, and the temperature change at 96 ° C. for 1 second and 58 ° C. for 30 seconds was performed for 40 cycles. The total reaction time was 50 minutes.

The detection results at five locations of each red tide plankton and the water temperature at a depth of 2 m are shown in FIGS. Each red tide plankton was successfully detected from seawater samples with high sensitivity.

Claims (5)

A primer set for detecting red tide comprising one or more of the following (a) to (e): (a) an oligonucleotide represented by SEQ ID NO: 1 and a primer set comprising an oligonucleotide represented by SEQ ID NO: 2 (b) Primer set consisting of the oligonucleotide shown in SEQ ID NO: 3 and the oligonucleotide shown in SEQ ID NO: 4 (c) The oligonucleotide set shown in SEQ ID NO: 5 and the primer set consisting of the oligonucleotide shown in SEQ ID NO: 6 (d) Primer set consisting of the oligonucleotide shown in SEQ ID NO: 7 and the oligonucleotide shown in SEQ ID NO: 8 A primer set comprising a probe used in a quantitative nucleic acid amplification method, comprising one or more of the following (e) to (h): a primer set for detecting red tide (e) an oligonucleotide represented by SEQ ID NO: 1 In addition to the primer set consisting of the oligonucleotide shown in SEQ ID NO: 2, in addition to the primer set containing the oligonucleotide shown in SEQ ID NO: 9, the primer set (f) the oligonucleotide shown in SEQ ID NO: 3 and SEQ ID NO: 4 In addition to the primer set consisting of the oligonucleotides shown in 1), the primer set (g) comprising the probe containing the oligonucleotide shown in SEQ ID NO: 10 and the oligonucleotide shown in SEQ ID NO: 6 and the oligonucleotide shown in SEQ ID NO: 6 In addition to the primer set consisting of In addition to a primer set comprising a probe comprising the oligonucleotide shown in 11 (h) the oligonucleotide shown in SEQ ID NO: 7 and the primer set consisting of the oligonucleotide shown in SEQ ID NO: 8, the oligo shown in SEQ ID NO: 12 Primer set containing probes containing nucleotides A primer set designed to specifically amplify a nucleic acid by the LAMP method, comprising one or a plurality of red tide detection primer sets (i) described in (i) to (l) below: A primer set comprising the oligonucleotide shown, the oligonucleotide shown in SEQ ID NO: 14, the oligonucleotide shown in SEQ ID NO: 15, and the oligonucleotide shown in SEQ ID NO: 16 (j) the oligonucleotide shown in SEQ ID NO: 17, SEQ ID NO: A primer set consisting of the oligonucleotide shown in Fig. 18, the oligonucleotide shown in SEQ ID NO: 19, and the oligonucleotide shown in SEQ ID NO: 20 (k) the oligonucleotide shown in SEQ ID NO: 21, the oligonucleotide shown in SEQ ID NO: 22, The oligonucleotide shown in SEQ ID NO: 23 Primer set consisting of nucleotide and oligonucleotide shown in SEQ ID NO: 24 (1) oligonucleotide shown in SEQ ID NO: 25, oligonucleotide shown in SEQ ID NO: 26, oligonucleotide shown in SEQ ID NO: 27, and SEQ ID NO: 28 Primer set consisting of the indicated oligonucleotides A primer set for detecting red tide, comprising an oligonucleotide in which one or a plurality of bases are deleted, added or substituted in an oligonucleotide constituting any one of the primer sets (a) to (l) Primer set for red tide detection that can function as A method for detecting a red tide comprising a step of specifically amplifying a red tide plankton nucleic acid using the primer set according to any one of claims 1 to 4.