CN1155720C

CN1155720C - Nucleic acid molecular probes and methods for identifying cyanobacteria

Info

Publication number: CN1155720C
Application number: CNB001172417A
Authority: CN
Inventors: 陈月琴; 周惠; 屈良鹄
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2000-07-07
Filing date: 2000-07-07
Publication date: 2004-06-30
Anticipated expiration: 2020-07-07
Also published as: CN1275717A

Abstract

The present invention belongs to the technical field of detecting environmental microbes by a molecular biology method. The present invention provides a nucleotide sequence of a blue-green alga rDNA interval area, a nucleic acid molecular probe derived from the sequence, and a method of identifying blue-green alga by the probe. By using the present invention, the blue-green alga (which comprises freshwater bloom blue-green alga, ocean red tide blue-green alga, etc.) can be conveniently, quickly and accurately identified. The present invention has the characteristics of objectiveness, accuracy, automatization, etc.

Description

Differentiate nucleic acid molecular probe and the method for blue-green algae

Technical field

The invention belongs to the technical field of utilizing molecular biology method testing environment (waters) miniature organism, specifically, relate to a kind of nucleotide sequence that is used to differentiate blue-green algae and nucleic acid molecular probe and differentiate the method for blue-green algae with it.

Background technology

Blue-green algae (Cyanophyta) claims cyanobacteria again, is one of main monoid that causes harmful poisonous wawter bloom of global fresh water water body and ocean harmful algal.Toxin that poisonous kind produces not only can cause neuron excitotoxicity, and can bring out liver cancer.The wildlife that cyanophycean toxin causes, domestic animal poison even the dead existing all over the world many reports of example, therefore, these poisonous kinds and distribution thereof are carried out identifying accurately rapidly become one of focus of present Study of Environmental Microbiology.

Belong to the of a great variety of blue-green algae, up to now, find that tens of kinds is the frequent species that causes fresh water bloom, and produce toxin.Poisonous blue-green algae is the essential items for inspection that fishery is imported and exported.Yet, the blue-green algae numerous species, its form has polytropy, some kind also contains the pigment that has only eukaryotic cell just to have, and shows the characteristic of eucaryon algae, has caused the confusion of identifying, add that its individuality is fine, separation and Culture is difficulty comparatively, and traditional classification differentiates that difficulty is bigger.

Up to now, research and the patent about aspects such as blue algae bloom control methods has many reports in the world, but standard and Study on Technology report that Shang Weijian differentiates relevant for the blue-green algae gene.In recent years, along with the aggravation of ocean and landlocked body eutrophication, the blue-green alga bloom distribution range and the frequency of occurrences obviously increase, and the research of blue-green algae have been caused people's great attention.In view of the problem that exists in the blue-green algae classification evaluation, adopt novel method new technology quick identification blue-green algae resource to have important practical significance.

Summary of the invention

The purpose of this invention is to provide standard and technology that the blue-green algae gene is differentiated, it can differentiate the blue-green algae monoid objective and accurately from inheritance.Specifically comprise: 1. be provided for blue-green algae genes identified fragment; 2. provide and derive from the segmental blue-green algae specificity of said gene nucleic acid molecular probe; 3. the method for utilizing above-mentioned nucleic acid molecular probe to differentiate blue-green algae is provided.

The present inventor is finding in different types of research blue-green algae, the rDNA transcribed spacer of blue-green algae has and the long nucleotide sequence of other biological diverse one section 20 oligonucleotide, this section nucleotides sequence is listed in tool high conservative in the blue-green algae, can be used as the characteristic nucleotide sequence of blue-green algae, be used to differentiate the blue-green algae monoid.According to this characteristic nucleotide sequence, can prepare the specificity nucleic acid molecular probe of blue-green algae rDNA transcribed spacer, set up the molecular biology method of fast, accurately differentiating blue-green algae.

Provided by the inventionly be used to differentiate that the nucleotide sequence of blue-green algae derives from microcystic aeruginosa (Microcystis aeruginosa, a kind of in the blue-green algae, be to bring out one of the red tide algae in China Xiamen west marine site in 97 years) rDNA transcribed spacer (being rrna rRNA internal gene transcribed spacers) sequence, the position of this sequence and constitutional features are shown in Fig. 1 and sequence table 1.

This sequence (SEQ ID NO1) can be by obtaining as embodiment 1 described method; Perhaps form and arrangement, on business-like automatic dna synthesizer, be synthesized into according to a conventional method by known this consecutive nucleotides.

Blue-green algae specificity nucleic acid molecular probe of the present invention is to classify the basis as with above-mentioned microcystic aeruginosa rDNA transcribed spacer nucleotides sequence, and relatively design by other blue-green algae kind in Internet (Internet) and the international molecular biology database.This molecular probe is oligonucleotide sequence or its complementary sequence that one section 20 Nucleotide in the rDNA transcribed spacer 2 (between tRNA gene and the 23S rRNA gene) is formed, or above-mentioned oligonucleotide sequence is modified again, change, and its Nucleotide variable quantity is no more than 10% sequence of total nucleotide amount.

Above-mentioned blue-green algae specificity nucleic acid molecular probe of the present invention is the sequence shown in the sequence table 2 (being called for short Cyano 20) or is the complementary sequence of this sequence: 5 ' CTATGCAGTTTTCAAGGTTC 3 '.

Above-mentioned blue-green algae specificity nucleic acid molecular probe of the present invention, can be according in advance according to the good sequence of microcystic aeruginosa rDNA transcribed spacer sequences Design, the DNA synthetic method by routine is synthesized into (for example can use business-like automatic dna synthesizer to synthesize).This probe can be incorporated into row labels by radio isotope, enzyme, vitamin H, fluorescent agent or chemiluminescence element.

The blue-green algae specificity nucleic acid molecular probe that the present invention is above-mentioned has high blue-green algae specificity, can with blue-green algae kind specificity reaction, but do not react with the DNA of other eucaryon algae or protokaryon bacterium.So, utilize this nucleic acid molecular probe, can differentiate blue-green algae (comprise the kind of bringing out fresh water bloom or marine red tide, perhaps have the ocean superminiature kind of eucaryon phycochrome etc.) quickly and accurately by PCR method or making nucleic acid molecular hybridization method.

Blue-green algae discrimination method provided by the present invention can adopt making nucleic acid molecular hybridization method or PCR method, and division is as follows:

Making nucleic acid molecular hybridization method: adopt foregoing nucleic acid molecular probe of the present invention, blue-green algae is identified with general making nucleic acid molecular hybridization method (for example Southern hybridization or dot blot).Concrete steps and process (comprising the pre-treatment of the mark and the testing sample of probe), molecular biology method routinely carries out.

This method can directly detect the blue-green algae sample of fresh water or seawater self-sow, also can be checked earlier with the amplification of the blue-green algae DNA extraction in the sample again.

PCR method: with foregoing nucleic acid molecular probe of the present invention is a PCR primer, with another prokaryotic organism universal PC R primer (for example 16N6:5 ' GAAGTCGTAACAAGGTAGCCG3 ' or 23R:5 ' NNGGGTTTCCCCATTCGGAAA3 ', wherein N represents base A, C, G or T) or the pairing of the specificity primer of a certain blue-green algae kind, carry out the PCR reaction.Concrete steps and the process operating process of PCR method are routinely carried out.

Adopt this PCR method to detect quickly and accurately and differentiate blue-green algae, and amount of samples is few.

Because the specificity sequence that blue-green algae nucleic acid molecular probe provided by the present invention is a blue-green algae, therefore under the prerequisite of sample the unknown, can by whether there being this section oligonucleotide sequence in the test sample, quick and convenient test sample, as accurately whether containing poisonous blue-green algae in rapid detection generation wawter bloom or red tide macrura reevesii, shrimp, the shellfish body, and the mark by probe, the number of individuals of detection by quantitative blue-green algae, play the effect of prevention forecast, avoid human poisoning to take place.

Description of drawings

Fig. 1 is a rrna rRNA gene structure synoptic diagram, and wherein ISR2 (transcribed spacer 2) is the zone of nucleotide sequence (comprising the blue-green algae specific molecular probe) existence involved in the present invention.

Fig. 2 is a microcystic aeruginosa rDNA transcribed spacer polynucleotide sequence structural representation, is blue-green algae specialized oligonucleotides molecular probe Cyano 20 of the present invention with the black matrix part (306-325bp) that goes up line wherein.

Embodiment

The present invention will be further described by the following examples:

The preparation of embodiment 1 microcystic aeruginosa rDNA transcribed spacer polynucleotide sequence

With toothpick or a small amount of microcystic aeruginosa frond of rifle point picking, put into and contain 100 microlitres extraction damping fluid (1%SDS, 10mM EDTA pH8.0,10mM Tris-Hcl pH7.6) in the little vial, shake up, 65 ℃ are incubated 0.5-2 hour, [comprise glass powder with the DPS purified reagent, 6M NaI, washing lotion (84mM Tris-Hcl, 40uM EDTA, 60% ethanol, 80mM KAC)] behind the purifying, airing adds 50-100 microlitre PCR reaction solution (100 microlitre PCR reaction solution compositions: 10X Taq dna polymerase buffer liquid 10 microlitres; 2mM dNTP solution 10 microlitres; Primer is M16:5 ' GGGGATGCCTAAGGCAGGGCT3 ' 0.5 microlitre; Primer is M23:5 ' CCACGTCCTTCTTCGCCTCTG3 ' 0.5 microgram; Taq archaeal dna polymerase 2.5 units add sterilized water and are settled to 100 microlitres), add 50 microlitre mineral oil, be put on the PCR instrument, carry out the PCR reaction by follow procedure: 94 ℃ of 5min, 94 ℃, 55 ℃ and 72 ℃ of each 1min, 30 circulations are 72 ℃ of 5min then.After reaction is finished, remove mineral oil, (QIAGEN, Germany) purified pcr product obtain required nucleotide sequence with the PCR purification kit.PCR product behind the purifying is measured on business-like automatic dna sequencer, obtains forming and arrangement as the Nucleotide of sequence table 1.

The preparation of embodiment 2 blue-green algae specificity nucleic acid molecular probes

On the basis that obtains Microcystis aeruginosa rDNA transcribed spacer nucleotide sequence, institute's calling sequence and other blue-green algae or eucaryon algae and protokaryon bacterium homologous sequence are arranged comparison, and the Nucleotide that draws Cyano 20 (306-325bp as shown in Figure 2) or its complementary sequence is formed and arranged is to be used for the good oligonucleotide fragment that the blue-green algae monoid is differentiated.According to the arrangement that the Nucleotide of Cyano 20 or its complementary sequence is formed, on business-like automatic dna synthesizer, carry out chemosynthesis, obtain nucleotide sequence or its complementary sequence shown in sequence table 2.

The discriminating of embodiment 3 blue-green algaes (conventional PCR method)

(1) sample pretreatment

With toothpick or rifle point picking small amount of sample (for example inclusion in water sample or fish, shrimp, the shellfish digestive tube), put into and contain 20 microlitres extraction damping fluid (1% SDS, 10mM EDTA pH8.0,10mM Tris-Hcl pH7.6), shake up, [comprise glass powder, 6M NaI, washing lotion (84mM Tris-Hcl with the DPS purified reagent, 40uM EDTA, 60% ethanol, 80mM Kac)] behind the purifying, airing, standby.

Composition in (2) the 100 microlitre PCR mixed solutions

Blue-green algae specific molecular probe Cyano 20 0.5 micrograms

Prokaryotic organism universal primer 1,6N6 0.5 microgram

10X Taq dna polymerase buffer liquid 10 microlitres

2mM dNTP solution 10 microlitres

Taq archaeal dna polymerase 2.5 units

Be 100 microlitres more than with the sterilized water constant volume

(3) PCR operation

Get 2 0.5 milliliter of little vials, add 20 microlitre PCR mixed solutions respectively, a pipe adds sample DNA then, another pipe only adds PCR mixed solution (contrast), adds 50 microlitre mineral oil in each pipe, is put on the PCR instrument, carry out PCR reaction by follow procedure: 94 ℃ 5 minutes, 94 ℃, 55 ℃ and 72 ℃ each 1 minute, and circulate continuously 30 times, then 72 ℃ 5 minutes.After reaction was finished, every pipe added 5 microlitre load sample liquid (30%Fcol, 0.25% bromjophenol blue).Get 4 microlitre point samples in 2% sepharose, electrophoresis result shows: adopt primer Cyano 20/ universal primer to carry out PCR and detect, if contain the positive reaction of blue-green algae (many pcr amplification bands of 1-in the sample, how many amplified band numbers depends on blue-green algae species number amount in the sample), do not contain the then negative reaction of blue-green algae (no pcr amplification band), the specificity of Cyano 20 has been described.

This method has following advantage:

(1) easy to be quick. Conventional DNA preparation needs 1 to a few hours, and the method is a demand when sample pretreatment Got final product in 20-30 minute (except the proliferation time).

(2) amount of samples is few, only needs a small amount of sample just can finish whole operation.

(3) accurate, highly sensitive, Cyano 20 is blue-green algae selectivity nucleic acid molecules pin, if other algae Class, negative reaction.

(4) do not use toxic reagent.

Sequence table 1

1. sequence signature: length: 359bp, type: nucleic acid, chain number: two strands, geometry: linearity;

2. molecule type: rDNA

3. originate: microcystic aeruginosa

4. sequence description: SEQ ID NO.1

AGGGAGACCT?AATTCAGGTA?GGAGACGAAA?AAAAAGTAGT?CGCTACCAAG 50

AATCAATCCC?AAAAGGTCGG?AGCGAGGCGA?AATTGGCTTT?CAAACTAGGT 100

TCTGGGCTCA?TAAAAGACCT?GAATCAGGAA?CAAGGGCTAT?TAGCTCAGGT 150

GGTTAGAGCC?GCCACCCCTG?ATAAGGGTGA?GGTCCCTGGT?TCGAGTCCAG 200

GATGGCCCAC?CTGCACAGGT?GGCAAAAACA?AAGAAGCGAG?GAATCAGCAC 250

CTTATCTTAC?TGACATAGTA?AGAGAGAATG?CTGGCTCTGA?GTAAAGAGTC 300

CAGAGGAACC?TTGAAAACTG?CATAGAGCTA?GGTGAAAAAG?CCAAAAAAGA 350

AGACCGCAA 359

Sequence table 2

1. sequence signature: length: 22bp, type: nucleic acid, chain number: strand, geometry: linearity;

2. molecule type: DNA

3. originate: blue-green algae

4. sequence description: SEQ ID NO.2

GAACCTTGAAAACTGCATAG

Claims

1. nucleic acid molecular probe of differentiating blue-green algae is characterized in that its nucleotides sequence classifies as:

Cyano20:5 ' GAACCTTGAAAACTGCATAG 3 ' or its complementary sequence: 5 ' CTATGCAGTTTTCAAGGTTC 3 '.

2. blue-green algae molecular biology identification method, it is characterized in that adopting nucleotides sequence to classify as: 5 ' GAACCTTGAAAACTGCATAG 3 ' or its complementary sequence: the nucleic acid molecular probe of 5 ' CTATGCAGTTTTCAAGGTTC 3 ', with the evaluation of testing of general nucleic acid molecular probe hybridizing method.

3. the molecular biology identification method of a blue-green algae, it is characterized in that adopting polymerase chain reaction method, classify as with nucleotides sequence: 5 ' GAACCTTGAAAACTGCATAG 3 ' or its complementary sequence: the nucleic acid molecular probe of 5 ' CTATGCAGTTTTCAAGGTTC 3 ' is a polymerase chain reaction primer, with another prokaryotic organism or the pairing of eukaryote typical zolymerization polymerase chain reaction primer, carry out the polymerase chain reaction again.

4. in accordance with the method for claim 3, the nucleotides sequence that it is characterized in that said nucleic acid molecular probe is classified as: 5 ' GAACCTTGAAAACTGCATAG 3 ' or its complementary sequence: 5 ' CTATGCAGTTTTCAAGGTTC 3 ', said prokaryotic organism or eukaryote typical zolymerization polymerase chain reaction primer are 16N6:5 ' GAAGTCGTAACAAGGTAGCCG3 ' or 23R:5 ' NNGGGTTTCCCCATTCGGAAA3 ', wherein N represents base A, C, G or T.