CN100560734C - The detection probes of middle Skeletonemacostatum - Google Patents
The detection probes of middle Skeletonemacostatum Download PDFInfo
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- CN100560734C CN100560734C CNB2004100755328A CN200410075532A CN100560734C CN 100560734 C CN100560734 C CN 100560734C CN B2004100755328 A CNB2004100755328 A CN B2004100755328A CN 200410075532 A CN200410075532 A CN 200410075532A CN 100560734 C CN100560734 C CN 100560734C
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Abstract
The invention discloses a kind of detection probes of middle Skeletonemacostatum, belong to the detection of nucleic acids field.Specifically synthetic at the rRNA specific region of middle Skeletonemacostatum is one group of probe of core with S1 enzyme protection analysis probe, and associating S1 enzyme protection analysis and sandwich hybridization technology realize the qualitative and quantitative detection of centering Skeletonemacostatum rRNA.Described detection probes comprises three oligonucleotide probes that are mutually related: S1 enzyme protection analysis probe, arrest probe, signal probe or linking probe.Probe suitability of the present invention is good, greatly facilitates the searching and the realization of specific probe.Stability of the present invention is higher than the sandwich hybridization technology far away.
Description
Technical field
The present invention relates to the oligonucleotide probe that one group of algae detects, specifically the specific probe that detects at middle Skeletonemacostatum qualitative and quantitative.Belong to the detection of nucleic acids field.
Background technology
Middle Skeletonemacostatum (Skeletonema costatum) is a kind of wide temperature eurysalinity plant plankton that worldwide extensively exists, and also is one of modal diatom sociales in China coastal waters.This kind is the secondary producer's in ocean good bait, be again common red tide plankton in China coastal waters and good indicator of pollution simultaneously, in the economical alga culture zone, normal fight for nutrition with sea-tangle, laver etc., the a large amount of wawter blooms that replant can cause the economic algae variable color, even rot and lose edible and commercial value; According to another report, be that the wawter blooms of sociales can cause the fish gill infringement of Atlantic Ocean Oncorhynchi and final dead with middle Skeletonemacostatum.Therefore, Skeletonemacostatum in detecting in time, is accurately and rapidly understood its population dynamics in the culture zone, in time controls when its quantity increases, and is significant to the sea farming industry.Traditional discrimination method that utilizes opticmicroscope direct viewing and counting, process is loaded down with trivial details, time-consuming, effort, and needs empirical systematicalian and participate.When sample size many, rich biodiversity, therefore workload was very big when monitoring range was wide, was mainly used in the qualitative of algae and separated, and be not suitable for the study on monitoring quantitatively and on a large scale of algae.So development is used for fast, accurately the novel method and the new technology of detection Skeletonemacostatum have realistic meaning.But at present international and domestic progress about this respect is little, does not form as yet very effectively to carry out qualitative and mature technology detection by quantitative to this algae.Because rRNA is considered to and the closely-related sequence of the phyletic evolution of algae, and rRNA content is very high in the eukaryotic cells, can reach 10
6～10
7Individual copy, rRNA are the good target spots of sandwich hybridization.
Find by prior art documents, Christopher A.Scholin etc. are in " utilizing rRNA southern rhombus algae of identification in full cell and " sandwich " hybridization " (U.S.'s " phycology magazine ", 1996,35 (3), 190～197) in the literary composition with the RNA that extracts or frustule lysate under certain condition with the oligonucleotide probe hybridization that is combined on 96 orifice plates after, hybridize with it with the signal probe of a mark again, carry out the enzyme labelled antibody color reaction at last, obtained satisfied result.Use to some extent in the monitoring of the present marine algae of this technology.But up to the present, use little algae that this technology can detect and be only limited to limited several (comprising middle Skeletonemacostatums).Its main drawback is: one, and because of the length of capture probe 10～30bp only, the capture probe quantity that therefore can effectively distinguish the nearer little algae of sibship is few; Two, in whole testing process, all need to prevent the RNA degraded, yet each step such as washing, antibody response very easily causes the RNA degraded, so operation easier is big, the detected result fluctuation is big.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art; one group of detection probes at the specific middle Skeletonemacostatum of middle Skeletonemacostatum rRNA is provided; according to this group probe this algae is carried out that the S1 enzyme protection is analyzed and sandwich hybridization detects, realize the fast qualitative of this algae and quantitative.
The present invention is used for this algae is carried out analysis of S1 enzyme protection and sandwich hybridization detection; specifically comprise three oligonucleotide probes that are mutually related: S1 enzyme protection analysis probe; arrest probe; signal probe or linking probe; described S1 enzyme protection analysis probe; be meant complementary and be used for the oligonucleotide probe of S1 enzyme protection analytical procedure with middle Skeletonemacostatum rRNA; the described probe of arresting; be meant with an end of S1 enzyme protection analysis probe and combine; this end is generally 3 ' end; an other end is marked with vitamin H; thereby will through S1 enzyme enzyme cut handle and denaturing treatment after be retained in S1 enzyme protection analysis probe in the solution and catch the probe that gets off; described signal probe or linking probe can arrested after probe catches S1 enzyme protection analysis probe; be incorporated into S1 enzyme protection analysis probe the other end probe be linking probe; this probe mark has detectable signal simultaneously; perhaps at one end have the signal probe land, can with the complementation of universal signal probe.
S1 enzyme protection analysis probe is when carrying out the information biology compare of analysis; only with middle Skeletonemacostatum small subunit ribosome 30～100 regional Nucleotide complementations; and do not have homology with other algae ribosome-RNA(rRNA); length is 60bp; on the rRNA of Skeletonemacostatum, sequence was: 5 '-GTCTCAAAGATTAAGCCATGCATGTGTAAGTATAAGATACTTTATCGAAACTGCGG ACGG during but specificity was incorporated under certain conditions.By the rRNA sequence of Skeletonemacostatum in measuring, carry out multisequencing relatively, the characteristic sequence of Skeletonemacostatum in the searching is as the special zone of the rRNA of this algae, then according to the special regional sequence of this rRNA (characteristic sequence) design S1 enzyme protection analysis probe.Seek the method in biologic specificity zone and all adopt prior art according to special zone design probe.Existing experiment shows, just can be used as the target sequence that specific probe designs if having the continuous mispairing of 2～3 above Nucleotide or lack between the different algae rRNA.
Arrest probe specificity and be incorporated into S1 enzyme protection analysis probe one end; length is 27bp; sequence signature is: 5 ' GTCTCAAAGATTAAGCCATGCATGTGT; be marked with vitamin H, phosphate group at 5 ' end, perhaps amino group and other any marks that can be used for can be for fixing to the marks of solid substrate.
Signal probe or linking probe at one end with the complementation of S1 enzyme protection analysis probe, and have the complementary district of universal signal probe at the other end, combine with the universal signal probe is complementary, length is 26bp, sequence is: 5 '-AGATACTTTATCGAAACTGCGGACGG; The signal of the various detections of mark on this sequence specifically comprises: fluorescein, digoxin, vitamin H are used for the sequence of signal detection, perhaps with general one section sequence of signal probe complementary.
One end of universal signal probe and linking probe (being different from and complementary bonded one end of S1 enzyme protection analysis probe) complementation.Marker commonly used comprises (but being not limited to): digoxin, fluorescein, vitamin H or horseradish peroxidase etc.
In brief, in The present invention be directed to the rRNA specific region of Skeletonemacostatum synthetic be one group of probe of core with S1 enzyme protection analysis probe, associating S1 enzyme protection is analyzed and the qualitative and quantitative detection of sandwich hybridization technology realization centering Skeletonemacostatum rRNA.
This group probe of the present invention can be used for Skeletonemacostatum in whether existing in analysis of S1 enzyme protection and the sandwich hybridization technology for detection sample and quantity thereof, and described testing process comprises the following steps:
(a) mixing of detected sample and S1 enzyme protection analysis probe
In this step, can handle the little algae sample to be detected that discharges rRNA through cracking and mix with S1 enzyme protection analysis probe.Also little algae sample to be detected directly can be mixed with S1 enzyme protection analysis probe, and then carry out cracking and handle, thereby discharge rRNA.In case the rRNA that discharges comprises the target sequence corresponding to S1 enzyme protection analysis probe,, then can form two strands with S1 enzyme protection analysis probe through hybridization.
(b) the S1 enzyme is handled
After the rRNA of S1 enzyme protection analysis probe and biology to be detected hybridization, with the excessive free probe of S1 enzymic digestion of proper concn (being generally 0.5U～2U/ μ l, preferably is 1U/ μ l); When the non-target rRNA reaction of this probe and other, because crossbred exists otch or small gap, the S1 enzyme also can cut off this probe; Finally retain and the S1 enzyme protection analysis probe of coupling and the DNA/RNA two strands that the rRNA of middle Skeletonemacostatum forms fully, and amount that should two strands has been represented the amount of corresponding rRNA in the sample.
(c) sex change of DNA-RNA heterozygote
With the double-stranded heterozygote sex change of DNA/RNA is dna single chain (being S1 enzyme protection analysis probe) and RNA single strand.Suitable denaturation method is not particularly limited, can be with this area various denaturation method commonly used, and thermally denature etc. for example.For example, add the neutralization solution post-heating, make the heterozygote sex change of the rRNA formation of S1 enzyme protection analysis probe and target organism, thereby discharge the S1 enzyme protection analysis probe that remains.Because the RNA instability, the RNA single strand of separating chain formation is easily by the RNA enzyme liberating of endogenous or external source.
(d) catch S1 enzyme protection analysis probe
On solid phase carrier, arrest probe hybridization with remaining with the solution of S1 enzyme protection analysis probe and predetermined fixed, S1 enzyme protection analysis probe is arrested on solid phase carrier specifically, and non-specific combination is removed in washing.
(e) probe of combined belt detectable signal
With linking probe and the hybridization of captive S1 enzyme protection analysis probe, and wash and remove non-specific combination, use signal probe to combine again, and non-specific combination is removed in washing with linking probe.
(f) signal detection
Available this area ordinary method is carried out signal detection to detectable signal.Fluorescent signal on for example can mark on the signal probe comes reading of data by fluorescence excitation like this; Also can be on mark on the signal probe digoxin, fluorescein or vitamin H etc., antibody or the avidin by horseradish peroxidase or alkali phosphatase enzyme mark reacts with it then; Add luminous substrate or chromogenic substrate (TMB or NPP etc.) at last, come the interpretation signal by surveying luminous power or absorbancy.
Major advantage of the present invention is:
(1) the probe suitability is good: the key of sandwich hybridization specific recognition is the specificity of arresting probe, generalized case is arrested long approximately 20～30 Nucleotide of probe, this probe will have specificity, must bigger difference to be arranged with the rRNA molecule of other nontarget organisms, although different biological rRNA can be variant on evolving, but find and the nearer biology of good authentication sibship between specific probe be not a nothing the matter, this may also be that the sandwich hybridization technical development only finds seldom amount to arrest one of reason of probe till now.The S1 enzyme protection analysis probe that relates among the present invention; although length is about 60 Nucleotide; but and do not require that the probe total length all will have specificity, and only require that the region intermediate of probe has the difference of several Nucleotide to get final product, this greatly facilitates the searching and the realization of specific probe.
(2) stability is high: one of key of sandwich hybridization technology successful execution is to guarantee that the RNA molecule is not degraded in whole process, will guarantee to arrest that section target RNA molecule (may arrive between the hundreds of Nucleotide tens) the standing intact in whole experiment between probe and the signal probe especially.But because experimentation relates to antigen antibody reaction and washing step repeatedly, this provides more chance for the RNA enzyme to the RNA molecular degradation, so will guarantee the target RNA molecule very large difficulty that has not been degraded.Even finally can realize qualitative analysis, the confidence level of quantitative analysis is also made a discount greatly.And the direct process relevant with the rRNA molecule has only S1 enzyme protection analysis probe and this step of rRNA molecular hybridization among the present invention; as long as the rRNA molecule was not degraded or seldom is degraded when the suitable RNA enzyme inhibitors of hybridization lysate adding just can guarantee hybridization; other steps of experimental implementation all are to carry out at stable dna molecular, carry out relatively easily.Therefore stability of the present invention is higher than the sandwich hybridization technology far away.
Description of drawings
Fig. 1 is the synoptic diagram that the detection probes of middle Skeletonemacostatum is used for surveying various different algaes.
Fig. 2 is the detection curve synoptic diagram to the middle Skeletonemacostatum of different cell count.
Embodiment
Below in conjunction with accompanying drawing, further set forth the present invention at specific embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment: use at the S1 enzyme protection of rRNA and analyze and sandwich hybridization technology centering Skeletonemacostatum qualitative and quantitative detection.
In the present embodiment, S1 enzyme protection analytical technology and sandwich hybridization technical tie-up are used for realizing qualitative and detection by quantitative to Skeletonemacostatum in the marine red tide biology (Skeletonema costatum.), step is as follows:
1.1 the searching in the special zone of middle Skeletonemacostatum rRNA and the design of S1 enzyme protection analysis probe are with synthetic
Carry out multisequencing relatively by Skeletonemacostatum small subunit ribosome 18S rRNA sequence in measuring and with the rRNA sequence of other algae, find its 30～100 zones different, determine that this regional sequence is a characteristic sequence with other algae at small subunit rRNA; According to characteristic sequence design with synthesize S1 enzyme protection analysis probe SKE_S1:5 '-GTCTCAAAGATTAAGCCATGCATGTGTAAGTATAAGATACTTTATCGAAACTGCGG ACGG (SEQ IDNO.1).
According to S1 enzyme protection analysis probe design with synthesize other required probes of sandwich hybridization:
Arrest probe SKE_CAPT:
5 '-Biotin-GTCTCAAAGATTAAGCCATGCATGTGT (SEQ ID NO.2), complementary with 3 ' end of S1 enzyme protection analysis probe, at 5 ' end vitamin H (Biotin) mark is arranged;
Linking probe SYM_LINK:
5 '-
AGATACTTTATCGAAACTGCGGACGGTAACAACTCACCTGCCGAATGAACTAGCCCTG (SEQID NO.3), complementary at 26 Nucleotide of 5 ' end with 5 ' end of S1 enzyme protection analysis probe, hold complementation at 32 Nucleotide of 3 ' end with 5 ' of universal signal probe.
1.2 arrest probe stationary in enzyme plate
Arrest the fixing means of probe: add the 40nM that 100 μ l are dissolved in carbonate buffer solution CBS (pH 7.2) and arrest probe in each hole of the enzyme plate that is coated with avidin in advance (Pierce company), 37 ℃ left standstill 2 hours.It is inferior standby to give a baby a bath on the third day after its birth with phosphate buffered saline buffer PBS.
1.3S1 enzyme protection analysis
After will counting with the marine alga sample of f2 culture medium culturing; centrifugal being collected in the 1.5ml plastics tubing; supernatant discarded; adding concentration is 1000 μ l lysate (0.8% sodium laurylsulfonate SDS of 50nM S1 enzyme protection analysis probe; 1.2M urea, 20% methane amide, 0.3M sodium-chlor; the 50mM Tutofusin tris, pH 7.0).
Specificity when checking of Skeletonemacostatum in carrying out, with unarmored dinoflagellate, Alexandrium tamarense, Chaetoceros, Heterosigma akashiwo, Scrippsiella trochoidea, globosa, red englena, ocean Prorocentrum, minisize Prorocentrum, spine rhombus algae etc. each about 10
6Individual cell carries out above-mentioned processing respectively.
When the detection curve of Skeletonemacostatum was analyzed in carrying out, the middle Skeletonemacostatum sample that filtration is collected obtained 7 samples for 6 times altogether with the 10 times of stepwise dilutions of lysate that contain S1 enzyme protection analysis probe.
For the mixture of marine alga sample and S1 enzyme protection analysis probe, ultrasonication two minutes is placed mixing solutions 10 minutes at 95 ℃, hybridizes 2 hours for 70 ℃ then.Add the S1 enzymolysis damping fluid (50mM zinc sulfate, 500mM sodium-acetate, 50mM sodium-chlor pH4.5) that 500 μ l contain 800US1 enzyme (Promega company) behind the naturally cooling, cut at 50 ℃ of enzymes and handled 30 minutes.
1.4DNA-RNA the sex change of heterozygote
Add 250 μ l sex change damping fluids (1.5M sodium hydroxide, 300mM EDTA), handled 10 minutes, and be used for sandwich hybridization behind the naturally cooling for 95 ℃.This moment, the sex change of DNA-RNA heterozygote formed single stranded DNA (promptly once being incorporated into the S1 enzyme protection analysis probe of target sequence) and single stranded RNA.Meanwhile, single stranded RNA is degraded, so only contains the S1 enzyme protection analysis probe that once was incorporated into target sequence in the solution.
1.5 sandwich hybridization
Arrest: the reaction mixture after the above-mentioned sex change of 100 μ l is moved into respectively pre-fixes in the enzyme plate micropore of arresting probe, 50 ℃ of hybridization 1 hour is washed 6 times with the 0.1 * SSC washings that contains 0.1%SDS.
Sandwich hybridization: every hole adds the hybridization buffer that contains 100 μ l 5nM linking probes, and (2 * SSC 0.1%SDS), in 50 ℃ of hybridization 30 minutes, washes 6 times with the 0.1 * SSC washings that contains 0.1%SDS; Every hole adds the hybridization buffer that contains 100ul 5nM signal probe, and (2 * SSC 0.5%SDS) in 50 ℃ of hybridization 30 minutes, washes 6 times with the 0.1 * SSC washings that contains 0.1%SDS.
1.6 signal detection
Enzyme plate is washed 6 times with phosphoric acid buffer (PBS); The adding of every hole has contained underlined the anti-fluorescein antibody of horseradish peroxidase (available from Roche company), hatches 30 minutes for 37 ℃, washes 6 times with PBS then; Every hole adds TMB (available from Pierce company) colour developing liquid 100 μ l, handles after 15 minutes for 37 ℃, and every hole adds 50 μ l2M sulfuric acid termination reactions.Enzyme plate is placed on the plate reading machine in 450nm mensuration absorbance value.
1.7 result
Detected result as depicted in figs. 1 and 2.
Fig. 1 has shown and has used the detected result of surveying various algae at the probe of middle Skeletonemacostatum, has proved that the inventive method has high feasibility and specificity.
Data are carried out match with conventional statistical method, obtain following relation
y＝1E-05x+0.0126；
R
2＝0.9935；
X is the cell count of middle Skeletonemacostatum in the sample, and y is the OD. of practical measurement
450, R
2Be relation conefficient.Following table has been listed with the middle Skeletonemacostatum sample size of present embodiment method mensuration and microscopic count result's comparison:
| OD. 450nm | The cell count (individual) that present method is measured | The cell count (individual) that microscopic count obtains |
| 0.602 | 58940 | 6.0E+04 |
| 0.320 | 30740 | 3.0E+04 |
| 0.064 | 5140 | 5.0E+03 |
Sequence table
＜110〉Chinese Marine University
＜120〉detection probes of Skeletonemacostatum in
<160>3
<210>1
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉complementary base sequence of Skeletonemacostatum rRNA sequence in.
<400>1
gtctcaaaga ttaagccatg catgtgtaag tataagatac tttatcgaaa ctgcggacgg 60
<210>2
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉5 ' end is marked with vitamin H, phosphate group, perhaps amino group.
<400>2
gtctcaaaga ttaagccatg catgtgt 27
<210>3
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉but 5 ' end mark digoxin, fluorescein, vitamin H or horseradish peroxidase etc.
<400>3
agatacttta tcgaaactgc ggacgg 26
Claims (4)
1. the detection probes of Skeletonemacostatum in a kind is characterized in that, is used for this algae carried out that the S1 enzyme protection is analyzed and the sandwich hybridization detection, specifically comprises three oligonucleotide probes that are mutually related: S1 enzyme protection analysis probe, arrest probe, signal probe;
Described S1 enzyme protection analysis probe, be meant complementaryly and be used for the oligonucleotide probe of S1 enzyme protection analytical procedure with middle Skeletonemacostatum rRNA, the sequence of described S1 enzyme protection analysis probe is: 5 '-GTCTCAAAGATTAAGCCATGCATGTGTAAGTATAAGATACTTTATCGAAACTGCGG ACGG;
The described probe of arresting, be meant with an end of S1 enzyme protection analysis probe and combine, an other end is marked with vitamin H, thereby will through S1 enzyme enzyme cut handle and denaturing treatment after be retained in S1 enzyme protection analysis probe in the solution and catch the probe that gets off, described sequence of arresting probe is: 5 '-GTCTCAAAGATTAAGCCATGCATGTGT;
Described signal probe can arrested after probe catches S1 enzyme protection analysis probe; be incorporated into the other end of S1 enzyme protection analysis probe; this signal probe is marked with detectable signal simultaneously; perhaps at one end has the signal probe land; can with the complementation of universal signal probe, the sequence of described signal probe is: 5 '-AGATACTTTATCGAAACTGCGGACGG.
2. the detection probes of Skeletonemacostatum in according to claim 1; it is characterized in that; described S1 enzyme protection analysis probe; when carrying out the information biology compare of analysis; only with middle Skeletonemacostatum small subunit ribosome 30～100 regional Nucleotide complementations; and do not have homology with other algae ribosome-RNA(rRNA), length is 60bp.
3. the detection probes of Skeletonemacostatum is characterized in that the described probe specificity of arresting is incorporated into S1 enzyme protection analysis probe one end in according to claim 1, and length is 27bp, is arresting probe 5 ' end all right labeled phosphorus acid groups, perhaps amino group.
4. the detection probes of Skeletonemacostatum is characterized in that in according to claim 1, is marked with fluorescein, digoxin or vitamin H on the described signal probe.
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| CN100372780C (en) * | 2006-05-26 | 2008-03-05 | 中国海洋大学 | Method for inhibiting red tide algae by ulva and its use |
| CN101130820B (en) * | 2007-09-07 | 2011-05-04 | 中国海洋大学 | Skeletonema costatum PCNA gene detecting method |
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Non-Patent Citations (6)
| Title |
|---|
| Identification of Pseudo-nitzschia australis (Bacillariophyceae)using rRNA-targeted probes in whole cell and sandwichhybridization formats. Scholin C A et al.Phycologia,Vol.35 No.3. 1996 |
| Identification of Pseudo-nitzschia australis(Bacillariophyceae)using rRNA-targeted probes in whole cell and sandwichhybridization formats. Scholin C A et al.Phycologia,Vol.35 No.3. 1996 * |
| Population genetics of Skeletonema costatum(Bacillariophyceae) in Narragansett Bay. Gallagher J C.J. Phycol.,Vol.16 . 1980 |
| Population genetics of Skeletonema costatum(Bacillariophyceae) in Narragansett Bay. Gallagher J C.J. Phycol.,Vol.16. 1980 * |
| 海藻分子生物学技术的应用与发展. 周志刚，孙育平.上海水产大学学报，第11卷第2期. 2002 |
| 赤潮生物检测的分子探针技术研究进展. 侯建军，陈纪新，黄邦钦.中国公共卫生，第20卷第9期. 2004 |
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