CN105349648B - For the primer and probe sequence of U. pertusa LAMP-LFD detections - Google Patents
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Abstract
The invention discloses the primer and probe sequences detected for U. pertusa LAMP LFD, feature is that the three pairs of primer sequences and a probe sequence of LAMP are designed according to the coded sequence for the U. pertusa internal transcribed spacer sequence that Genbank accession number is HQ902008, the nucleotide sequence of primer is as shown in SEQ ID NO.1 6, the nucleotide sequence of probe is as shown in SEQ ID NO.7, utilize above-mentioned primer and probe, pass through LAMP reaction system amplification steps, probe hybridizes LAMP reaction product steps, the detection of U. pertusa is realized using LFD detecting steps, advantage is that detection speed is fast, testing cost is low, detection sensitivity and accuracy are high.
Description
Technical field
The present invention relates to a kind of primer and probe sequences for detecting U. pertusa, and U. pertusa is used for more particularly, to one kind
The primer and probe sequence of LAMP-LFD detections.
Background technology
U. pertusa（Ulva pertusa）Belong to Chlorophyceae, Ulvales, Ulvaceae, Ulva.Ulva (Ulva) algae
In global distribution, most types are lived in temperate zone to subtropical zone ocean, and some growth is brought to low tide band and dry greatly in climax
Damp line is nearby on rock or Shi Zhaozhong.Ulva algae is rich in albumen and various trace elements, has very high edible and medicinal
Value.As important economic algae, U. pertusa（U. pertusa）, Enteromorpha（Ulva prolifera）Deng having obtained increasingly
More utilization.However, when Ulva algae is detached from adhesive substrate, when forming a large amount of floating proliferating population, green tide will be led to
Outburst, destroy Benthic ecology system.Since the coastal report for the first time of early 1970s France's Brittany, green tide occurs
Range has spread Coast of Europe, U.S.'s thing seashore, East Asia and Southeast Asia is coastal and the states such as Australian, becomes international
Marine Environmental Problems.Therefore, establish the new method of quick, accurate, sensitive detection U. pertusa, carly fruit drop for green tide and
Early warning, in real time monitoring and prevention have great importance.
For a long time, the taxonomic identification of the green tides algae such as Ulva depends on the morphological observation under microscope, usually
In the form of it, structure feature etc. is as main classification foundation.However, Ulva algae is in its form of growth period and cell knot
There is good plasticity on structure, can change with the variation of season and environment, cause morphological classification relatively difficult.Cause
This, has scholar to attempt the Protocols in Molecular Biology using PCR as representative being introduced into the detection of green tide algae, to tradition point
Class method is assisted.Wherein, rDNA-ITS sequences, rDNA -18s sequences, diphosphoribulose carboxylase large subunit gene
（rbcL）Deng because of the larger otherness of its inter-species, be typically used as the target of identification, applied to algae genealogical classification and identified
Journey.However, round pcr needs to be operated by professional in certain circumstances, is only suitable for opening in laboratory to instrument dependence height
Exhibition.
Ring mediated isothermal amplification（loop-mediated isothermal amplification, LAMP）It is 2000
A kind of new nucleic acid amplification method to grow up.Relative to only needing normal PCR method of the 1 pair of upstream and downstream primer of design to extend,
The different zones that this method need to be directed to one section of target sequence design 2-3 to primer, these primers need completely and target during the reaction
Sequences match could complete amplified reaction.Therefore, LAMP has high specific and high sensitivity.LAMP is reacted under constant temperature
It carries out, without heating and cooling repeatedly so that template unwinding, shortens detection time to a certain extent.In addition, the technology is to instrument
Equipment requirement is low, and only a thermostat water bath or metal bath can meet reaction condition.Thus, since the technological invention extensively
It is general to be applied in the Pathogen tests such as bacterium, virus, parasite.But the detection of LAMP products with agarose gel electrophoresis method or
Based on Turbidity measurement, putting it at this still can not break away from gel imaging system or transmissometer.LAMP-LFD technologies are reacted with LAMP
Based on, then the LAMP products of biotin labeling are hybridized with the specific probe of marked by fluorescein isothiocyanate, most
It completes to detect and carry out result judgement in lateral flow test strips eventually.Compared to LAMP product detection methods before, LFD is just
The property taken is preferable, and the false positive that can be avoided non-specific amplification and occur.At present, which successfully applies to infectious spleen
Kidney necrosis virus（Infectious spleen and kidney necrosis virus, ISKNV）, infectivity muscle necrosis
Virus（Infectious myonecrosis virus, IMNV）, Vibrio vulnificus（Vibrio vulnificus）And dolphin
Streptococcus（Strepstococcus iniae）The detection of aquatic products pathogenic microorganism is waited, having not been reported both at home and abroad should by the technology
For the diagnosis and detection of U. pertusa.
Invention content
The technical problems to be solved by the invention are to provide that a kind of detection speed is fast, and testing cost is low, detection sensitivity and
The high primer and probe sequence for U. pertusa LAMP-LFD detections of accuracy.
Technical solution is used by the present invention solves above-mentioned technical problem：It is a kind of to be used for what U. pertusa LAMP-LFD was detected
Primer and probe sequence is set according to the coded sequence for the U. pertusa internal transcribed spacer sequence that Genbank accession number is HQ902008
Three pairs of primer sequences and a probe sequence, the primer sequence for counting LAMP are specific as follows：
UpeITS-F3：5’- TAGCCTCACCTGAACTCAG-3’
UpeITS-B3：5 '-ACGGATATCTCGGCTCTC-3 ',
UpeITS-FIP：5 '-CGGCTGAAATACAGAGGCTCGTATCCTTCGTGGCTCACA-3 ',
UpeITS-BIP：5 '-GTTATTCCGACGCTGAGGCAGCAGAATTCCGTGAGTCAT-3 ',
UpeITS-LF：5 '-TAGGTAGCTCGCTACTCCTAC-3 ',
UpeITS-LB：5 ' the ends of 5 '-GTGGTCTCATCCGAAGACTC-3 ', UpeITS-FIP are biotin labeling；
Probe sequence is as follows：
UpeITS-HP：5 '-GCAAGCGCGTGAGGGGTTAT-3 ', 5 ' ends are marked by fluorescein isothiocyanate.
The LAMP reaction systems of primer work：The final concentration of each ingredient of reaction system is respectively：Outer primer UpeITS-F3 and
UpeITS-B3 each 0.2 μm of ol/L, inner primer UpeITS-FIP and UpeITS-BIP each 1.6 μm of ol/L, ring primer UpeITS-
LF and UpeITS-LB each 0.4 μm of ol/L, the Tris-HCl 20mmol/L, KCl of dNTPs 1.4mmol/L, pH 8.8
10mmol/L, MgSO46.5mmol/L, (NH4)2SO410mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerases
Large fragment and 2 μ L sample templates add distilled water that reaction system total volume is made to be 25 μ l.
LAMP reaction conditions：Above-mentioned reaction system is subjected to amplified reaction, amplified reaction temperature is 63 DEG C, amplified reaction
Time is 50 min.
Compared with prior art, the advantage of the invention is that
1st, detection sensitivity is high, and the detection sensitivity to U. pertusa genome is 3.04 × 10-2 Pg/ μ L, are Standard PCRs
1000 times of detection.
2nd, detection time is short, and amplified reaction only needs 50 min, judges since nucleic acid amplification to completion result, whole flow process
60 min are only needed, shorten about 1.5 h than Standard PCR detection technique.
3rd, high specificity, eight different zones of specific primer used in U. pertusa the Internal Transcribed Spacer are set
The methods of counting, and the also specific probe of DNA, can effectively avoid using agarose gel electrophoresis, fluorescent dye is caused
False positive issue.
4th, instrument and equipment requirement is low, without PCR instrument, gel electrophoresis and imaging system used in Standard PCR etc., only needs one
Detection can be completed in water-bath.
5th, easy to operate, as a result significantly, entire detection process is not related to complex instrument and equipment, slightly has a molecular biology base
Operation can be completed in the personnel of plinth；Testing result is clearly apparent, visually observes and can determine whether.
6th, it is safer to the person and environment, it is not related to the toxic reagents such as EB in detection process.
In conclusion being detected using the primer and probe of the present invention using LAMP-LFD methods to U. pertusa, have
Higher agility, specificity and sensitivity, instrument demand is simple, is conducive to the early diagnosis and detection of U. pertusa, can meet
The needs of testing agency of base.
Description of the drawings
Fig. 1 is LAMP specificity experiments results.M：100 bp Plus DNA ladder (Fermentas, the U.S.)；
Upe：Using the genomic DNA of U. pertusa S66 as template；Ufl、Uli、Uoh、Upr、Ata、Gin、Hak、Pdo、Str：Respectively with song
Enteromorpha SDF12, marginal tuber HS42,UlvaohnoiFJ4, Enteromorpha XS5, Alexandrium tamarense NMBjah048, without line circular groove
Algae NMBjah046, Heterosigma akashiwo H1, Prorocentrum donghaiense NMBjah045, taper Si Kelipu algaes NMBjah044 genome
DNA is template；NC：Using aseptic deionized water as template；
Fig. 2 is LAMP-LFD specificity experiments results.Upe：Using the genomic DNA of U. pertusa S66 as template；Ufl、Uli、
Uoh、Upr、Ata、Gin、Hak、Pdo、Str：Respectively with bent Enteromorpha SDF12, marginal tuber HS42,UlvaohnoiFJ4, Enteromorpha
XS5, Alexandrium tamarense NMBjah048, without line circular groove algae NMBjah046, Heterosigma akashiwo H1, Prorocentrum donghaiense
NMBjah045, taper Si Kelipu algaes NMBjah044 genomic DNA be template；NC：Using aseptic deionized water as template；
Fig. 3 is the sensitivity results of LAMP detections.M：100 bp Plus DNA ladder (Fermentas, the U.S.)；
3.04×102、3.04×101、3.04×100、3.04×10-1、3.04×10-2、3.04×10-3：With U. pertusa various concentration
Genomic DNA be template（Unit：pg/µL）；NC：Using aseptic deionized water as template；
Fig. 4 is the sensitivity results of LAMP-LFD detections.3.04×102、3.04×101、3.04×100、3.04×10-1、
3.04×10-2、3.04×10-3：Using the genomic DNA of U. pertusa various concentration as template（Unit：pg/µL）；NC：With sterile
Deionized water is as template；
Fig. 5 is the sensitivity results of PCR detections.M：100 bp Plus DNA ladder (Fermentas, the U.S.)；
3.04×102、3.04×101、3.04×100、3.04×10-1、3.04×10-2、3.04×10-3：With U. pertusa various concentration
Genomic DNA be template（Unit：pg/µL）；NC：Using aseptic deionized water as template.
Specific embodiment
The present invention is described in further detail below in conjunction with attached drawing embodiment.
Embodiment 1
The foundation of the method for LAMP-LFD technologies detection U. pertusa
1. design of primers：According to the U. pertusa the Internal Transcribed Spacer delivered in NCBI（GenBank accession number：
HQ902008）Coded sequence be designed, wherein, primer sequence is specific as follows：
UpeITS-F3：5’- TAGCCTCACCTGAACTCAG-3’
UpeITS-B3：5 '-ACGGATATCTCGGCTCTC-3 ',
UpeITS-FIP：5 '-CGGCTGAAATACAGAGGCTCGTATCCTTCGTGGCTCACA-3 ',
UpeITS-BIP：5 '-GTTATTCCGACGCTGAGGCAGCAGAATTCCGTGAGTCAT-3 ',
UpeITS-LF：5 '-TAGGTAGCTCGCTACTCCTAC-3 ',
UpeITS-LB：5 ' the ends of 5 '-GTGGTCTCATCCGAAGACTC-3 ', UpeITS-FIP are biotin labeling；
Probe UpeITS-HP：5 '-GCAAGCGCGTGAGGGGTTAT-3 ', 5 ' ends are marked by fluorescein isothiocyanate.
2. sample DNA extracts：According to tissue gene group DNA extraction kit（Centrifuge column type）The step of D3396-0, carries
Take U. pertusa S66 separation strain gene group DNA (Qiagen, Hilden, Germany).The genomic DNA of acquisition is dissolved in 50
The ddH of μ L2O, through 2000 spectrophotometers of Nanodrop（Thermo Fisher Scientific, USA）After measured concentration
As standard items, -30 DEG C of storages are spare.Measure a concentration of 3.04 × 105Pg/ μ L, through ddH2After O gradient dilutions, 3.04 are chosen
×102、3.04×101、3.04×100、3.04×10-1、3.04×10-2、3.04×10-36 concentration gradients such as pg/ μ L
Genomic DNA is verified for LAMP and PCR.
3. U. pertusa LAMP reacts
The specific primer designed using step 1 carries out LAMP amplifications by template of U. pertusa DNA.
3.1 LAMP reaction systems, the final concentration of each ingredient are respectively：Outer primer UpeITS-F3 and UpeITS-B3 each 0.2
μm ol/L, inner primer UpeITS-FIP and UpeITS-BIP each 1.6 μm of ol/L, ring primer UpeITS-LF and UpeITS-LB are each
0.4 μm of ol/L, dNTPs 1.4 mmol/L, Tris-HCl（pH 8.8）20 mmol/L, KCl 10 mmol/L, MgSO4 6.5
Mmol/L, (NH4)2SO410 mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerase large fragments (New
England Biolabs) and 2 μ L sample templates, add distilled water that reaction system total volume is made to be 25 μ L；
3.2 LAMP reaction conditions：Above-mentioned reaction system is subjected to amplified reaction, amplified reaction temperature is 63 DEG C, and amplification is anti-
It is 50 min between seasonable.
4. probe hybridizes and LFD detections：The UpeITS-HP probes of 20 pmol are added in reaction system after amplification, 63
DEG C incubate 5 min, hybridized, take 5 μ L hybridization solutions add in 100 μ L buffer solutions in mixing, then LFD test strips are immersed
It adds in the buffer solution of hybridization solution and develops the color, judge the amplification situation of LAMP.
Embodiment 2
The specific assay of U. pertusa LAMP-LFD detections is carried out using the primer and probe of the present invention
Using designed specific primer and probe, respectively to select U. pertusa S66, bent Enteromorpha SDF12, marginal tuber
HS42、UlvaohnoiThe common Ulva green alga such as FJ4, Enteromorpha XS5 and Alexandrium tamarense NMBjah048, without line ring
Ditch algae NMBjah046, Heterosigma akashiwo H1, Prorocentrum donghaiense NMBjah045 and taper Si Kelipu algaes NMBjah044 etc. are domestic
The genomic DNA of common microalgae type is template, carries out LAMP-LFD detections by the step 3 and step 4 of above-described embodiment 1, tests
The specificity of primer and probe is demonstrate,proved, distilled water is as negative control.As a result as depicted in figs. 1 and 2, electrophoresis is utilized（Fig. 1）With
LFD（Fig. 2）It can only all be expanded from the genome DNA sample of U. pertusa and obtain purpose band, other samples are said without amplified band
It is bright to carry out LAMP-LFD detections using primer and probe provided by the invention, there is good specificity.
Embodiment 3
The sensitivity determination of U. pertusa LAMP-LFD detections is carried out using the primer and probe of the present invention
Using the genomic DNA of the method extraction U. pertusa of the step 2 of above-described embodiment 1,10 times of gradient dilutions, choosing are carried out
Select 3.04 × 102、3.04×101、3.04×100、3.04×10-1、3.04×10-2、3.04×10-3Pg/ μ L etc. are used as mould
Plate carries out LAMP-LFD detections by the step 3 and step 4 of above-described embodiment 1, verifies the sensitivity of primer and probe, sterile to go
Ionized water is as negative control.As a result it as shown in Fig. 3, Fig. 4 and Fig. 5, is carried out using primer and probe provided by the invention
The sensitivity of LAMP-LFD detections is 3.04 × 10-2pg/μL（Fig. 4）, agarose gel electrophoresis is utilized with the amplified production of LAMP
It is consistent to detect the sensitivity obtained（Fig. 3）, it is the Standard PCR detection method that UpeITS-F3 and UpeITS-B3 are established as primer
1000 times（Fig. 5）.
Embodiment 4
Algae sample in seawater is detected with LAMP-LFD technologies of the present invention.
1. algal gene group DNA is extracted
17 parts of seawater sample is collected altogether, respectively after the bolting silk net filtration of 100 mesh, respectively takes 400 mL water samples in 500 mL
Conical flask in, add in f/2 nutritive salt, go to that temperature is 20 DEG C, intensity of illumination is 8 000 Lux, the photoperiod is 12 h
Several days are cultivated under the conditions of the h of (daytime)/12 (night).Ripe frond is taken to be used for microexamination.Separately took water after filter
250 mL of sample is directly used in extracting genome DNA.Seawater sample extracts genomic DNA by 1 step 2 the method for embodiment.Respectively
2 μ L are taken for LAMP-LFD and PCR amplification.
2. LAMP reaction systems are prepared and reaction condition, carried out according to 1 step 3 of embodiment.
3. LFD develops the color, testing result judges, is carried out according to 1 step 4 of embodiment.
11 seawater samples of acquisition are identified using the method for traditional algae separation, culture, microexamination,
It was found that ZQ-5, TD-4, S73, S77 and S78 from Qingdao are U. pertusa（Table 1）.LAMP-LFD's the result shows that Qingdao
ZQ-4, ZQ-5, TD-2, TD-4, S73, S77 and S78 are U. pertusa（Table 1）.Qingdao is equally obtained using the method for PCR
ZQ-4, ZQ-5, TD-2, TD-4, S73, S77 and S78 are U. pertusa（Table 1）.
Note："+" represents that testing result is positive；"-" represents that testing result is negative.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Technical staff is in the essential scope of the present invention, the variations, modifications, additions or substitutions made, should also belong to the protection of the present invention
Range.
Claims (3)
1. a kind of primer and probe sequence for U. pertusa LAMP-LFD detections, it is characterised in that be according to Genbank accession number
The three pairs of primer sequences and a probe sequence of the coded sequence design LAMP of the U. pertusa internal transcribed spacer sequence of HQ902008
Row, primer sequence are specific as follows：
UpeITS-F3：5’- TAGCCTCACCTGAACTCAG-3’
UpeITS-B3：5 '-ACGGATATCTCGGCTCTC-3 ',
UpeITS-FIP：5 '-CGGCTGAAATACAGAGGCTCGTATCCTTCGTGGCTCACA-3 ',
UpeITS-BIP：5 '-GTTATTCCGACGCTGAGGCAGCAGAATTCCGTGAGTCAT-3 ',
UpeITS-LF：5 '-TAGGTAGCTCGCTACTCCTAC-3 ',
UpeITS-LB：5 ' the ends of 5 '-GTGGTCTCATCCGAAGACTC-3 ', UpeITS-FIP are biotin labeling；
Probe sequence is as follows：
UpeITS-HP：5 '-GCAAGCGCGTGAGGGGTTAT-3 ', 5 ' ends are marked by fluorescein isothiocyanate.
2. the primer and probe sequence according to claim 1 for U. pertusa LAMP-LFD detections, it is characterised in that draw
The LAMP reaction systems of object work：The final concentration of each ingredient of reaction system is respectively：Outer primer UpeITS-F3 and UpeITS-B3
Each 0.2 μm of ol/L, inner primer UpeITS-FIP and UpeITS-BIP each 1.6 μm of ol/L, ring primer UpeITS-LF and
Tris-HCl 20mmol/L, the KCl 10mmol/L of UpeITS-LB each 0.4 μm of ol/L, dNTPs 1.4mmol/L, pH 8.8,
MgSO46.5mmol/L, (NH4)2SO410mmol/L, Triton X-100 0.1%, 8U Bst archaeal dna polymerases large fragment and 2
μ L sample templates add distilled water that reaction system total volume is made to be 25 μ l.
3. the primer and probe sequence according to claim 1 or 2 for U. pertusa LAMP-LFD detections, it is characterised in that
LAMP reaction conditions：Above-mentioned reaction system is subjected to amplified reaction, amplified reaction temperature is 63 DEG C, and the amplified reaction time is 50
min。
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| molecular biotechnology of marine algae in China;Song Qin等;《hydrobiologia》;20040131;21-26 * |
| 环介导等温扩增联合横向流动试纸条快速检测扁浒苔（Ulva compressa）的研究;陈先锋等;《海洋与湖沼》;20150731;摘要、第819页左栏第1段、第819-820页跨页段、第820页表1、第821页1.3-1.5、表2、第822页1.7、第824页左栏第1-2段、第825页跨栏段 * |
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