CN103866020A - Primer, kit and method for detecting Ichthyophthirius multifiiis - Google Patents

Primer, kit and method for detecting Ichthyophthirius multifiiis Download PDF

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CN103866020A
CN103866020A CN201410090981.3A CN201410090981A CN103866020A CN 103866020 A CN103866020 A CN 103866020A CN 201410090981 A CN201410090981 A CN 201410090981A CN 103866020 A CN103866020 A CN 103866020A
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detection
primer
melon seeds
result
little melon
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CN103866020B (en
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郭书林
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INSPECTION AND QUARANTINE CENTER OF XIAMEN ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU
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    • C12Q1/6844—Nucleic acid amplification reactions

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Abstract

The invention proposes a primer, a kit and a method for detecting Ichthyophthirius multifiiis. The primer sequence is formed by four sequences in SEQ ID NO:1-4. The invention also protects the kit and the method for detecting the Ichthyophthirius multifiiis. LAMP rapid detection is carried out by using the primer, so that the primer has high sensitivity, strong specificity and short detection time, the detection result can be obtained within about an hour, the detection time is shortened by 2-4 hours in comparison with that of common PCR detection, the operation is simple, and the result is obvious. The overall detection process does not relate to complicated apparatus and equipment, detection can be finished by general technical personnel, the result is easily distinguished, the result can be observed by naked eyes through different colors, fussy electrophoresis and ultraviolet observation are not required, and the primer and the kit are safe.

Description

A kind of primer, test kit and method thereof that detects how sub little melon seeds
Technical field
The present invention relates to biological technical field.Relate to more specifically a kind of primer, test kit and method thereof that detects how sub little melon seeds.
Background technology
The ichthyophthirius multifiliis that parasitizes freshwater fish belongs to Protozoa (Portozea), Ciliata (Ciliata), and Hymenostomatida (Holotricha), recess section (Ophryoglenidae), Ichthyophthirius ( ichthyophthirius).At present only ichthyophthirius multifiliis one ( i.multifiiis).Little melonworm is distributed in the torrid zone, subtropics and Temperate Region in China, can extend to the north polar circle northwards.
PCR detection technique is the special gene fragment according to organism, organism is detected to the technology of identifying.The method is widespread use in the mankind and vegeto-animal cause of disease detect, has high specificity, highly sensitive, plurality of advantages conveniently.Sun etc. (2006) have reported that application special primer is to amplification ichthyophthirius multifiliis part ITS-1 sequence, complete 5.8S rDNA sequence, and the PCR method of part ITS-2 sequence.The forward primer sequence that the method is used is 5 '-GTA CTT TAT TTA GGA GGA GGA CT-3 ', reverse primer sequence is 5 '-TGT TTA ACG AGA GAA AAT CAT AAA T-3 ', the target fragment of energy specific amplification 326 bp left and right, in conjunction with product order-checking, can identify accurately ichthyophthirius multifiliis.
Real time fluorescent PCR method is because having reduced pollution section, and PCR detects and result judges that a step completes, sensitiveer and quick than regular-PCR, is also applicable to high throughput testing, has therefore obtained widespread use at cause of disease detection field.Olivier(2005) reported that SYBR Green fluorescent PCR method detects the detection technique that is free in the ichthyophthirius multifiliis in water body.The method is with primers IMRf1 5 '-AGTGACAAGAAATAGCAAGCCAGGAG-3 ' and the IMRr1 5 '-ACCCAGCTAAATAGGCAGAAGTTCAA-3 ' between 455-647 base in ichthyophthirius multifiliis 18 S rDNA genes, not only can carry out quantitative analysis to the infection of ichthyophthirius multifiliis, can also be issued in the situation of avoiding damaging fish body the object of detection.
Overcome the defect on traditional form is identified take PCR as basic molecular assay method, but in application process, it is found that round pcr comes with some shortcomings, as easily contaminated in target gene, may cause non-specific amplification, many factors can produce suppression of amplification reaction, and the amplified reaction time is longer, generally needs several hours, need more expensive PCR instrument, be unfavorable for applying at laboratories etc.2000, Japanese scholars Notomi etc. (Loop-mediated isothermal amplification of DNA [J]. Nucleic Acids Research, 2000,28:E63) set up a kind of new nucleic acid amplification method---ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) technology, LAMP technology is to adopt 4 Auele Specific Primers setting for the special positions of 6 of target gene (inner primer: FIP is made up of F1C and F2; BIP is made up of BIC and B2, outer primer: F3, B3) and there is the Bst archaeal dna polymerase of strand displacement activity, under 60-65 ℃ of constant temperature, nucleic acid being increased, short period of time amplification efficiency can reach 10 9-10 10individual copy.This technology has overcome some shortcomings of round pcr.In the detection of the mankind and animals and plants bacterium, virus, parasite, fungi etc., make important progress at present, demonstrate wide application prospect in disease diagnosis field.
The current detection diagnostic method of ichthyophthirius multifiliis has morphology detection method, regular-PCR detection method, and three kinds of fluorescence PCR detecting methods.Above-mentioned these three kinds detection diagnostic methods need respectively to use the more accurate plant and instrument such as opticmicroscope, regular-PCR instrument, fluorescent PCR instrument, computer.These plant and instrument are not only worth higher, and it deposits and use environmental requirement highlyer, are unwell in culture environment of aquatic products and settle and application.Meanwhile, regular-PCR detection method and fluorescence PCR detecting method, testing process is more complicated, does not reach the object of rapid detection diagnosis.At present, at home and abroad except exploitation has the regular-PCR and Fluorescence PCR assay method that is suitable for laboratory environment detection diagnosis ichthyophthirius multifiliis, not yet develop more fast and conveniently, the ichthyophthirius multifiliis being suitable in the extensive environment such as aquaculture detects diagnostic techniques.
Summary of the invention
The object of the present invention is to provide one easier, and result detect the method for how sub little melon seeds accurately.
For achieving the above object, the invention provides a kind of primer for detection of how sub little melon seeds, it is characterized in that, described primer sequence is to be made up of 4 sequences of SEQ ID NO:1-4.
For detection of a test kit for how sub little melon seeds, it is characterized in that, comprise described primer.
For detection of a method for how sub little melon seeds, it is characterized in that, described method has been applied described primer or described test kit.
Described method, comprises the steps,
Extract DNA profiling;
LAMP pcr amplification: the primer of application rights requirement 1 or the test kit of claim 2 increase;
Result judgement.
Described extraction DNA profiling can extract from the things such as fin, the fish gill, fish body surface scraping thing, raising water body centrifugal collecting precipitation.
Described LAMP pcr amplification system is, the each 0. 8 μ mol/L of sequence of SEQ ID NO:1-2, the each 0.2 μ mol/L of sequence of SEQ ID NO:3-4, I U DNA template, 1.2 mmol/L dNTP, 1 mol/L trimethyl-glycine, 8U Bsm DNA polysaccharase, 1 × Bsm buffer, adds the completion of sterilizing bi-distilled water to 25 μ l.
Described LAMP pcr amplification program is that 95 ℃ are incubated 5 min, 60 ℃ of insulation 1 h, 80 ℃ of insulation 5 min.
Described result is judged as and adds developer, and light rolling mixes, and reaction solution is lurid positive result, and reaction solution is orange-yellow negative result.
Described developer is SYBR green I.
Although LAMP technology is a disclosed technological method, but its concrete effectiveness must be passed through researchist, through the creative thinking of self, scientific attainments, research and development, the processes such as repetition test, innovation is set up out in research, have the achievement in research that scientific research is worth, just can make LAMP technology concrete, specific field plays a role.Even if otherwise people are familiar with LAMP technology, but still cannot reach the object that detects identification of organism body, cannot solving practical problems.
Innovative point of the present invention, not in LAMP technology itself, and is that research and development person, by which kind of means, is the detection diagnostic method that can be used for detecting ichthyophthirius multifiliis by LAMP technical change, to fill up the blank of ichthyophthirius multifiliis Site Detection diagnostic field.At present, investigator both domestic and external, all detects diagnostic means as target fast and accurately to set up.Research and development that detect fast and accurately diagnostic means and foundation need investigator's continuous exploration and effort, also can create huge social value and economic worth.So the present invention is take LAMP technology as ultimate principle, exploitation creates that other investigators fail to realize on this basis, many new primers, thus realize LAMP method to the qualitative leap that detects diagnosis ichthyophthirius multifiliis LAMP technology.If do not had, contriver is creationary develops special primer, and LAMP technology can only be a concept, can not bring into play any value.
Ichthyophthirius multifiliis LAMP method for quick provided by the present invention has the following advantages:
One, highly sensitive, can reach 1 larva level to the limit of detection of ichthyophthirius multifiliis.
Two, high specificity, special primer used is 4 primers designing according to six different zones of 18S rDNA of ichthyophthirius multifiliis, has strengthened the specificity of reaction.
Three, detection time short, about 1 hour, can obtain detected result, than regular-PCR detect shorten 24h.
Four, do not need PCR instrument, low to plant and instrument requirement, without any need for PCR instrument, gel electrophoresis and imaging system, only need a water-bath or vacuum flask just can complete detection.
Five, simple to operate, result is obvious, whole testing process does not relate to complex instrument and equipment, those skilled in the art can complete detection, result easily distinguishes, by different colours, naked eyes get final product observations, do not need loaded down with trivial details electrophoresis and ultraviolet visualization.
Six, testing process does not need to use the toxic reagents such as EB, very safe to human and environment.
In sum, the present invention has than the method for existing round pcr detection ichthyophthirius multifiliis and has higher specificity, sensitivity and portability.Can in actual production, rig-site utilization detect.
Can find out from Fig. 1 of embodiment gained, result is clear and legible, simple and clear.
Accompanying drawing explanation
Fig. 1 adds detection of fluorescent dyes result figure.
Embodiment
Describe embodiments of the invention below in detail, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has the element of identical or similar functions from start to finish.Be exemplary below by the embodiment being described with reference to the drawings, be intended to for explaining the present invention, and can not be interpreted as limitation of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
embodiment 1:the detection of many sub little melon seeds
1) DNA extraction: get fish body surface scraping thing to be measured as sample, extract by DNA kit method extracting method or classical DNA phenol chloroform extraction method the DNA profiling that DNA detects as LAMP.The DNA of the fish body surface scraping thing of known infection ichthyophthirius multifiliis is as positive control, and the DNA of the fish body surface scraping thing of known health is as negative control.
2) design of primers
Ichthyophthirius multifiliis LAMP design of primers
According to ichthyophthirius multifiliis 18S rDNA(sequence number Sequence ID:gb|DQ270016.1|) sequencing result is template, following 4 primers of design, sequence is as follows:
3 × GCF3:5 '-GAT TAT GTC CCT GCC GTT-3 ' sequence SEQ ID NO:1
3 × GCB3:5 '-TCC TCC TCC TAA ATA AAG TAC A-3 ' sequence SEQ ID NO:2
3×GCFIB: 5’-ACT TCC CAA AGC CTT GCG ACC CGT CGC TTG TAG TAA CG-3’
Sequence SEQ ID NO:3
3 × GCBIP:5 '-AAG TAA ACC CTA CCA TTT GGA ACA TTG TGT TAA TGA TCC ATC TGC-3 ' sequence SEQ ID NO:4 (primer is synthetic: Xiamen Jing Ju Science and Technology Ltd.)
3) LAMP detects
LAMP reaction system configuration: the each 0. 8 μ mol/L of primer 3 × GCF3 and 3 × GCB3, the each 0.2 μ mol/L of primer 3 × GCFIB and 3 × GCBIP, 1.2 mmol/L dNTP, 1 mol/L trimethyl-glycine, 8U Bsm DNA polysaccharase, Thermo company of 1 × Bsm buffer(U.S.).
LAMP reacts amplification condition: by primer, dNTP, trimethyl-glycine, Bsm buffer, Bsm DNA polysaccharase, and after 1 U DNA profiling (being step 1) gained) is mixed in proportion, 95 ℃ of insulation 5 min, 60 ℃ of insulation 1 h, 80 ℃ of insulation 5 min.
LAMP result detects:
The developer SYBR green I of 1 μ l will be added in system complete above-mentioned reaction.Light rolling mixes, i.e. observable, the results are shown in Figure 1.Wherein 1 positive result, it is light yellow that reaction solution is, with coming to the same thing of positive control.2 negative results (negative control), it is orange-yellow that reaction solution is.
Although illustrated and described embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention in the situation that not departing from principle of the present invention and aim, modification, replacement and modification.
SEQUENCE LISTING
<110> Inspection and Quarantine Center of Xiamen Entry-Exit Inspection and Quarantine Bureau
Primer, test kit and the method thereof of the <120> how sub little melon seeds of detection
<130> 346
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> synthetic
<400> 1
gattatgtcc ctgccgtt
18
<210> 2
<211> 22
<212> DNA
<213> synthetic
<400> 2
tcctcctcct aaataaagta ca
22
<210> 3
<211> 38
<212> DNA
<213> synthetic
<400> 3
acttcccaaa gccttgcgac ccgtcgcttg tagtaacg
38
<210> 4
<211> 45
<212> DNA
<213> synthetic
<400> 4
aagtaaaccc taccatttgg aacattgtgt taatgatcca tctgc
45

Claims (9)

1. for detection of a primer for how sub little melon seeds, it is characterized in that, described primer sequence is to be made up of 4 sequences of SEQ ID NO:1-4.
2. for detection of a test kit for how sub little melon seeds, it is characterized in that, comprise primer claimed in claim 1.
3. for detection of a method for how sub little melon seeds, it is characterized in that, described method has been applied the primer of claim 1 or the test kit of claim 2.
4. the method for the how sub little melon seeds of detection of claim 3, is characterized in that, comprise the steps,
Extract DNA profiling;
LAMP pcr amplification: the primer of application rights requirement 1 or the test kit of claim 2 increase;
Result judgement.
5. the method for the how sub little melon seeds of detection of claim 4, is characterized in that, described extraction DNA profiling can extract from the things such as fin, the fish gill, fish body surface scraping thing, raising water body centrifugal collecting precipitation.
6. the method for the how sub little melon seeds of detection of claim 4, it is characterized in that, described LAMP pcr amplification system is, the each 0. 8 μ mol/L of sequence of SEQ ID NO:1-2, the each 0.2 μ mol/L of sequence of SEQ ID NO:3-4, I U DNA template, 1.2 mmol/L dNTP, 1 mol/L trimethyl-glycine, 8U Bsm DNA polysaccharase, 1 × Bsm buffer, adds the completion of sterilizing bi-distilled water to 25 μ l.
7. the method for the how sub little melon seeds of detection of claim 4, is characterized in that, described LAMP pcr amplification program is that 95 ℃ are incubated 5 min, 60 ℃ of insulation 1 h, 80 ℃ of insulation 5 min.
8. the method for the how sub little melon seeds of detection of claim 4, is characterized in that, described result is judged as and adds developer, and light rolling mixes, and reaction solution is lurid positive result, and reaction solution is orange-yellow negative result.
9. the method for the how sub little melon seeds of detection of claim 8, is characterized in that, described developer is SYBR green I.
CN201410090981.3A 2014-03-13 2014-03-13 Primer, kit and method for detecting Ichthyophthirius multifiiis Expired - Fee Related CN103866020B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105524996A (en) * 2016-01-15 2016-04-27 中国长江三峡集团公司中华鲟研究所 Method for rapid detection of ichthyophthirius multifiliis in water body
CN115807112A (en) * 2022-11-30 2023-03-17 中国科学院水生生物研究所 Primer group for identifying and quantitatively detecting Polychachis magna and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2375276A1 (en) * 2001-03-09 2002-09-09 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Methods to isolate gene coding and flanking dna
CN102912013A (en) * 2012-09-19 2013-02-06 长沙三济生物科技有限公司 Sequencing primer for qualitative detection of cytochrome oxidase CYP2C19 genetic typing and kit of sequencing primer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2375276A1 (en) * 2001-03-09 2002-09-09 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Methods to isolate gene coding and flanking dna
CN102912013A (en) * 2012-09-19 2013-02-06 长沙三济生物科技有限公司 Sequencing primer for qualitative detection of cytochrome oxidase CYP2C19 genetic typing and kit of sequencing primer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
王国良等: "刺激隐核虫LAMP检测方法的建立", 《中国预防兽医学报》, vol. 35, no. 7, 31 July 2013 (2013-07-31) *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105524996A (en) * 2016-01-15 2016-04-27 中国长江三峡集团公司中华鲟研究所 Method for rapid detection of ichthyophthirius multifiliis in water body
CN105524996B (en) * 2016-01-15 2019-05-10 中国长江三峡集团公司中华鲟研究所 A kind of method of small melonworm in quick detection water body
CN115807112A (en) * 2022-11-30 2023-03-17 中国科学院水生生物研究所 Primer group for identifying and quantitatively detecting Polychachis magna and application thereof

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