CN105154576A - Primer pair for rapidly detecting silkworm microsporidia and reagent kit and detection method thereof - Google Patents
Primer pair for rapidly detecting silkworm microsporidia and reagent kit and detection method thereof Download PDFInfo
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Abstract
The invention relates to a primer pair for rapidly detecting silkworm microsporidia and a reagent kit and a detection method thereof. The nucleotide sequence of the primer pair is shown as SEQ ID NO.7 and SEQ ID NO.8. By the utilization of the primer pair, different nucleic acid molecular markers are added into the upstream primer and the downstream primer respectively, PCR products of the marked primers can be directly determined through immuno-gold test strips, the operation steps are simplified, convenience is achieved, specificity is good, and sensitivity is high. Compared with a traditional gel electrophoresis method, the sensitiveness is increased by 10 times, and the primer pair can serve as an ideal detection means for silkworm microsporidia in silkworm finished product eggs and has good application prospects.
Description
Technical field
The invention belongs to molecular biology and field of immunology, the primer pair of rapid detection nosema bombycis, also relate to the primer pair test kit of rapid detection nosema bombycis and utilize this test kit to detect the detection method of nosema bombycis.
Background technology
For effectively controlling because of the pebrine disease that nosema bombycis causes in sericultural production, in order to avoid it causes silkworm mortality, need that exploitation is sensitive, convenient, nosema bombycis detection method accurately.Nosema bombycis (Nosemabombycis), also Bm cell line is claimed, main parasitic is in economic insects Silkworm, Bombyx mori, the pebrine disease caused is destructive disease in sericulture there, its cause of disease nosema bombycis is classified as the legal Quarantine Objects on sericultural production by each sericulture countries and regions, the world, and successfully detects nosema bombycis period at Product eggs and have vital role for the nosema bombycis prevention and control in sericultural production.
In traditional sericultural production, the propagation of nosema bombycis is controlled in order to produce nontoxic silkworm seed, what silkworm egg sanitary authority often adopted is female moth Microscopical Method For Detection, it is a kind of indirect judgement method, its main process is: after grinding female moth, the detection of nosema bombycis is carried out by the method for opticmicroscope microscopy, the ovum that poisonous female moth produces is considered as poisonous ovum, thus whether containing nosema bombycis in indirect judgement Product eggs, whole checkout procedure labor intensive, and the skill requirement of final detection result to blood slide examiners is higher, subjectivity is strong; On the other hand, female moth of seed station censorship may not represent the truth of Product eggs product by the gross.Based on above reason, a kind ofly can the detection method of nosema bombycis in direct-detection Product eggs to be badly in need of setting up.Detected object based on nucleic acid amplification method is the DNA from nosema bombycis, after specific amplification, carry out Product Identification, and these class methods are highly sensitive, and specificity is good.
Although nucleic acid amplification detection method has highly sensitive, specificity and reliability is strong, the usual device dependence of these class methods is comparatively strong, and amplification need could obtain final detection result with agarose gel electrophoresis after carrying out IMAQ, consuming time longer.
Another kind of detection method is based on the immunological detection method of double antibodies sandwich, its detected object is pathogenic proteins matter mainly, the specificity of detected result is ensured by the antibody of specificity for this protein, it is a kind of ordinary method of rapid detection, this method can combine with colloidal gold strip detection technique, has advantage with low cost, simple to operate.In immunity colloidal gold test paper strip detects, often utilize the feature of Ag-Ab specific binding, build the complex body of Ag-Ab-colloid gold particle, when on the detection line that this complex body concentrates on test strip, colloid gold particle can form red precipitate line, but the sensitivity of this kind of method depends on the antigenic content in the acquisition of high-affinity and antibody with high specificity and sample, microsporidium quantity in bombyx mori egg is few, the antigen amount of its correspondence is also seldom corresponding, also limits the application of the method in quarantine of commercial eggs.
In sum, if special marker can be added in nucleic acid amplification product, the product of pcr amplification just can be made also can to detect with immunity colloidal gold test paper strip, can reduce and instrument is relied on, and accelerate detection time.Therefore in the PCR process of this detection method, employ the primer that a pair marked two kinds of compounds simultaneously, this test strip detects in the mode of dual anti-(or part) sandwich assay.
Summary of the invention
In view of this, an object of the present invention is the primer pair providing a kind of rapid detection nosema bombycis, and specificity is good; Two of object of the present invention is the primer pair test kit providing rapid detection nosema bombycis, specificity is good, highly sensitive, three of object of the present invention is to provide the primer pair test kit utilizing rapid detection nosema bombycis to detect the detection method of nosema bombycis in bombyx mori egg, simple operating steps.
For achieving the above object, the invention provides following technical scheme:
1, the primer pair of rapid detection nosema bombycis, the nucleotide sequence of described primer pair is as shown in SEQIDNO.7 and SEQIDNO.8.This primer pair selected zone is suitable, avoids the formation of primer dimer, thus has stopped false-positive generation.
2, the test kit of rapid detection nosema bombycis, described test kit comprises two kinds of different IPs acid molecule markers, mark primer pair and the colloidal gold immuno-chromatography test paper strip of SEQIDNO.7 and SEQIDNO.8 respectively, the point sample district of described colloidal gold immuno-chromatography test paper strip is coated with the colloid gold particle of connection wherein a kind of labeled nucleic acid molecule thing antibody, and detection line is fixed with the part with another kind of labeled nucleic acid molecule thing specific combination.Owing to introducing special marker in primer, the product of pcr amplification can be detected with immunity colloidal gold test paper strip, simplify detecting step, and the primer pair specificity that the present invention uses is good, highly sensitive, compared with traditional gel electrophoresis, its susceptibility increases by 10 times.
As the preferred technical scheme of the present invention, described labeled nucleic acid molecule thing is vitamin H, digoxin, Fluoresceincarboxylic acid or fluorescein isothiocyanate.This several marker can be identified by the antibody of its correspondence or ligand specificity, and specificity is high, thus is detected by immunologic method.
As the preferred technical scheme of the present invention, described labeled nucleic acid molecule thing is vitamin H or Fluoresceincarboxylic acid, and part is that Streptavidin molecule and anti-Fluoresceincarboxylic acid rabbit resist.
Also comprise developping solution in test kit of the present invention, each concentration of component of described developping solution is as follows: Tris10mM, BSA1%, NaCl137mM, KCl2.7mM, Na
2hPO
410mM and KH
2pO
42mM.Tris composition in developping solution can realize the removing to primer dimer and non-specific amplification product to a certain extent.
In the present invention, described test kit also comprises measuring samples treatment solution and lysate, described measuring samples treatment solution to be massfraction be 4% KOH; Described lysate containing volume fraction be 1% NP-40 (Nonidet P40) and volume fraction be 2% TritonX-100 (Triton X-100).Measuring samples treatment solution can make nosema bombycis surface portion albumen come off, and lysate main purpose is that cracking nosema bombycis discharges DNA, using the template as pcr amplification reaction.
Owing to not carrying out purifying to extracted From Nosema bombycis molitor genomic dna in DNA extraction of the present invention, therefore have more impurity, its common PCR nucleic acid polymerase is not also suitable for this detection system; Select the PCR nucleic acid polymerase of warm start effectively can reduce the generation of primer dimer in PCR process and non-specific amplification product, so employ the heat start PCR polysaccharase MightyAmpDNApolymeraseVer.3 being applicable to template source complexity in the present invention.
As the present invention's preferred technical scheme further, measuring samples treatment solution and lysate is utilized to extract the step of DNA as follows: to get silkworm seed and add water centrifugal after grinding, collecting precipitation, measuring samples treatment solution is added in precipitation, clean with water after mixing 18 ~ 25 DEG C of placement 10min, centrifugal collecting precipitation, then adds lysate in precipitation, places 1hr for 100 DEG C.
As the most preferred technical scheme of the present invention, described colloidal gold immuno-chromatography test paper strip is prepared by following methods: be first cross-linked with colloid gold particle with anti-Fluoresceincarboxylic acid rabbit anti-(anti-FAM rabbit resists), form Radioactive colloidal gold-anti-mixture of anti-Fluoresceincarboxylic acid rabbit, be then sprayed at point sample district; On tunica fibrosa, press wire-like spray with the part Streptavidin of vitamin H again, form detection line; Last and absorbent filter, sample pad and base plate assemble colloidal gold immuno-chromatography test paper strip.In order to prove that test strip is in the inherent detection line downstream of validity period and fixes nature controlling line, nature controlling line wrapping and is resisted, as goat anti-rabbit igg antibody by two of anti-rabbit IgG.
3, utilize the test kit of described rapid detection nosema bombycis to detect the method for nosema bombycis, comprise the steps:
A. extract testing sample DNA, the primer pair then marking SEQIDNO.7 and SEQIDNO.8 respectively with labeled nucleic acid molecule thing carries out pcr amplification, collects amplified production;
B. by steps A) whether gained amplified production point in the sample pad of described colloidal gold immuno-chromatography test paper strip, and with containing the developping solution chromatography of nucleic acid denaturation agent, have bands visible according to detection line, judges negative or positive; If there is bands visible, be judged to be the positive, if without bands visible, be judged to be feminine gender.
Cleaning Principle is as follows: when there is specific amplification products to be checked, amplified production is with different markers, form mark A-amplified production-mark B mixture, mark A-amplified production-mark B mixture is combined with Radioactive colloidal gold-anti-mark A antibody complex, form the red granules mixture of colloid gold particle-anti-mark A antibody-marker A-amplified production-mark B, red granules mixture is launching in damping fluid along test strip direction chromatography, ligand binding at detection line position with mark B, form macroscopic red lines, be judged to be the positive; When specific amplification products does not exist, the red granules mixture of colloid gold particle-anti-mark A antibody-mark A-amplified production-mark B can not be formed, detection line with the ligand binding marking B, can not can not be formed red lines, be judged to be feminine gender.
In steps A of the present invention, described pcr amplification condition is 98 DEG C of denaturation 5min, 98 DEG C of sex change 10sec, 64 DEG C of annealing 15sec, and 68 DEG C extend 35sec, 30 circulations of increasing; Last 68 DEG C extend 3min.
The explanation of technical terms used in specification sheets of the present invention:
PCR: the abbreviation of polymerase chain reaction (polymerasechainreaction), can amplify the Protocols in Molecular Biology of amplification specific DNA fragments.
Primer: guide DNA synthetic enzyme to carry out the material of DNA synthesis, be generally a pair single stranded oligonucleotide, after combining with amplification template, synthesis from 3 ' end of primer.
Mark: the method together with the molecule that can be detected is coupled at single stranded oligonucleotide.
Antibody: the protein molecule that can be combined with antigen or hapten specificity.
Primer dimer: in PCR process, between upper and lower primer, or the connection phenomenon between unidirectional primer self is called primer dimer, and the primer dimer formed between unidirectional primer self can not form false positive signal.
Nucleic acid polymerase: the enzyme catalyzing and synthesizing nucleotide chain, is mainly divided into archaeal dna polymerase and RNA polymerase.
Beneficial effect of the present invention is: the present invention is the Auele Specific Primer that marks to the suitable gene fragment of amplification, can ensure the specificity of amplification, thus ensure that the accuracy of detected result; In order to simplify trace routine, the special primer of design is carried out double-tagging, thus avoid the cumbersome procedure of the probe of additional tape label; Amplified production directly uses immunity colloidal gold test paper strip to judge, makes whole testing process simplify operation steps, whole testing process fast and easy on the basis keeping detected result accuracy; Present invention also offers the sample treatment that in a kind of bombyx mori egg, nosema bombycis nucleic acid amplification detects, the method and tradition are extracted compared with the genomic method of nosema bombycis, fast, efficiently.Therefore the present invention is the detection means of nosema bombycis in a kind of desirable bombyx mori egg.
This patent also provides the test kit of nosema bombycis in rapid detection bombyx mori egg, and tool has the following advantages:
Simple to operate: all testing process only needs amplified production to be added in and launches after in damping fluid, is dripped in test strip by mixture, without the need to professional's operation, is applicable to seed station recruitment environment;
Quick: amplified production gets final product result of determination after being added drop-wise to test strip 2-5min;
Sensitivity: sensitiveer than agarose gel electrophoresis 10 times, the nosema bombycis that cannot detect in opticmicroscope can be detected;
Specificity is high: because employ the Auele Specific Primer pair of mark, its detected result is more accurately objective.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is primer screening result figure (A:LSU149; B:LSU151; C:LSU153; D:LSU323; E:LSU3238 and LSU397; F:LSU480; G:LSU482; Nb in figure: nosema bombycis; Ser: Serratia; BmNPV: Bombyx mori nuclear polyhydrosis virus; Bp: beauveria bassiana; S.au: streptococcus aureus).
Fig. 2 detects beauveria bassiana colloidal gold immuno-chromatography test paper strip result for adopting specificity labeled primers LSU480.
Fig. 3 detects nosema bombycis specific detection result (A: agarose gel electrophoresis for adopting specificity labeled primers; B: colloidal gold immuno-chromatography test paper strip).
Fig. 4 detects nosema bombycis susceptibility detected result (A: agarose gel electrophoresis for adopting specificity labeled primers; B: colloidal gold immuno-chromatography test paper strip, 1 ~ 8 is respectively 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10 nosema bombycis and 100 normal bombyx mori eggs be mixed with the amplified production of template).
Fig. 5 is nosema bombycis detected result (A: agarose gel electrophoresis; B: colloidal gold immuno-chromatography test paper strip).
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.
Embodiment 1, primer screening
Extract From Nosema bombycis molitor genomic dna, concrete grammar to add water centrifugal 5 minutes of 9000r/min after grinding for getting silkworm seed, collecting precipitation, measuring samples treatment solution is added in precipitation, clean with water after mixing 18 ~ 25 DEG C of placement 10min, 9000r/min is collecting precipitation after centrifugal 5 minutes, then in precipitation, adds lysate, place 1hr for 100 DEG C, wherein said measuring samples treatment solution to be massfraction be 4% KOH; Described lysate be containing volume fraction be 1% NP-40 and volume fraction be the TritonX-100 of 2%.
Extract Serratia, nuclear polyhedrosis virus, beauveria bassiana, staphylococcus aureus gene group DNA in contrast simultaneously.
Then according to large ribosomal subunit RNA (LargesubunitrRNA) encoding sequence of nosema bombycis, design of amplification primers, concrete primer is as shown in table 1.
The large ribosomal subunit RNA amplification primer of table 1, nosema bombycis
With From Nosema bombycis molitor genomic dna for template, the sequence shown in table 1 is that primer carries out pcr amplification, and PCR amplification system is as shown in table 2 time:
Table 2, PCR amplification system
| Template DNA | 10μL |
| Primer F | 0.3μL |
| Primer R | 0.3μL |
| 2×MightyAmp Buffer Ver.3 | 12.5μL |
| MightyAmp DNA polymerase Ver.3 | 0.5μL |
| ddH 2O | 1.4μL |
| Cumulative volume | 25μL |
Increase by following condition, 98 DEG C of denaturation 5min; 98 DEG C of sex change 10sec, 64 DEG C of annealing 15sec, 68 DEG C extend 35sec, and 30 circulations, extend 3min after last 68 DEG C.Amplified production gets 10 μ L for agarose gel electrophoresis, and Gel electrophoresis conditions is: 1 × TAE damping fluid, voltage 200V, electrophoresis time 17min, and result as shown in Figure 1.Result shows, in 8 pairs of primers of design, for template has, obvious amplified production produces with nosema bombycis DNA for primer pair LSU323, LSU480 and LSU482, wherein primer LSU480 and LSU482 primer sequence similarity very high, but the brightness of LSU480 amplified production is a little more than the amplified production brightness of LSU482, therefore chooses LSU323 and LSU480 and carry out Modify to primer.
LSU323 and LSU480 primer is used respectively vitamin H and Fluoresceincarboxylic acid (FAM) mark, prepare colloidal gold immuno-chromatography test paper strip simultaneously, be specially: be cross-linked with colloid gold particle with anti-FAM rabbit is anti-, form Radioactive colloidal gold-anti-mixture of anti-FAM rabbit, then spray paint in point sample district; Press wire-like spray with the part Streptavidin of vitamin H again, form detection line, and fix nature controlling line in detection line downstream, on nature controlling line, bag quilt is as goat anti-rabbit igg antibody; Last and absorbent filter, sample pad and base plate assemble colloidal gold immuno-chromatography test paper strip by the preparation method of colloidal gold immuno-chromatography test paper strip.
By biotin labeled primer amplification beauveria bassiana DNA, then get 10 μ L amplified production points in test strip sample pad, join 90 μ L and complete chromatography in test strip with the developping solution of nucleic acid denaturation agent, developping solution composition is: 10mMTris, 1%BSA, 137mMNaCl, 2.7mMKCl, 10mMNa
2hPO
4and 2mMKH
2pO
4, observe detected result after 5min, in 15min, result is effective, and result as shown in Figure 2.Result shows, the PCR primer that primer LSU480 after modification is template with beauveria bassiana DNA has the weak positive in test strip, see Fig. 2, and LSU323 and all no positive band of all the other silkworm pathogenic bacterias except nosema bombycis produce, show that the specificity of LSU323 primer is better, therefore the present invention is using LSU323 primer pair as detection primer.
Embodiment 2, specificity experiments
Detect respectively as lower bolster according to the method LSU323 upstream primer of embodiment 1 and downstream primer: 1. with the positive plasmid built after not tagged LSU323-F and LSU323-R primer pair amplifies; 2. nosema bombycis DNA; 3. blank water contrast; 4. Bombyx mori nuclear polyhydrosis virus DNA; 5. beauveria bassiana DNA; 6. Serratia DNA; 7. L-form staphylococcus aureus; Adopt the MightyAmpDNApolymeraseVer.3 of TAKARA company, total reaction system is 25 μ L.Increase with AppliedBiosystemsPCR instrument, reaction parameter is: 98 DEG C of denaturation 5min; 98 DEG C of sex change 10sec, 64 DEG C of annealing 15sec, 68 DEG C extend 35sec, 30 circulations; Last 68 DEG C extend 3min, 12 DEG C of preservations.Get 10 μ L amplified productions and carry out electrophoresis in 2% agarose gel electrophoresis, then by ethidium bromide staining, under ultraviolet lamp, whether observe and decide has specific band to produce, and result is as shown in A in Fig. 3; Amplified production colloidal gold immuno-chromatography test paper strip prepared by embodiment 1 detects simultaneously, and result is as shown in B in Fig. 3.Result shows, nosema bombycis DNA and positive plasmid are that the detection line of template is for positive, negative control group is that in other common causative microorganisms of silkworm or environment, common microbiological DNA does template, it is that template detection line is negative that negative control bacterium is respectively Bombyx mori nuclear polyhydrosis virus, beauveria bassiana, Serratia, streptococcus aureus and blank water, show that LSU323 detects nosema bombycis and has good specificity, to other silkworm pathogenic micro-organism no cross reactions.
Embodiment 3, susceptibility are tested
For detecting the sensitivity of colloidal gold immuno-chromatography test paper strip, be 10 by dilution after nosema bombycis counting
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10 nosema bombycis, mix with 100 normal bombyx mori eggs respectively, extract DNA according to the method for embodiment 1, and increase by the PCR system set up.10 μ L are got for agarose gel electrophoresis after having increased.Gel electrophoresis conditions is: 1 × TAE damping fluid, voltage 200V, electrophoresis time 17min.Get 10 μ L ELISA test strips, then join 90 μ L and launch to detect in damping fluid, observe detected result after 5min, in 15min, result is effective, and result as shown in Figure 4 simultaneously.Result shows, the positive is not detected in the sample that agarose gel electrophoresis mixes with 100 normal bombyx mori eggs 10 nosema bombycis, and ELISA test strip goes out the positive, show that the sensitivity of the gel electrophoresis that colloidal gold immuno-chromatography test paper strip is more traditional adds 10 times.
Embodiment 4, nosema bombycis detect
Colloidal gold immuno-chromatography test paper strip for be with malicious female moth lay eggs middle nosema bombycis detect, 100 ovum that 100 normal ovum and the female moth of headband poison produce are carried out pcr amplification by the method for embodiment 1 respectively, amplified production gets 10 μ L for agarose gel electrophoresis, Gel electrophoresis conditions is: 1 × TAE damping fluid, voltage 200V, electrophoresis time 17min.Get 10 μ L amplified production ELISA test strips, then join 90 μ L and launch to detect in damping fluid, observe detected result after 5min, in 15min, result is effective, and detected result as shown in Figure 5 simultaneously.Result shows, and be with malicious female moth product silkworm seed and positive plasmid result to be positive, and blank and normal silkworm seed result is negative, and show that colloidal gold immuno-chromatography test paper strip of the present invention can be used in nosema bombycis in silkworm seed and detects.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (10)
1. the primer pair of rapid detection nosema bombycis, is characterized in that: the nucleotide sequence of described primer pair is as shown in SEQIDNO.7 and SEQIDNO.8.
2. the test kit of rapid detection nosema bombycis, it is characterized in that: described test kit comprises primer pair and the colloidal gold immuno-chromatography test paper strip that two kinds of different IPs acid molecule markers mark SEQIDNO.7 and SEQIDNO.8 respectively, the point sample district of described colloidal gold immuno-chromatography test paper strip is coated with the colloid gold particle of connection wherein a kind of labeled nucleic acid molecule thing antibody, and detection line is fixed with the part with another kind of labeled nucleic acid molecule thing specific combination.
3. the test kit of rapid detection nosema bombycis according to claim 2, is characterized in that: described labeled nucleic acid molecule thing is vitamin H, digoxin, Fluoresceincarboxylic acid or fluorescein isothiocyanate.
4. the test kit of rapid detection nosema bombycis according to claim 3, is characterized in that: described labeled nucleic acid molecule thing is vitamin H or Fluoresceincarboxylic acid, and part is that Streptavidin molecule and anti-Fluoresceincarboxylic acid rabbit resist.
5. the test kit of rapid detection nosema bombycis according to claim 2, it is characterized in that: described test kit also comprises developping solution, each concentration of component of described developping solution is as follows: Tris10mM, BSA1%, NaCl137mM, KCl2.7mM, Na
2hPO
410mM and KH
2pO
42mM.
6. the test kit of rapid detection nosema bombycis according to claim 2, is characterized in that: described test kit also comprises measuring samples treatment solution and lysate, described measuring samples treatment solution to be massfraction be 4% KOH; Described lysate containing volume fraction be 1% Nonidet P40 and volume fraction be the TritonX-100 of 2%.
7. the test kit of rapid detection nosema bombycis according to claim 6, it is characterized in that: utilize measuring samples treatment solution and lysate to extract the step of DNA as follows: get silkworm seed and add water centrifugal after grinding, collecting precipitation, measuring samples treatment solution is added in precipitation, clean with water after mixing 18 ~ 25 DEG C of placement 10min, centrifugal collecting precipitation, then adds lysate in precipitation, places 1hr for 100 DEG C.
8. according to the test kit of any one of claim 2 ~ 7 rapid detection nosema bombycis, it is characterized in that: described colloidal gold immuno-chromatography test paper strip is prepared by following methods: be first cross-linked with colloid gold particle with anti-Fluoresceincarboxylic acid rabbit is anti-, form Radioactive colloidal gold-anti-mixture of anti-Fluoresceincarboxylic acid rabbit, be then sprayed at point sample district; On tunica fibrosa, press wire-like spray with the part Streptavidin of vitamin H again, form detection line, last and absorbent filter, sample pad and base plate assemble colloidal gold immuno-chromatography test paper strip.
9. utilize the test kit of rapid detection nosema bombycis described in any one of claim 2 ~ 8 to detect the method for nosema bombycis in bombyx mori egg, it is characterized in that, comprise the steps:
A, extract testing sample DNA, the primer pair then marking SEQIDNO.7 and SEQIDNO.8 respectively with labeled nucleic acid molecule thing carries out pcr amplification, collects amplified production;
B, by steps A) whether gained amplified production point in the point sample district of described colloidal gold immuno-chromatography test paper strip, and with containing the developping solution chromatography of nucleic acid denaturation agent, have bands visible according to detection line, judges negative or positive; If there is bands visible, be judged to be the positive, if without bands visible, be judged to be feminine gender.
10. method according to claim 9, is characterized in that: in steps A, and described pcr amplification condition is 98 DEG C of denaturation 5min, 98 DEG C of sex change 10sec, 64 DEG C of annealing 15sec, and 68 DEG C extend 35sec, 30 circulations of increasing; Last 68 DEG C extend 3min.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946747A (en) * | 2015-05-29 | 2015-09-30 | 中国水产科学研究院东海水产研究所 | Nested primer for early warning of cultured and wild Portunus trituberculatus microsporidium infection, and application thereof |
| CN106591492A (en) * | 2016-12-21 | 2017-04-26 | 东北农业大学 | LAMP detection kit and application |
| CN107312869A (en) * | 2017-08-31 | 2017-11-03 | 西南大学 | The kit and its detection method of PCR ELISA method detection nosema bombycis |
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| CN104946747B (en) * | 2015-05-29 | 2018-05-01 | 中国水产科学研究院东海水产研究所 | A kind of nested primer and its application for infecting early warning with Wild Portunus trituberculatus microsporidian for China's cultivation |
| CN106591492A (en) * | 2016-12-21 | 2017-04-26 | 东北农业大学 | LAMP detection kit and application |
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| CN107400668B (en) * | 2017-09-07 | 2021-03-02 | 辽宁省农业科学院大连生物技术研究所 | Extraction method of tussah microsporidian template DNA and application of tussah microsporidian template DNA in molecular diagnosis |
| CN108490171A (en) * | 2018-03-02 | 2018-09-04 | 北京库尔科技有限公司 | High-sensitivity saliva antibody detection kit |
| WO2019170709A1 (en) * | 2018-03-06 | 2019-09-12 | Centro Di Sperimentazione Laimburg | Oligonucleotides and methods for internal control of eukaryotic dna amplification reactions |
| IT201800003299A1 (en) * | 2018-03-06 | 2019-09-06 | Centro Di Sperimentazione Laimburg | Oligonucleotides and methods for the internal control of nucleic acid amplification reactions. |
| US11427862B2 (en) | 2018-03-06 | 2022-08-30 | Centro Di Sperimentazione Laimburg | Oligonucleotides and methods for internal control of eukaryotic DNA amplification reactions |
| CN108548800A (en) * | 2018-03-16 | 2018-09-18 | 华侨大学 | The kit and detection method of yapamicin relict amount in a kind of detection food |
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| CN111269999A (en) * | 2020-02-09 | 2020-06-12 | 西南大学 | Universal primer, kit, test strip and application for simultaneously detecting four kinds of human microsporidia |
| CN114277177A (en) * | 2021-12-30 | 2022-04-05 | 广西壮族自治区蚕业技术推广站 | Probe, kit and detection method for rapidly detecting nosema bombycis |
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