WO2016065983A1 - Ensemble amorces de détection lamp et kit de réactifs pour identifier une microsporidie chez les œufs de vers à soie - Google Patents

Ensemble amorces de détection lamp et kit de réactifs pour identifier une microsporidie chez les œufs de vers à soie Download PDF

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WO2016065983A1
WO2016065983A1 PCT/CN2015/088349 CN2015088349W WO2016065983A1 WO 2016065983 A1 WO2016065983 A1 WO 2016065983A1 CN 2015088349 W CN2015088349 W CN 2015088349W WO 2016065983 A1 WO2016065983 A1 WO 2016065983A1
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silkworm
primers
microsporidia
detection
dna
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Chinese (zh)
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刘吉平
程伟
晏育伟
宋小景
杨思佳
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华南农业大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/90—Protozoa ; Processes using protozoa

Definitions

  • the invention belongs to the field of biotechnology. More specifically, it relates to a group of LAMP detection primers and kits for the microsporidia of the silkworm, Bombyx mori.
  • Bombyx mori disease is a devastating disease caused by pathogenic infection of Nosema bombycis (Nb) through infection or embryonic egg (fetal), which causes the infection of silkworm, and is also affecting the sustainable development of silk industry in China. Important epidemics, economic losses caused by microscopic disease every year are very heavy. At the same time, the wild insect microsporidia can cross-infect the silkworm, and can spread during the silkworm and between different silkworms, resulting in the scrapping of a large number of silkworm species, which seriously restricts the silkworm trade and the sustainable development of the silkworm industry. China has listed silkworm granule disease as a quarantine list for import and export animal quarantine.
  • the silkworm owners' management departments and related production units in various regions have invested a large amount of manpower, material resources and financial resources.
  • the traditional microscopic microscopic examination of the female moth and the elimination of the silkworm species produced by the poisonous female moth have been carried out to prevent and control the silkworm granule disease, but the effect is not good.
  • the designed PCR primers have low detection sensitivity for the microsporidia, and have many operation steps, which are not suitable for field detection, and are not suitable for widespread promotion and use in actual production.
  • a larger problem is that the currently disclosed detection primers are based on the DNA of microsporidia, but the separation and collection of microsporidia are complicated, time consuming, and extremely unfavorable for large-scale detection or on-site detection.
  • microsporidia are parasitic in silkworm eggs, and the egg content of silkworm eggs is significantly higher than that of microsporidia to be detected.
  • DNA samples obtained by extraction both DNAs exist simultaneously, and the DNA of silkworm eggs is seriously detected. Interference, therefore, if you want to directly use the silkworm egg DNA as a template for microsporidia detection, put forward higher requirements for detection.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the existing technology for detecting the infection of the silkworm microsporidia in the silkworm eggs, and design the LAMP detection primers for detecting the microsporidia of the silkworm eggs by using the septin3 gene of the microsporidia of the silkworm, and provide a A simpler and faster detection method than the PCR detection method.
  • Another object of the invention is to provide the use of said LAMP detection primers.
  • Still another object of the present invention is to provide a method and kit for detecting a microsporidia of Bombyx mori established using the above-described LAMP detection primer set.
  • the present invention provides a set of LAMP detection primer sets of Microsporidium larvae, the primer set comprising a pair of lateral primers Sep3F3/Sep3B3 and a pair of inner primers Sep3FIP/Sep3BIP, the nucleotide sequences of the primers are respectively SEQ ID NO: 1 to 4.
  • the invention also provides the use of the LAMP detection primer set of the above-mentioned silkworm, Bombyx mori, in the preparation of a microsporidia detection kit for silkworm eggs.
  • the invention also provides a detection kit for the microsporidia LAMP of Bombyx mori, comprising a pair of outer primers Sep3F3/Sep3B3 and a pair of inner primers Sep3FIP/Sep3BIP, the nucleotide sequences of the primers are respectively SEQ ID NO: 1. ⁇ 4 is shown.
  • the kit further includes 2X reaction buffer, a positive control, a negative control, a chromogenic solution or a fluorescent staining solution, Bst DNA polymerase, a sealing solution, and sterile water.
  • the components of the 2X reaction buffer are as follows: 20 mM Tris-HCl, pH 8.8; 10 mM KCl; 2 mM MgSO 4 ; 20 mM (NH 4 ) 2 SO 4 ; 0.1% Triton X-100; 2.8 mM dNTPs ; 1 M betaine; 25 mM MgCl 2 .
  • the positive control is a Septin3-DNA-pMD recombinant plasmid; the negative control is a normal home Silkworm DNA.
  • the nucleotide sequences of the primers Sep3F and Sep3R are shown in SEQ ID NOS: 5-6.
  • the color developing solution is 10000 ⁇ SYBR Green I or a fluorescent indicator 1 ⁇ SYBR Green I.
  • the sealing liquid is glycerin.
  • reaction system of the kit is as follows:
  • reaction system When the detection result is determined by staining or agarose gel electrophoresis, the reaction system is:
  • the reaction system is:
  • the concentration of the primer Sep3F3/Sep3B3 is 5 pmol/ ⁇ L
  • the concentration of the primer Sep3FIP/Sep3BIP is 20-40 pmol/ ⁇ L.
  • a (N + 3) volume reaction system (including a negative control, a positive control, and a packing error) should be prepared to ensure uniform dispensing of each reaction tube.
  • the LAMP reaction conditions of the kit are: 63 ° C constant temperature reaction for 60 min; then 95 ° C, placed for 2 min inactivation.
  • the method for detecting microsporidia of silkworm eggs by using the kit of the present invention comprises the following steps:
  • Disposition reaction system configured according to the above reaction system
  • reaction tube For the detection of the staining method and the agarose gel electrophoresis determination result, the reaction tube is placed in a constant temperature water bath or other constant temperature equipment, and placed at a constant temperature of 63 ° C for 30 to 60 minutes, then 95 ° C, placed for 2 minutes to inactivate; For the detection of the real-time fluorescence determination result, the reaction tube is placed in a Deaou-308C constant temperature fluorescence detector or other fluorescence detector, and the observation result is kept at a constant temperature of 63 ° C for 00 min;
  • results can be judged by staining (chromogenic method), agarose gel electrophoresis and real-time fluorescence;
  • the preparation of the microsporidia DNA template of the silkworm used in the positive control kit of the present invention is carried out by using the Qiagen plant mini-rabble kit according to the method. Specifically, the following steps are included:
  • the mixed solution is incubated at 65 ° C for 10 min (during the test tube 2 to 3 times upside down);
  • Step S13 Step S12 is repeated, and the refrigerator is kept at -20 ° C for use.
  • Loop-mediated isothermal amplification is a novel technique for in vitro isothermal amplification of specific nucleic acid fragments invented by Japanese scholar Notomi et al. (2000).
  • the LAMP method has the advantages of high specificity, rapidity, high efficiency, high sensitivity, simple operation, simple detection method, and the like, and the result can be judged by the naked eye, thereby simplifying the detection process and greatly shortening the detection time.
  • primers are the most critical factor for the LAMP detection method.
  • the selection of target gene sequences, the design and selection of primers directly affect the quality of the test results. Even for the same target gene sequence, how to select the appropriate six regions and design the appropriate four primer sequences is very important and very significant for detection sensitivity.
  • the inventors designed a series of multiple sets of LAMP primers, and considered various factors and combined with the judgment of the experimental results to screen out three sets of LAMP primers. Further, the inventors finally determined the optimal four specific primers, namely four specific primers of primer set III as a primer set for LAMP detection.
  • the four primer sequences of the primer set III are:
  • Sep3B3 (shown in SEQ ID NO: 2): 5'-AATTTGTTACAGAAGGACTCC-3'
  • the above-mentioned LAMP detection primer set for microsporidia detection is obtained by the present inventors on the basis of obtaining the septin3 gene of the microsporidia of the silkworm, and has been obtained through extensive exploration and research, which not only overcomes the complexity of ordinary PCR operation, but also relies on expensive.
  • the defects of the instrument more importantly, the biggest innovations of the present invention are: 1) Compared with the ordinary PCR method, the invention is short in time, the detection result is easier to judge; 2) the method relative to the existing LAMP is applied to the micro Primers used for the detection of sporozoans, the primers used in this method directly use the silkworm egg DNA as a template, which not only overcomes the interference caused by the silkworm egg DNA, but also makes the detection result more reliable and the sensitivity is very good.
  • the method is simple to operate and detect It takes a short time, is easy to judge, and has strong specificity. It can detect 1 silkworm egg infected with silkworm microbe disease.
  • the invention discloses a group of LAMP primers for detecting microsporidia of Bombyx mori.
  • the Sep3F/3R primer is designed by using the septin3 gene sequence as a target gene, and the PCR primer is used to verify the specificity of the target gene, and the result indicates that the primer has specificity.
  • the septin3 gene was designed as a target gene to design multiple sets of LAMP primers.
  • Three groups of LAMP primers were screened according to the design principle of LAMP primers and the amplified sequence positions of each set of primers, and then further screened according to the validity of the primers.
  • a set of four specific primers were used as primers for LAMP detection, namely the outer primer Sep3F3/Sep3B3 and the inner primer Sep3FIP/Sep3BIP; and amplification was performed only when only four specific primers fully recognized the six regions of the target gene. Therefore, it ensures the specificity and detection of the specificity and detection of the speptin3 gene of Bombyx mori.
  • the invention utilizes the LAMP primer set to further establish a rapid detection method and a kit for microsporidia LAMP, and comprises four LAMP primers, a LAMP reagent, a color developing solution or a fluorescent staining liquid, etc., to form a detection reaction system, and the constant temperature condition at 63 ° C
  • the rapid amplification of the template DNA can detect the silkworm egg DNA produced by the silkworm infected with Bombyx mori, which is 5.0 ⁇ 10 -3 ng/ ⁇ L, and can detect 103 copies/ ⁇ L of the recombinant plasmid Septin3-DNA-pMD.
  • the detection sensitivity of microsporidia has increased by an order of magnitude, which is of great significance for the detection of microsporidia.
  • microsporidium samples are parasitic in the silkworm eggs, and the silkworm egg content is significantly higher than the microsporidia to be detected.
  • both DNAs exist simultaneously, which raises the detection higher. Claim. Therefore, the biggest innovation of the present invention is that microsporidia detection is carried out directly on the silkworm egg DNA produced by the silkworm infected with the microsporidia of the silkworm, and the complicated time-consuming steps of microsporidia separation are eliminated.
  • the primers used in the method not only avoid the interference caused by the DNA detection of the silkworm silkworm eggs, but also make the detection result more reliable and the detection sensitivity is very good.
  • the invention adopts the LAMP technology and adopts constant temperature amplification, and does not need to amplify a complicated and expensive amplification instrument like a PCR instrument.
  • the invention optimizes the reaction system, and the whole reaction can be completed within 30 to 60 minutes, which greatly shortens the detection time. The human and material cost of microsporidia detection is further reduced.
  • the detection method of the invention has multiple choices for the determination of the detection result, and is easy to observe and judge: (1) color development method: adding a color developing liquid to the reaction product, and visually observing the color reaction, and can be visually observed under natural light. Detection results; (2) agarose gel electrophoresis: according to the electrophoresis strips, the reaction results can be observed more intuitively, and more convincing, the false positive test results can also be easily eliminated, but the cover before the electrophoresis operation
  • the sample loading should be carried out in different rooms with the configuration reaction system to reduce the unnecessary contamination of the subsequent experiments after opening the cover; (3) Real-time fluorescence method, the result can be judged intuitively through the amplification curve, and for the sample with high concentration The result can be judged early, reducing unnecessary time wastage, but the instrument is more expensive and can be used by laboratories or companies with sufficient funds.
  • the rapid detection method of the LAMP of the silkworm pathogenic microsporidia of the present invention is simple in operation, short in time, does not require complicated instruments and complicated amplification procedures, and the interference of impurities on the amplification is small and reacts.
  • the results are easy to judge and have strong specificity. It provides an important basis and technical reserve for the detection of microsporidia of silkworm eggs and the quality of silkworms by using LAMP technology. It is not only suitable for on-site or field testing, but also widely promoted in actual production. It can also better meet the needs of scientific research institutions, silkworm production units and silkworm quality inspection centers, and is easy to promote and apply in a wide range.
  • Figure 1 is an electrophoresis pattern of two PCR primers for detecting healthy silkworm eggs and infected silkworm eggs, M: DL 2000 DNA Marker; 1: Adding DNA to silkworm eggs; 2: Microsporidium DNA of silkworm; 3: DNA of normal silkworm eggs ; 4: ddH 2 O.
  • Figure 2 is a schematic representation of the position of a common PCR primer and the three selected LAMP primer sets of the present invention on the septin3 gene.
  • Figure 3 is a schematic representation of the position of four LAMP primers of the present invention on a target gene sequence.
  • Fig. 4 is a graph showing the results of detection of different concentrations of positive control (recombinant plasmid Septin3-DNA-pMD) by the rapid detection method of the microsporidia LAMP of the silkworm, Bombyx mori L. (the result of agarose gel electrophoresis).
  • Fig. 5 is a diagram showing the results of detection of different concentrations of positive control (recombinant plasmid Septin3-DNA-pMD) by the rapid detection method of the microsporidia LAMP of the silkworm, Bombyx mori L. (color detection method).
  • Fig. 6 shows the results of detection of different concentrations of positive control (recombinant plasmid Septin3-DNA-pMD) by the rapid detection method of the microsporidia LAMP of the silkworm, Bombyx mori L. (real-time fluorescence detection results).
  • Fig. 7 is a graph showing the sensitivity of the rapid detection of the microsporidia LAMP of the silkworm eggs of the silkworm eggs of the silkworm eggs of the silkworm eggs of the silkworm eggs of different concentrations of the silkworm eggs of the silkworm (the results of agarose gel electrophoresis).
  • Fig. 8 is a graph showing the sensitivity results of the silkworm egg DNA produced by the silkworm larvae of the silkworm, Bombyx mori L., by the rapid detection method of the microsporidia of the silkworm, Bombyx mori L. (color detection method).
  • Fig. 9 is a graph showing the sensitivity of the rapid detection of the microsporidia LAMP of the silkworm eggs of the silkworm eggs of the silkworm eggs of the silkworm eggs of the silkworm eggs of different concentrations of the silkworm eggs of the silkworm (the real-time fluorescence detection results).
  • Fig. 10 is a view showing the detection result of the microparticles of the larvae of the larvae of the larvae of the silkworm, the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larvae of the larva
  • Fig. 11 is a view showing the detection result of the granule-added silkworm eggs of different numbers of microsporidia LAMP in the silkworm larvae of the present invention (the color detection method).
  • Fig. 12 is a diagram showing the detection result of the microparticles of the microsporidia LAMP visualized rapid detection kit of the silkworm, Bombyx mori, in the detection of different number of eggs on the silkworm eggs (real-time fluorescence detection results).
  • the invention designed a set of PCR primers using the gene as a target gene: the primer Sep3F/Sep3R.
  • the sequence of the primer Sep3F (shown in SEQ ID NO: 5) is:
  • the sequence of the primer Sep3R (shown in SEQ ID NO: 6) is:
  • the position of the fragment was amplified, and the target gene designed with the septin3 gene as the LAMP primer was used to design multiple sets of LAMP primers using the online software Primer ExplorerV4 (http://primerexplorer.jp/elamp4.0.0/index.htmL).
  • Three sets of LAMP primers namely primer set I, primer set II and primer set III, were screened according to the design principle of LAMP primers and the positions of amplified sequences of each set of primers.
  • Primer group I Four primer sequences are:
  • Primer III Four primer sequences are:
  • Sep3B3 (shown in SEQ ID NO: 2): 5'-AATTTGTTACAGAAGGACTCC-3'
  • Fig. 2 The position of the above common PCR primers and the three groups of LAMP primer sets selected by the present invention on the septin3 gene are shown in Fig. 2.
  • the mixed solution is incubated at 65 ° C for 10 min (during the test tube 2 to 3 times upside down);
  • Step S13 Step S12 is repeated, and the refrigerator is kept at -20 ° C for use.
  • PCR was carried out using the DNA of Bombyx mori
  • the amplified product was cloned into the pMD-19T vector to obtain the Septin3-DNA-pMD plasmid and used as a positive control for the detection.
  • the extracted normal silkworm DNA was used as a negative control.
  • the components were as follows: 20 mM Tris-HCl, pH 8.8; 10 mM KCl; 2 mM MgSO 4 ; 20 mM (NH 4 ) 2 SO 4 ; 0.1% Triton X-100; 2.8 mM dNTPs; 1 M betaine; 25 mM MgCl 2 .
  • the primer set Sep3F3/Sep3B3 was used as the external primer and Sep3FIP/Sep3BIP was used as the internal primer (Sep3F3/Sep3B3 concentration was 5 pmol/ ⁇ L, and Sep3FIP/Sep3BIP concentration was 20-40 pmol/ ⁇ L), 2 ⁇ reaction buffer, positive control, negative control, Bst DNA polymerase (8 U/ ⁇ L), sealing solution, color developing solution (10000 ⁇ SYBR Green I); fluorescent indicator (1 ⁇ SYBR Green I) ; Sterilize ddH 2 O. It is the detection reagent for the LAMP rapid detection kit for the microsporidia of Bombyx mori.
  • the LAMP reaction condition of the kit is: constant temperature reaction at 63 ° C for 30 to 60 min; then 95 ° C, placed for 2 min inactivation.
  • the reaction system of the kit is:
  • the reaction system is:
  • the concentration of the primer Sep3F3/Sep3B3 is 5 pmol/ ⁇ L
  • the concentration of the primer Sep3FIP/Sep3BIP is 20-40 pmol/ ⁇ L.
  • a (N + 3) volume reaction system (including a negative control, a positive control, and a packing error) should be prepared to ensure uniform dispensing of each reaction tube.
  • Disposition reaction system configured according to the above reaction system
  • reaction tube For the detection of the staining method and the agarose gel electrophoresis determination result, the reaction tube is placed in a constant temperature water bath or other constant temperature equipment, and placed at a constant temperature of 63 ° C for 30 to 60 minutes, then 95 ° C, placed for 2 minutes to inactivate; For the detection of the real-time fluorescence determination result, the reaction tube is placed in a Deaou-308C constant temperature fluorescence detector or other fluorescence detector, and the observation result is kept at a constant temperature of 63 ° C for 30 to 60 minutes;
  • results can be judged by staining (chromogenic method), agarose gel electrophoresis and real-time fluorescence;
  • the sample to be tested in the step S1 the silkworm egg and the silkworm egglet DNA of the silkworm added with the microsporidia of the silkworm (QIAGEN plant small kit):
  • the supernatant in the centrifuge tube was moved to a new tube (do not agitate the residue), 1.5 times the volume of AP3/E was added, and the pipette was mixed; 650 ⁇ L of the mixture was transferred to a silica gel adsorption column, and centrifuged at 4200 rpm for 1 min; Repeat this step for the remaining liquid; place the silica column into the new collection tube.
  • the results show that the primer of the present invention and the kit thereof are capable of detecting 10 3 copies/ ⁇ L of the recombinant plasmid Septin3-DNA-pMD.
  • M is DL2000 DNA Marker
  • 1-7 is: 5.0 ⁇ 10 0 ng / ⁇ L, 5.0 ⁇ 10 -1 ng / ⁇ L, 5.0 ⁇ 10 -2 ng / ⁇ L, 5.0 ⁇ 10 -3 ng / ⁇ L, 5.0 ⁇ 10 -4 ng/ ⁇ L, 5.0 ⁇ 10 -5 ng/ ⁇ L, 5.0 ⁇ 10 -6 ng/ ⁇ L;
  • 8 is a ddH 2 O blank control.
  • the primer of the present invention and the kit thereof were capable of detecting the silkworm egg DNA of the silkworm infected with the microsporidia of Bombyx mori at a concentration of 5.0 ⁇ 10 -3 ng / ⁇ L.
  • M is DL2000DNA Marker
  • No. 1-4 tube is a silkworm egg produced by the silkworm infected with the microsporidia of Bombyx mori, which is infected with 5, 10, 20, and the DNA extracted
  • Silkworm egg DNA 6 is normal silkworm egg DNA
  • 7 is ddH 2 O blank control.
  • the primers and the kits of the invention have excellent detection sensitivity and short time for the microsporidia of the silkworm, and are of great significance for rapid detection of the silkworm microbial disease in the original production.

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Abstract

Cette invention concerne un ensemble amorces de détection LAMP et un kit de réactifs pour identifier une microsporidie chez les œufs de vers à soie. L'ensemble amorces comprend des amorces externes Sep3F3/Sep3B3 et des amorces internes Sep3FIP/Sep3BIP, dont les séquences sont telles que représentées par SEQ ID No : 1-4. L'invention utilise également les amorces pour établir un procédé de détection LAMP et le kit de réactifs pour identifier une microsporidie chez les œufs de vers à soie. Le kit de réactifs comprend l'ensemble amorces, un tampon de réaction 2x, un témoin de référence positif, un témoin de référence négatif, une solution chromogène (ou une solution de coloration fluorescente), l'ADN polymérase Bst, une barrière fluidique, et de l'eau stérile. Pour la détermination, le résultat de la détection peut être observé à l'œil nu sous une lumière naturelle ou observé par électrophorèse sur gel d'agarose ou encore à l'aide de la courbe de fluorescence en temps réel.
PCT/CN2015/088349 2014-10-29 2015-08-28 Ensemble amorces de détection lamp et kit de réactifs pour identifier une microsporidie chez les œufs de vers à soie WO2016065983A1 (fr)

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CN201410592795.XA CN104372081B (zh) 2014-10-29 2014-10-29 一组家蚕蚕卵微孢子虫的lamp检测引物及试剂盒
CN201410592795.X 2014-10-29

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115628961A (zh) * 2022-10-26 2023-01-20 山东大学 一种微孢子虫荧光染色标记方法及应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104372081B (zh) * 2014-10-29 2016-08-17 华南农业大学 一组家蚕蚕卵微孢子虫的lamp检测引物及试剂盒
CN105219875A (zh) * 2015-11-04 2016-01-06 吉林出入境检验检疫局检验检疫技术中心 一种用于鉴定新孢子虫的lamp引物组及其应用
CN105695566A (zh) * 2015-11-06 2016-06-22 华南农业大学 用于检测家蚕蚕卵微孢子虫的lamp引物及快速检测方法
CN105755158A (zh) * 2016-05-12 2016-07-13 福建农林大学 一种东方蜜蜂微孢子虫的lamp检测引物及其检测试剂盒
CN108715905A (zh) * 2018-06-01 2018-10-30 华南农业大学 一组桑花叶型萎缩病的lamp检测引物及试剂盒
CN114277177B (zh) * 2021-12-30 2023-05-16 广西壮族自治区蚕业技术推广站 快速检测家蚕微孢子虫的探针、试剂盒及检测方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009149166A2 (fr) * 2008-06-03 2009-12-10 Children's Hospital Medical Center Procédés et compositions pour le diagnostic et le traitement de troubles prolifératifs
CN103131773A (zh) * 2013-01-24 2013-06-05 华南农业大学 一种家蚕病原微孢子虫lamp可视化快速检测试剂盒及其检测方法
CN104372081A (zh) * 2014-10-29 2015-02-25 华南农业大学 一组家蚕蚕卵微孢子虫的lamp检测引物及试剂盒

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102260737B (zh) * 2011-05-30 2013-01-09 华南农业大学 一组引物及其在家蚕微孢子虫的快速检测方面的应用
CN104017891B (zh) * 2014-06-20 2015-05-20 华南农业大学 septin1基因在检测家蚕微孢子虫中的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009149166A2 (fr) * 2008-06-03 2009-12-10 Children's Hospital Medical Center Procédés et compositions pour le diagnostic et le traitement de troubles prolifératifs
CN103131773A (zh) * 2013-01-24 2013-06-05 华南农业大学 一种家蚕病原微孢子虫lamp可视化快速检测试剂盒及其检测方法
CN104372081A (zh) * 2014-10-29 2015-02-25 华南农业大学 一组家蚕蚕卵微孢子虫的lamp检测引物及试剂盒

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115628961A (zh) * 2022-10-26 2023-01-20 山东大学 一种微孢子虫荧光染色标记方法及应用

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