WO2016065982A1 - 一种家蚕蚕卵微孢子虫的lamp检测引物及其应用 - Google Patents

一种家蚕蚕卵微孢子虫的lamp检测引物及其应用 Download PDF

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WO2016065982A1
WO2016065982A1 PCT/CN2015/088335 CN2015088335W WO2016065982A1 WO 2016065982 A1 WO2016065982 A1 WO 2016065982A1 CN 2015088335 W CN2015088335 W CN 2015088335W WO 2016065982 A1 WO2016065982 A1 WO 2016065982A1
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primers
silkworm
microsporidia
detection
lamp
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French (fr)
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刘吉平
程伟
宋小景
晏育伟
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华南农业大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology. More specifically, it relates to a LAMP detection primer for the microsporidia of the silkworm, Bombyx mori, and an application thereof.
  • 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 micro-sporozoites in the wild can cross-infect the silkworm, and can spread between the silkworms and different silkworms, resulting in the scrapping of a large number of silkworms, seriously restricting the silkworm trade and the sustainable development of the silkworm industry. China has listed silkworm micro-particle disease as Quarantine list of import and export animal quarantine diseases.
  • 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.
  • PCR primers designed using the gene have many steps for detecting the microsporidia of the silkworm, and take a long time, which is not suitable for field detection and the like, and is 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 detection technology of the microsporidia of Bombyx mori, and to design a LAMP detection primer for detecting the microsporidia of the silkworm egg with the EB1 gene of the Bombyx mori, and provide a specific PCR detection method. A simpler and faster method of detection.
  • 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 group of LAMP detection primers for the microsporidia of Bombyx mori, the primer set comprising a pair of lateral primers EB1-F3/EB1-B3 and a pair of inner primers EB1-FIP/EB1-BIP, the primers
  • the nucleotide sequences are shown in SEQ ID NOS: 1-4, respectively.
  • the invention also provides the application of the LAMP detection primer set of the microsporidia of the silkworm, Bombyx mori, in the preparation of a microsporidia detection kit for silkworm eggs.
  • the invention provides a kit for detecting LAMP of Bombyx mori, including the above outer primers EB1-F3/EB1-B3 and the inner primers EB1-FIP/EB1-BIP, wherein the nucleotide sequences of the primers are respectively SEQ ID NO: 1 to 4.
  • the kit further includes 2 x reaction buffer, a positive control, a negative control, a chromogenic solution (or fluorescent stain), 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 an EB1-DNA-pMD recombinant plasmid; the negative control is a normal silkworm DNA.
  • the nucleotide sequences of the primers EB1F and EB1R 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.
  • the reaction system of the kit is:
  • the reaction system of the kit is:
  • the concentration of the primer EB1F3/B3 was 5 pmol/ ⁇ L, and the concentration of the primer EB1FIP/BIP was 20 to 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: constant temperature reaction at 63 ° C for 40 to 90 min; then inoculation at 95 ° C for 2 min.
  • the method for detecting microsporidia of silkworm eggs using the method or 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 results of staining and agarose gel electrophoresis, the reaction tube is placed in a constant temperature water bath or other constant temperature equipment, placed at a constant temperature of 63 ° C for 60 min, then 95 ° C, placed for 2 min inactivation; for real time The detection result of the fluorescence method is detected, and the reaction tube is placed in a Deaou-308C constant temperature fluorescence detector or other fluorescence detector, and the temperature is placed at a constant temperature of 63 ° C for 40 to 90 minutes.
  • 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.
  • PCR primers designed for the target sequence of Bombyx mori EB1 gene can be used to detect microsporidia of silkworm eggs without non-specific strips. Band and false positive results also achieved good detection sensitivity.
  • the designed PCR primers have many steps for detecting the microsporidia of the silkworm, and it takes a long time. On the other hand, we are not satisfied with the specificity and sensitivity of microsporidia detection.
  • 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:
  • EB1-F3 (SEQ ID NO: 1): 5'-GGTCAACAGTAGAAAAGAGT-3'
  • EB1-B3 (SEQ ID NO: 2): 5'-TGCAATTAAAAAGGCTTGAA-3'
  • the above-mentioned LAMP detection primer set for microsporidia detection is based on the application of the EB1 gene of Bombyx mori, and the application of the silkworm egg to the silkworm microsporidia in the early stage, and a large number of The exploration and research not only overcome the defects of common PCR operation and rely on expensive instruments, but more importantly, the biggest innovations of the present invention are: 1) Compared with the ordinary PCR method, the invention is short in time and the detection result is Easier to judge; 2) relative The existing LAMP method is applied to the primers used for the detection of microsporidia, and the primers used in the method directly use the silkworm egg DNA of the silkworm as a template, which not only overcomes the interference caused by the detection of the silkworm egg DNA, and enables the detection. The result is more reliable and the sensitivity is very good. In short, the method is simple in operation, short in detection time, easy to judge and highly specific, and capable of detecting one silkworm egg infected with silkworm microbe disease.
  • the invention discloses a group of LAMP primers for detecting microsporidia of Bombyx mori.
  • the EB1F/1R primer is designed by using the EB1 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 EB1 gene was designed as a target gene to design multiple sets of LAMP primers. According to the design principle of LAMP primers and the position of the amplified sequences of each set of primers, three sets of LAMP primers were screened, 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 primers EB1-F3/EB1-B3 and the inner primers EB1-FIP/EB1-BIP; and at the same time, only four specific primers completely recognized the target gene in six regions.
  • the specificity and detection of the EB1 gene amplification of the microsporidia of Bombyx mori were ensured.
  • 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 10 2 copies/ ⁇ L of the recombinant plasmid EB1-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 40 to 90 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 Adding samples to the configuration reaction system is not In the same room, to reduce the unnecessary contamination of the subsequent experiments after opening the cover; (3) real-time fluorescence method, the results can be judged intuitively through the amplification curve, and the determination of the sample results with high concentration can be terminated early, reducing Unnecessary time wasted, 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.
  • FIG. 2 is a schematic diagram showing the positions of common PCR primers and three primary LAMP primer sets of the present invention on the EB1 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 view showing the results of detection of different concentrations of positive control (recombinant plasmid EB1-DNA-pMD) by the rapid detection method of the microsporidia LAMP of the silkworm, Bombyx mori L. (agarose gel electrophoresis detection result).
  • Fig. 5 is a diagram showing the results of detection of different concentrations of positive control (recombinant plasmid EB1-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 EB1-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).
  • Figure 12 is a visual rapid detection kit for the microsporidia LAMP of the silkworm, Bombyx mori L. Egg test results (real-time fluorescence test results).
  • EB1 gene (Accession number: KF421134.1) sequence of Bombyx mori, which was verified by transcriptome sequencing method and cloned sequencing method, the homology analysis was carried out by BLAST software, and it was found that there is no similarity sequence.
  • the invention designed a set of PCR primers using the gene as a target gene: primers EB1F and EB1R.
  • primer EB1F shown in SEQ ID NO: 5 is:
  • primer EB1R shown in SEQ ID NO: 6 is:
  • the target gene designed with the EB1 gene as a LAMP primer was designed using the online software Primer Explorer V4 ( http://primerexplorer.jp/elamp4.0.0/index.htmL ) to design multiple sets of LAMP primers.
  • Primer group I Four primer sequences are:
  • 2BIP 5'-ATGCAAGATGAAGAAAAAAAGAG-CTTTTTCATTAATGGTCTCT-3'
  • Primer III Four primer sequences are:
  • EB1-F3 (SEQ ID NO: 1): 5'-GGTCAACAGTAGAAAAGAGT-3'
  • EB1-B3 (SEQ ID NO: 2): 5'-TGCAATTAAAAAGGCTTGAA-3'
  • 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.
  • the upstream primer EB1F and the downstream primer EB1R were used as the reaction primers, and the DNA of the microsporidia of the silkworm was used as a template for PCR amplification.
  • the amplified product was cloned into the pMD-19T vector to obtain the EB1-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 .
  • primer set EB1F3/B3 as external primer and EB1FIP/BIP as internal primer
  • EB1F3/B3 concentration was 5pmol/ ⁇ L
  • EB1FIP/BIP concentration was 20 ⁇ 40pmol/ ⁇ L
  • 2 ⁇ reaction buffer positive control, negative control
  • Bst DNA polymerase 8 U / ⁇ L
  • sealing solution coloring solution (10000 ⁇ SYBR Green I
  • fluorescent indicator (1 ⁇ SYBR Green I
  • Bacteria 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 40 to 90 min; then inoculation at 95 ° C for 2 min.
  • the reaction system of the kit is:
  • the reaction system of the kit is:
  • the concentration of the primer EB1F3/B3 was 5 pmol/ ⁇ L, and the concentration of the primer EB1FIP/BIP was 20 to 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 results of staining and agarose gel electrophoresis, the reaction tube is placed in a constant temperature water bath or other constant temperature equipment, placed at a constant temperature of 63 ° C for 60 min, then 95 ° C, placed for 2 min inactivation; for real time The detection result of the fluorescence method is detected, and the reaction tube is placed in a Deaou-308C constant temperature fluorescence detector or other fluorescence detector, and the temperature is placed at a constant temperature of 63 ° C for 40 to 90 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):
  • M is a DL2000 DNA Marker
  • 1-7 is a recombinant plasmid EB1-DNA-pMD diluted 10 6 - 10 0 copies/ ⁇ L, respectively
  • 8 is a ddH 2 O blank control.
  • the results show that the primer of the present invention and the kit thereof are capable of detecting 10 2 copies/ ⁇ L of the recombinant plasmid EB1-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

本发明提供了一种家蚕蚕卵微孢子虫的LAMP检测引物及其应用。所述引物组包括外侧引物EB1-F3/EB1-B3和内侧引物EB1-FIP/EB1-BIP，序列如SEQ ID NO:1-4所示。本发明还利用该引物建立了家蚕蚕卵微孢子虫的LAMP检测方法和试剂盒，所述试剂盒包括上述引物组、2×反应缓冲液、阳性对照品、阴性对照品、显色液（或荧光染色液）、Bst DNA聚合酶、密封液和无菌水。该方法检测的结果可在自然光下肉眼观察或琼脂糖凝胶电泳观察或实时荧光曲线观察判定。

Description

一种家蚕蚕卵微孢子虫的LAMP检测引物及其应用 技术领域
本发明属于生物技术领域。更具体地，涉及一种家蚕蚕卵微孢子虫的LAMP检测引物及其应用。
背景技术
家蚕微粒子病是病原性的家蚕微孢子虫(Nosema bombycis，N.b)经食下传染或胚卵(胎)传染，使家蚕感染发病的一种毁灭性疫病，也是目前影响我国蚕丝业持续稳定发展的重要疫病，每年各地因微粒子病危害而造成的经济损失十分惨重。同时，野外昆虫微孢子虫可交叉感染家蚕，并能在蚕体间以及不同蚕期间传播，导致大批蚕种报废，严重制约蚕种贸易和蚕业可持续发展，我国已将家蚕微粒子病列为进出口动物检疫二类疫病的检疫名录。各地区蚕业主管部门和相关生产单位投入大量人力、物力和财力，采取传统显微镜镜检母蛾，淘汰带毒母蛾产下的蚕种的常规方法进行防治家蚕微粒子病，但效果不佳。
最初人们判别家蚕是否感染家蚕微粒子病主要根据显微镜下组织研磨液中的微孢子虫的形态和大小进行的，这种方法也有效地遏制了世界各地蚕区微粒子病的流行，拯救了世界的养蚕业。而对于家蚕蚕卵微孢子虫的检测主要是通过将产卵24h后的蚕卵浸酸处理，待蚕卵孵化收蚁后进行显微镜检测，这种方法不仅耗费了大量时间，而且显微镜镜检的缺点是对操作人员的技术及经验要求较高，且很难将其他孢子类似物与家蚕微孢子虫孢子区别开来。随着PCR技术的普及，人们开始使用PCR方法来检测家蚕微孢子虫并且达到了较高的灵敏度。目前用于家蚕微粒子病PCR检测的引物多是针对靶基因SSUrRNA的(Terry R S,Smith JE,Bouchon D,et al.Ultrastructural characterization and molecular taxonomy of Nosema granulosis sp.n.,a transovarially transmitted,feminizing(TTF)microsporidium.J.Eukaryot.Microbiol.1999,(46):492-499；Huang Wei-Fone,Tsai Shu-Jen,Lo Chu-Fang,et al.The novel organization and complete sequence of the ribosomal RNA gene of Nosema bombycis.Fungal Genetics and Biology,2004.41(5):473-481)。但是刘吉平等(2004)(刘吉平，曹阳，Smith J.E.等.模拟感染家蚕微粒子病的PCR分子诊断技术研究.中国农业科学，2004，37(12)：1925－1931)研究发现，基于16SrDNA保守区段设计的引物检测蚕卵微孢子虫，经常会有非目的条带及假阳性结果出现，推测蚕卵抽提物中可能存在某种抑制物质干扰了对病原微孢子虫基因组DNA有效扩增。2013年本实验室根据家蚕微孢子虫转录组测序的结果，经测序验证发现了家蚕微孢子虫EB1基因(登录号：KF421134.1)(专利201410279223.6)，经实验验证发现以EB1基因为靶序列设计的PCR引物检测蚕卵微孢子虫并无非特异性条带及假 阳性结果，也达到了很好的检测灵敏度。但是使用该基因设计的PCR引物对于家蚕微孢子虫检测操作步骤较多，且花费时间较长，不适于野外检测等缺点，不利于实际生产上广泛地推广和使用。更大的一个问题是现有公开的检测引物均是以微孢子虫的DNA为模板，但是微孢子虫的分离和收集较复杂，耗时，极不利于大量检测或现场检测。
另一方面，微孢子虫寄生于蚕卵中，且蚕卵含量明显高于要检测的微孢子虫，在提取获得的DNA样品中，两者DNA同时存在，家蚕蚕卵DNA对检测造成严重的干扰，因此，如果想要直接以蚕卵DNA为模板进行微孢子虫的检测，对检测提出了更高的要求。
发明内容
本发明要解决的技术问题是克服现有家蚕微孢子虫检测技术的不足，以家蚕微孢子虫EB1基因为靶基因设计LAMP检测引物用于检测蚕卵微孢子虫，提供一种比PCR检测方法更加简单和快速的检测方法。
本发明的目的是提供一种家蚕蚕卵微孢子虫的LAMP检测引物组。
本发明另一目的是提供所述LAMP检测引物的应用。
本发明再一目的是提供一种利用上述LAMP检测引物组建立的家蚕微孢子虫的检测方法和试剂盒。
本发明上述目的通过以下技术方案实现：
本发明提供了一组家蚕蚕卵微孢子虫的LAMP检测引物组，所述引物组包括一对外侧引物EB1-F3/EB1-B3和一对内侧引物EB1-FIP/EB1-BIP，所述引物的核苷酸序列分别如SEQ ID NO:1～4所示。
本发明还提供所述家蚕蚕卵微孢子虫的LAMP检测引物组在制备家蚕蚕卵微孢子虫检测试剂盒方面的应用。
本发明提供了一种家蚕蚕卵微孢子虫LAMP检测试剂盒，包括上述外侧引物EB1-F3/EB1-B3和内侧引物EB1-FIP/EB1-BIP，所述引物的核苷酸序列分别如SEQ ID NO:1～4所示。
所述试剂盒还包括2×反应缓冲液、阳性对照品、阴性对照品、显色液(或荧光染色液)、Bst DNA聚合酶、密封液和无菌水。
优选地，所述2×反应缓冲液的组分如下：20mM Tris-HCl，pH8.8；10mM KCl；2mM MgSO4；20mM(NH4)2SO4；0.1％Triton X-100；2.8mM dNTPs；1M甜菜碱；25mM MgCl2。
优选地，所述阳性对照品为EB1-DNA-pMD重组质粒；所述阴性对照品为正常家蚕 DNA。
所述EB1-DNA-pMD重组质粒的制备：利用引物EB1F和EB1R，以家蚕微孢子虫DNA为模板进行PCR扩增，扩增产物克隆入pMD-19T载体得到；
所述引物EB1F和EB1R的核苷酸序列如SEQ ID NO:5～6所示。
优选地，所述显色液为10000×SYBR Green I或荧光指示剂1×SYBR Green I。
优选地，所述密封液为甘油。
另外，当检测结果的判定采用染色法或琼脂糖凝胶电泳法时，所述试剂盒的反应体系为：
Figure PCTCN2015088335-appb-000001
当检测结果的判定采用实时荧光法时，所述试剂盒的反应体系为：
Figure PCTCN2015088335-appb-000002
其中，所述引物EB1F3/B3的浓度为5pmol/μL，引物EB1FIP/BIP的浓度为20～40pmol/μL。
建议：使用试剂盒时，对于N个样品，应配制(N+3)倍体积的反应体系(包括阴性对照、阳性对照各一个，以及分装耗费误差)，保证各反应管分装均匀。
所述试剂盒的LAMP反应条件为：63℃恒温反应40～90min；然后95℃，放置2min灭活。
采用本发明的方法或试剂盒检测家蚕蚕卵微孢子虫的方法包含以下步骤：
S1.提取待测样品的模板DNA；
S2.配置反应体系：按照上述反应体系进行配置；
S3.加样：反应体系分装后，分别依次向反应管加入ddH2O(空白对照)、各模板DNA、阳 性对照品、阴性对照品；然后加入20μL密封液(对于染色法判定结果，要在反应管管盖内侧加入1μL显色液，将管盖盖紧，注意避免显色液掉入反应液中)；
S4.反应：对于染色法和琼脂糖凝胶电泳法判定结果的检测，将反应管置于恒温水浴箱或其他恒温设备中，63℃恒温放置60min，然后95℃，放置2min灭活；对于实时荧光法判定结果的检测，将反应管放入Deaou-308C恒温荧光检测仪或其他荧光检测仪中，63℃恒温放置40～90min观察结果；
S5.结果判读：结果判读可以有染色法(显色法)、琼脂糖凝胶电泳法和实时荧光法；
(1)染色法：将反应管管盖内侧的显色液弹入反应管，与反应产物混合；在自然光下通过肉眼观察颜色变化，反应产物颜色变为绿色，说明待测样品含有家蚕蚕卵微孢子虫；反应产物变为橙色，则不含有家蚕蚕卵微孢子虫；
(2)琼脂糖凝胶电泳法：取2μL反应后的产物，与1μL 6×loading buffer和8μL ddH2O混合，于2％琼脂糖凝胶电泳；若出现大小不等的众多梯形条带，说明待测样品含有家蚕蚕卵微孢子虫；若无大小不等的众多梯形条带，说明待测样品不含有家蚕蚕卵微孢子虫；
(3)实时荧光法：若有“S”型扩增曲线且扩增值超过设定的阈值为阳性，若扩增曲线的扩增值未超过设定的阈值则为阴性。
本发明制备试剂盒的阳性对照品所采用的家蚕微孢子虫DNA模板的制备，采用Qiagen公司植物小提试剂盒，方法参照说明书进行。具体地包括以下步骤：
S1.取微孢子虫样品200μL，12,000rpm，离心5min，弃上清；然后加50～100μL ddH2O重悬；
S2.吸取重悬的孢子液至预冷的研钵中，液氮充分研磨(3次以上)；
S3.将研磨后孢子放入1.5mL离心管中，加入400μL裂解缓冲液AP1和4μL RNase A，涡旋混匀(400μL裂解缓冲液AP1和4μL RNase A勿在使用前混合)；
S4.混匀后的溶液，65℃，孵育10min(期间上下颠倒试管2～3次)；
S5.加130μL缓冲液P3，混合后冰浴5min；然后14,000rpm，离心5min；
S6.吸取上清于过滤柱中，14,000rpm，离心2min；
S7.将离心管中上清液移至新的回收管(勿搅动出现的残渣)，加入1.5倍体积的缓冲液AW1，移液器混合；
S8.将650μL混合液移至硅胶吸附柱中，4,200rpm，离心1min；剩下的液体重复此步骤；
S9.将硅胶吸附柱放入新回收管中，加入500μL缓冲液AW2，4,200rpm，离心1min；弃上清；
S10.再加入500μL缓冲液AW2，14,000rpm，离心2min(保证收集管不接触到底部上清)；
S11.移吸附柱至1.5mL或2mL离心管中；
S12.加入40μL洗脱缓冲液AE，室温放置5min；4,200rpm，1min；
S13.重复步骤S12，-20℃冰箱保存备用。
本课题组前期研究发现(专利201410279223.6)，以家蚕微孢子虫EB1基因(登录号：KF421134.1)为靶序列设计的PCR引物可很好的用于检测蚕卵微孢子虫，无非特异性条带及假阳性结果，也达到了很好的检测灵敏度。但是一方面其所设计的PCR引物对于家蚕微孢子虫检测操作步骤较多，且花费时间较长。另一方面我们对于微孢子虫检测的特异性和灵敏性并不满足。
环介导等温扩增法(Loop-mediated isothermal amplification，LAMP)是日本学者Notomi等(2000)发明的一种新型体外等温扩增特异核酸片段的技术。LAMP法具有特异性强、快速、高效、敏感性高、操作简便、检测方法简单等优点，肉眼即可判断结果，从而简化了检测过程，检测时间也大大缩短。其中，对于LAMP检测方法，引物是最关键的一个因素。靶基因序列的选择、引物的设计及选择的好坏直接影响到检测结果的好坏。即使是针对同一种靶基因序列，如何选择合适的六个区域，并设计出合适的四条引物序列，对检测灵敏度具有非常至关重要的作用和很大的意义。本发明人前期就是设计了一系列的多组LAMP引物，又考虑了各种因素并结合实验结果的判断，筛选出3组LAMP引物。进一步地，本发明人最终确定了最优的四条特异性引物，即引物组Ⅲ的四条特异性引物作为LAMP检测用引物组。
所述引物组Ⅲ四条引物序列为：
EB1-F3(SEQ ID NO:1所示)：5'-GGTCAACAGTAGAAAAGAGT-3'
EB1-B3(SEQ ID NO:2所示)：5'-TGCAATTAAAAAGGCTTGAA-3'
EB1-FIP(F1c+F2)(SEQ ID NO:3所示)：
5'-AGCATTTCCTTTCCCTAAATCTTCT-TAGTGAATTGGTTTAATGATCTCGG-3'
EB1-BIP(B1c+B2)(SEQ ID NO:4所示)：
5'-TTCTCAAATCCACCATACTTTCCCT-GATACTCGTATTCATTGGAAGGTT-3'
因此，上述用于微孢子虫检测的LAMP检测引物组是本发明人在前期获得家蚕微孢子虫EB1基因及其在检测家蚕蚕卵是否感染家蚕微孢子虫中的应用的基础上，通过大量的探索和研究得到的，不仅克服了普通PCR操作复杂、依靠昂贵仪器的缺陷，更重要的是，本发明最大的创新点在与：1)相对于普通PCR法，本发明耗时短，检测结果更易判定；2)相对 于现有的LAMP的方法应用于微孢子虫的检测所使用的引物，本方法所用引物直接以家蚕蚕卵DNA为模板，不仅很好的克服了家蚕蚕卵DNA对检测造成的干扰，使检测结果更加可靠，且灵敏度非常好。总之，该方法操作简单、检测耗时短、结果易于判定，特异性强，能够检测感染了家蚕微粒子病的1粒蚕卵。
本发明具有以下有益效果：
本发明公开了一组用于家蚕微孢子虫检测的LAMP引物组，是以EB1基因序列为靶基因设计EB1F/1R引物，使用该PCR引物验证靶基因的特异性，结果表明该引物具有特异性，再以该引物为参考，EB1基因为靶基因设计多组LAMP引物，根据LAMP引物的设计原则以及各组引物的扩增序列位置筛选出3组LAMP引物，然后根据引物的有效性实验进一步筛选出一组即四条特异性引物作为LAMP检测用引物，即外侧引物EB1-F3/EB1-B3和内侧引物EB1-FIP/EB1-BIP；同时在只有四条特异性引物完全识别靶基因的6个区域的情况下，才能进行扩增，从而保证了其对家蚕微孢子虫EB1基因扩增的特异性和检测的全面性。
本发明利用该LAMP引物组进一步建立了微孢子虫的LAMP快速检测方法及试剂盒，以四条LAMP引物、LAMP试剂和显色液或荧光染色液等，构成检测反应体系，在63℃的恒温条件下，快速扩增模板DNA，能够检测浓度为5.0×10-3ng/μL的感染家蚕微孢子虫的家蚕所产的蚕卵DNA，能够检测102拷贝/μL的重组质粒EB1-DNA-pMD，添毒后感染家蚕微孢子虫的家蚕所产的蚕卵1粒时，即可检测到。对微孢子虫的检测灵敏度又提高了一个数量级，这对微孢子虫的检测具有重要的意义。
更重要的是，微孢子虫样品寄生于蚕卵中，且蚕卵含量明显高于要检测的微孢子虫，在提取获得的DNA样品中，两者DNA同时存在，对检测提出了更高的要求。因此，本发明最大的创新点就在与：直接以添毒后感染家蚕微孢子虫的家蚕所产的蚕卵DNA为模板进行微孢子虫检测，省去了微孢子虫分离的复杂耗时步骤，本方法所用引物不仅很好地避免克服了家蚕蚕卵DNA对检测造成的干扰，使检测结果更加可靠，且检测灵敏度非常好。
本发明采用LAMP技术，采用恒温扩增，不需要像PCR仪一样扩增程序复杂且价格昂贵的扩增仪器。本发明对反应体系进行优化，整个反应可在40～90min内完成，大大缩短了检测时间。进一步降低了微孢子虫检测的人力物力成本。
本发明的检测方法对检测结果的判定有多重选择，且均易于观察判定：(1)显色法：在反应产物中加入显色液，通过直观的显色反应，可以在自然光下肉眼观察判读检测结果；(2)琼脂糖凝胶电泳法：根据电泳条带可以更加直观的观察反应结果，且更有说服力，对于假阳性检测结果也可以轻易地排除掉，但是电泳操作前的开盖加样要与配置反应体系在不 同房间进行，以减少开盖后对以后的实验造成不必要的污染；(3)实时荧光法，可以通过扩增曲线直观的判断结果，且对于浓度高的样品结果的判定可以提前结束，减少了不必要的时间浪费，但是仪器价格较为昂贵，对于经费比较充足的实验室或者公司可以使用。
综上所述，本发明所述的家蚕病原微孢子虫的LAMP快速检测方法，操作简单、耗时短、不需要复杂的仪器及复杂的扩增程序、杂质对扩增的干扰较小且反应结果易于判定，特异性强，为应用LAMP技术进行家蚕蚕卵微孢子虫的检测和保证蚕种的质量提供重要基础和技术储备，不仅适合于现场或野外检测，利于实际生产上广泛地推广和使用，还能较好地满足科研院校、蚕种生产单位及蚕种质检中心的检毒需要，易于大范围推广应用。
附图说明
图1为两条PCR引物检测健康蚕卵和添毒蚕卵的电泳图，M：DL 2000DNA Marker；1：添毒家蚕蚕卵DNA；2：家蚕微孢子虫DNA；3：正常家蚕蚕卵DNA；4：ddH2O。
图2为普通PCR引物及本发明初选的三组LAMP引物组在EB1基因上的位置示意图。
图3为本发明的四条LAMP引物在靶基因序列上的位置示意图。
图4为本发明家蚕蚕卵微孢子虫LAMP快速检测法检测不同浓度阳性对照(重组质粒EB1-DNA-pMD)的结果(琼脂糖凝胶电泳法检测结果)。
图5为本发明家蚕蚕卵微孢子虫LAMP快速检测法检测不同浓度阳性对照(重组质粒EB1-DNA-pMD)的结果(显色法检测结果)。
图6为本发明家蚕蚕卵微孢子虫LAMP快速检测法检测不同浓度阳性对照(重组质粒EB1-DNA-pMD)的结果(实时荧光法检测结果)。
图7为本发明家蚕蚕卵微孢子虫LAMP快速检测法检测不同浓度感染家蚕微孢子虫的家蚕所产蚕卵DNA的灵敏性结果(琼脂糖凝胶电泳法检测结果)。
图8为本发明家蚕蚕卵微孢子虫LAMP快速检测法检测不同浓度感染家蚕微孢子虫的家蚕所产蚕卵DNA的灵敏性结果(显色法检测结果)。
图9为本发明家蚕蚕卵微孢子虫LAMP快速检测法检测不同浓度感染家蚕微孢子虫的家蚕所产蚕卵DNA的灵敏性结果(实时荧光法检测结果)。
图10为本发明家蚕蚕卵微孢子虫LAMP可视化快速检测试剂盒检测不同粒数添毒蚕卵的检测结果(琼脂糖凝胶电泳法检测结果)。
图11为本发明家蚕蚕卵微孢子虫LAMP可视化快速检测试剂盒检测不同粒数添毒蚕卵的检测结果(显色法检测结果)。
图12为本发明家蚕蚕卵微孢子虫LAMP可视化快速检测试剂盒检测不同粒数添毒蚕 卵的检测结果(实时荧光法检测结果)。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，本发明所用试剂和材料均为市购。
实施例1引物设计
1、根据实验室通过转录组测序方法并通过克隆测序方法验证的家蚕微孢子虫EB1基因(Accession number：KF421134.1)序列，通过BLAST软件进行同源性分析，发现并无相似性序列，本发明以该基因为靶基因设计了一组PCR引物：引物EB1F和EB1R。
引物EB1F的序列(SEQ ID NO:5所示)为：
5'GGTCAACAGTAGAAAAGAGTTAGTG 3'
引物EB1R的序列(SEQ ID NO:6所示)为：
5'CTTCTTCTAAATCCTCAATTCTCTT 3'
2、使用上述PCR引物检测健康蚕卵和感染了家蚕微孢子虫的蚕卵，结果显示，该引物对于家蚕蚕卵微孢子虫的检测具有特异性(如附图1所示)。
因此基于该PCR引物扩增片段位置，以EB1基因为LAMP引物设计的靶基因，使用在线软件Primer ExplorerV4(http://primerexplorer.jp/elamp4.0.0/index.htmL)，设计多组LAMP引物，根据LAMP引物的设计原则以及各组引物的扩增序列位置筛选出3组LAMP引物，即引物组Ⅰ、引物组Ⅱ和引物组Ⅲ。
(1)引物组Ⅰ四条引物序列为：
1F3：5’-CTTCCAATGAATACGAGTA-3'
1B3：5'-CATCTTGTCTTCTTCTGTT-3'
1FIP：5'-CTCTACTGGAAAATACAATTCAA-AGAATATGAAGATAATTCAAGCC-3'
1BIP：5'-AGAAGTAGCTCAAAAGTTGT-ACTTTACTTTTCCCATAACTCA-3'
(2)引物组Ⅱ四条引物序列为：
2F3：5'-GAGTAAATCTATCTCAGAATGT-3'
2B3：5'-CGCCTTAAATTTACAAATCTCAAGA-3'
2FIP：5'-CAAGCTTCTCGATCAAGTG-CCAAAAGAATCTCCTAAAAAACCG-3'
2BIP：5'-ATGCAAGATGAAGAAAAAAAGAG-CTTTTTCATTAATGGTCTCT-3'
(3)引物组Ⅲ四条引物序列为：
EB1-F3(SEQ ID NO:1所示)：5'-GGTCAACAGTAGAAAAGAGT-3'
EB1-B3(SEQ ID NO:2所示)：5'-TGCAATTAAAAAGGCTTGAA-3'
EB1-FIP(F1c+F2)(SEQ ID NO:3所示)：
5'-AGCATTTCCTTTCCCTAAATCTTCT-TAGTGAATTGGTTTAATGATCTCGG-3'
EB1-BIP(B1c+B2)(SEQ ID NO:4所示)：
5'-TTCTCAAATCCACCATACTTTCCCT-GATACTCGTATTCATTGGAAGGTT-3'
上述普通PCR引物及本发明初选的三组LAMP引物组在EB1基因上的位置示意图如附图2所示。
3、然后根据引物的有效性实验，又考虑了各种因素并结合实验结果的判断，进一步筛选出引物组Ⅲ的四条特异性引物作为LAMP检测用引物，该引物组在靶基因序列上的位置示意图如附图3所示。
实施例2试剂盒制备
1、家蚕微孢子虫DNA模板的制备，采用Qiagen公司植物小提试剂盒，方法参照说明书进行。具体地包括以下步骤：
S1.取微孢子虫样品200μL，12,000rpm，离心5min，弃上清；然后加50～100μL ddH2O重悬；
S2.吸取重悬的孢子液至预冷的研钵中，液氮充分研磨(3次以上)；
S3.将研磨后孢子放入1.5mL离心管中，加入400μL裂解缓冲液AP1和4μL RNase A，涡旋混匀(400μL裂解缓冲液AP1和4μL RNase A勿在使用前混合)；
S4.混匀后的溶液，65℃，孵育10min(期间上下颠倒试管2～3次)；
S5.加130μL缓冲液P3，混合后冰浴5min；然后14,000rpm，离心5min；
S6.吸取上清于过滤柱中，14,000rpm，离心2min；
S7.将离心管中上清液移至新的回收管(勿搅动出现的残渣)，加入1.5倍体积的缓冲液AW1，移液器混合；
S8.将650μL混合液移至硅胶吸附柱中，4,200rpm，离心1min；剩下的液体重复此步骤；
S9.将硅胶吸附柱放入新回收管中，加入500μL缓冲液AW2，4,200rpm，离心1min；弃上清；
S10.再加入500μL缓冲液AW2，14,000rpm，离心2min(保证收集管不接触到底部上清)；
S11.移吸附柱至1.5mL或2mL离心管中；
S12.加入40μL洗脱缓冲液AE，室温放置5min；4,200rpm，1min；
S13.重复步骤S12，-20℃冰箱保存备用。
2、制备阳性和阴性对照品
用上游引物EB1F和下游引物EB1R为反应引物，以家蚕微孢子虫DNA为模板进行PCR扩增，扩增产物克隆入pMD-19T载体获得EB1-DNA-pMD质粒并作为检测的阳性对照品。
提取的正常家蚕DNA作为阴性对照品。
3、其他试剂
(1)2×反应缓冲液
组分如下：20mM Tris-HCl，pH8.8；10mM KCl；2mM MgSO4；20mM(NH4)2SO4；0.1％Triton X-100；2.8mM dNTPs；1M甜菜碱；25mM MgCl2。
(2)显色液10000×SYBR Green I(或荧光指示剂1×SYBR Green I)、Bst DNA聚合酶、密封液甘油和无菌水。
4、试剂盒组装
按照一定的规格，将以下组分进行分装：用引物组EB1F3/B3作为外部引物，EB1FIP/BIP作为内部引物(EB1F3/B3浓度为5pmol/μL，EB1FIP/BIP浓度为20～40pmol/μL)，2×反应缓冲液，阳性对照品，阴性对照品，Bst DNA polymerase(8U/μL)，密封液，显色液(10000×SYBR Green I)；荧光指示剂(1×SYBR Green I)；灭菌ddH2O。即为家蚕蚕卵微孢子虫的LAMP快速检测试剂盒的所有检测试剂。
实施例3试剂盒检测方法
1、所述试剂盒的LAMP反应条件为：63℃恒温反应40～90min；然后95℃，放置2min灭活。
2、当检测结果的判定采用染色法或琼脂糖凝胶电泳法时，所述试剂盒的反应体系为：
Figure PCTCN2015088335-appb-000003
当检测结果的判定采用实时荧光法时，所述试剂盒的反应体系为：
Figure PCTCN2015088335-appb-000004
Figure PCTCN2015088335-appb-000005
其中，所述引物EB1F3/B3的浓度为5pmol/μL，引物EB1FIP/BIP的浓度为20～40pmol/μL。
建议：使用试剂盒时，对于N个样品，应配制(N+3)倍体积的反应体系(包括阴性对照、阳性对照各一个，以及分装耗费误差)，保证各反应管分装均匀。
3、检测步骤如下：
S1.提取待测样品的模板DNA；
S2.配置反应体系：按照上述反应体系进行配置；
S3.加样：反应体系分装后，分别依次向反应管加入2μL的ddH2O(空白对照)、各模板DNA、阳性对照品；然后加入20μL密封液，(对于染色法判定结果，要在反应管管盖内侧加入1μL显色液，将管盖盖紧(注意避免显色液掉入反应液中)；
S4.反应：对于染色法和琼脂糖凝胶电泳法判定结果的检测，将反应管置于恒温水浴箱或其他恒温设备中，63℃恒温放置60min，然后95℃，放置2min灭活；对于实时荧光法判定结果的检测，将反应管放入Deaou-308C恒温荧光检测仪或其他荧光检测仪中，63℃恒温放置40～90min观察结果；
S5.结果判读：结果判读可以有染色法(显色法)、琼脂糖凝胶电泳法和实时荧光法；
(1)染色法：将反应管管盖内侧的显色液弹入反应管，与反应产物混合；在自然光下通过肉眼观察颜色变化，反应产物颜色变为绿色，说明待测样品含有家蚕蚕卵微孢子虫；反应产物变为橙色，则不含有家蚕蚕卵微孢子虫；
(2)琼脂糖凝胶电泳法：取2μL反应后的产物，与1μL 6×loading buffer和8μLddH2O混合，于2％琼脂糖凝胶电泳；若出现大小不等的众多梯形条带，说明待测样品含有家蚕蚕卵微孢子虫；若无大小不等的众多梯形条带，说明待测样品不含有家蚕蚕卵微孢子虫；
(3)实时荧光法：若有“S”型扩增曲线且扩增值超过设定的阈值为阳性，若扩增曲线的扩增值未超过设定的阈值则为阴性。
其中，步骤S1所述待测样品，家蚕蚕卵及添加家蚕微孢子虫的家蚕所产蚕卵模板DNA的制备方法(QIAGEN公司植物小提试剂盒)：
取健康家蚕蚕卵或添食家蚕微孢子虫的家蚕所产蚕卵样品10～50粒，液氮充分研磨(3次 以上)；将研磨后孢子放入1.5mL离心管中，加入400μL裂解缓冲液AP1和4μL RNase A，涡旋混匀(400μL裂解缓冲液AP1和4μL RNase A勿在使用前混合)；混匀后的溶液，65℃，孵育10min(期间上下颠倒试管2～3次)；加130μL缓冲液P3，混合后冰浴5min；然后14,000rpm，离心5min；吸取上清于过滤柱中，14,000rpm，离心2min；将离心管中上清液移至新的回收管(勿搅动出现的残渣)，加入1.5倍体积的缓冲液AW1，移液器混合；将650μL混合液移至硅胶吸附柱中，4,200rpm，离心1min；剩下的液体重复此步骤；将硅胶吸附柱放入新回收管中，加入500μL缓冲液AW2，4,200rpm，离心1min；弃上清；再加入500μL缓冲液AW2，14,000rpm，离心2min(保证收集管不接触到底部上清)；移吸附柱至1.5mL或2mL离心管中；加入40μL洗脱缓冲液AE，室温放置5min；4,200rpm，1min；重复步骤S12，-20℃冰箱保存备用。
实施例4应用试剂盒检测家蚕病原微孢子虫
1、不同浓度阳性对照品检测
利用实施例3的检测方法，对不同浓度阳性对照(重组质粒EB1-DNA-pMD)进行检测，结果如附图4～6所示。图中，M为DL2000DNA Marker；1-7分别为稀释106-100拷贝/μL的重组质粒EB1-DNA-pMD；8为ddH2O空白对照。
结果显示，本发明的引物及其试剂盒能够检测102拷贝/μL的重组质粒EB1-DNA-pMD。
2、检测灵敏度
(1)利用实施例3的检测方法，对不同浓度的感染微孢子虫的家蚕所产蚕卵的DNA进行检测，结果如附图7～9所示。图中，M为DL2000DNA Marker；1-7分别为：5.0×100ng/μL、5.0×10-1ng/μL、5.0×10-2ng/μL、5.0×10-3ng/μL、5.0×10-4ng/μL、5.0×10-5ng/μL、5.0×10-6ng/μL；8为ddH2O空白对照。
结果显示，本发明的引物及其试剂盒能够检测浓度为5.0×10-3ng/μL的感染家蚕微孢子虫的家蚕所产蚕卵DNA。
(2)利用实施例3的检测方法，对不同粒数添毒蚕卵进行检测，结果如附图10～12所示。图中，M为DL2000DNA Marker；1-4号管分别为添毒后感染家蚕微孢子虫的家蚕所产的蚕卵1粒，5粒，10粒，20粒所提的DNA；5为添毒家蚕蚕卵DNA；6为正常家蚕蚕卵DNA；7为ddH2O空白对照。
结果显示，本发明的引物及其试剂盒检测特异性和灵敏性都很优异，针对添毒后感染家蚕微孢子虫的家蚕所产的一粒蚕卵所提的DNA都能够很特异清晰的检测出来。
综上所述，本发明的引物及其试剂盒对家蚕蚕卵微孢子虫的检测灵敏度较优异且耗费时间较短，这对原种生产中家蚕微粒子病的快速检测具有重要的意义。

Claims (10)

  1. 一组家蚕蚕卵微孢子虫的LAMP检测引物组，其特征在于，所述引物组包括一对外侧引物EB1-F3/EB1-B3和一对内侧引物EB1-FIP/EB1-BIP，所述引物的核苷酸序列分别如SEQ ID NO:1～4所示。
  2. 权利要求1所述家蚕蚕卵微孢子虫的LAMP检测引物组在制备家蚕蚕卵微孢子虫检测试剂盒方面的应用。
  3. 一种家蚕蚕卵微孢子虫LAMP检测试剂盒，其特征在于，包括一对外侧引物EB1-F3/EB1-B3和一对内侧引物EB1-FIP/EB1-BIP，所述引物的核苷酸序列分别如SEQ ID NO:1～4所示。
  4. 根据权利要求3所述试剂盒，其特征在于，所述试剂盒还包括2×反应缓冲液、阳性对照品、阴性对照品、显色液或荧光染色液、Bst DNA聚合酶、密封液和无菌水。
  5. 根据权利要求4所述试剂盒，其特征在于，所述2×反应缓冲液的组分如下：20mM Tris-HCl，pH 8.8；10mM KCl；2mM MgSO4；20mM (NH4)2SO4；0.1％ Triton X-100；2.8mM dNTPs；1M甜菜碱；25mM MgCl2。
  6. 根据权利要求4所述试剂盒，其特征在于，所述阳性对照品为EB1-DNA-pMD重组质粒；所述阴性对照品为正常家蚕DNA；
    所述EB1-DNA-pMD重组质粒的制备：利用引物EB1F和EB1R，以家蚕微孢子虫DNA为模板进行PCR扩增，扩增产物克隆入pMD-19T载体得到；
    所述引物EB1F和EB1R的核苷酸序列分别如SEQ ID NO:5～6所示。
  7. 根据权利要求4所述试剂盒，其特征在于，所述显色液为10000×SYBR Green I或者荧光指示剂1×SYBR Green I。
  8. 根据权利要求4所述试剂盒，其特征在于，所述密封液为甘油。
  9. 根据权利要求3～8任一所述试剂盒，其特征在于，所述试剂盒的反应体系如下：
    当检测结果的判定采用染色法或琼脂糖凝胶电泳法时，反应体系为：
    Figure PCTCN2015088335-appb-100001
    当检测结果的判定采用实时荧光法时，反应体系为：
    Figure PCTCN2015088335-appb-100002
    Figure PCTCN2015088335-appb-100003
    其中，所述引物EB1F3/B3的浓度为5pmol/μL，引物EB1FIP/BIP的浓度为20～40pmol/μL。
  10. 根据权利要求3～8任一所述试剂盒，其特征在于，所述试剂盒的LAMP反应条件为：63℃恒温反应40～90min；然后95℃，放置2min灭活。
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