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WO2016115903A1 - 家蚕微孢子虫Met-AP2基因及其应用 - Google Patents

家蚕微孢子虫Met-AP2基因及其应用 Download PDF

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WO2016115903A1
WO2016115903A1 PCT/CN2015/088367 CN2015088367W WO2016115903A1 WO 2016115903 A1 WO2016115903 A1 WO 2016115903A1 CN 2015088367 W CN2015088367 W CN 2015088367W WO 2016115903 A1 WO2016115903 A1 WO 2016115903A1
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microsporidia
seq
primer
gene
detection
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刘吉平
杨思佳
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华南农业大学
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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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    • C12N1/10—Protozoa; Culture media therefor
    • C12N1/105—Protozoal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/90—Protozoa ; Processes using protozoa
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    • 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 the Microsporidium Met-AP2 gene of Bombyx mori and its application.
  • the pathogen of Bombyx mori microsporidia is a class of intracellular obligate parasitic eukaryotes that have been previously classified as protozoa and have been classified as fungi in recent years. Because of its vertical transmission characteristics, it has become the most concerned one among the pathogenic microorganisms of silkworm, and it is also the only legally quarantined disease among silkworm pathogenic microorganisms. From the discovery of the 19th century to the present, the microsporidia of silkworms has caused huge losses to the sericulture industry in various countries.
  • microsporidia can also invade other insects such as bees, aphids, aquatic products such as fish, mammals such as rabbits, dogs, and even humans, causing human keratitis, encephalitis, enteritis and other diseases. . No matter what kind of microsporidia treatment, no good results have been obtained, and accurate detection indicates that microsporidia is a prerequisite for treatment.
  • microsporidia were mainly observed by the naked eye according to the onset characteristics of microparticle disease.
  • microscopic examination based on the morphology and size of the microsporidia increased the sensitivity and efficiency of the detection to a certain extent, and thus curbed the silkworm microparticle disease prevalent in European sericulture countries such as France.
  • microscopic microscopic examination has obvious shortcomings, such as high technical and experience requirements for operators, and microsporidia individuals and their tiny, commonly used microscopic microscopic examination methods have low specificity and sensitivity, and it is difficult to distinguish microsporidia. Things.
  • the “molecular clock” is an effective means of molecular level analysis in the evolution of biological systems.
  • SSU rRNA (16S rDNA) is a "molecular clock” commonly used in microbial evolution studies (Pei A Y, et al., 2010).
  • Most of the target genes targeted by primers designed for PCR detection of silkworm microparticle disease are also SSU rRNA, and primers designed for other microsporidia genes have fewer primers or less sensitivity, and thus have rarely been reported.
  • Microsporidia has a wide host domain and can invade not only various insects, but also water-producing organisms and terrestrial mammals. Microsporidia infects humans, causing diseases such as human keratitis, encephalitis, enteritis, and diarrhea. The same species may also be infected by different species of microsporidia. Therefore, in the actual work, especially the inspection and quarantine work, it is very important to detect the presence of multiple microsporidia pathogens in the sample, and there is no possibility of missed detection. Therefore, it is necessary to find a test that can be used universally. Microsporidia, which has a good detection sensitivity group, has wide application value and significance in the practical detection of microsporidia and microsporidia.
  • the technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing universal detection technology of microsporidia of insects, and to provide a Met-AP2 gene of Bombyx mori, and its application in the detection of microsporidia.
  • Another object of the invention is to provide a set of microsporidia universal detection primers and uses thereof.
  • the present invention obtains the Met-AP2 gene of Bombyx mori, and the nucleotide sequence of the DNA is shown in SEQ ID NO.
  • the nucleotide sequence of its cDNA is shown in SEQ ID NO.
  • the amino acid sequence of the biologically active protein encoded by the M. striata Met-AP2 gene is shown in SEQ ID NO.
  • the invention also provides the application of the Met-AP2 gene of the silkworm, Bombyx mori, in the detection of microsporidia.
  • the Met-AP2 gene was used as a target gene to design primers for PCR amplification, and based on the results of amplification of DNA fragments, it was determined whether the sample contained microsporidia.
  • the present invention also provides a set of universal detection primers for microsporidia, comprising an upstream primer MC-F and a downstream primer MC-R, and the nucleotide sequence of the upstream primer MC-F is as shown in SEQ ID NO. 4, a downstream primer.
  • the nucleotide sequence of MC-R is shown in SEQ ID NO.
  • the above primers are designed with the Met-AP2 gene as a target gene, and have been obtained through a large number of experiments and studies.
  • the primers can be used for the detection of a variety of microsporidia, and have a good detection sensitivity, in the actual microsporidia. It has a wide range of application value and significance in detection applications.
  • the primers are especially suitable for simultaneously detecting a variety of mulberry and common insect microsporidia in the wild, such as the microsporidia, the mulberry microsporidia, the microsporidia, the spores of Spodoptera litura, the microspore of Plutella xylostella. Insects, microsporidia, or microsporidia.
  • the invention also provides a universal detection kit for microsporidia, comprising an upstream primer MC-F and a downstream primer MC-R, the nucleotide sequence of the upstream primer MC-F is as shown in SEQ ID NO. 4, and the downstream primer MC The nucleotide sequence of -R is shown in SEQ ID NO.
  • the kit is used as follows:
  • the PCR reaction was carried out using the primers MC-R and MC-F. After the reaction, the amplified product was detected by gel electrophoresis. According to the result of the amplification product, when the specific agarose gel appeared 1077 bp.
  • the DNA fragment product ie the sample is infected with microsporidia.
  • the PCR reaction system (total volume 20 ⁇ L):
  • reaction buffer 2 ⁇ Taq Master Mix (reaction buffer)
  • the components of 2 ⁇ Taq Master Mix were Taq DNA polymerase, 160 mM Tris-HCl, 40 mM (NH 4 ) 2 SO 4 , 3.0 mM MgCl 2 , 400 ⁇ M dNTP.
  • the PCR reaction procedure was: 94 ° C for 5 min; 94 ° C for 30 s, 53 ° C for 45 s, 72 ° C for 90 s, 32 cycles; 72 ° C for 10 min.
  • the invention discloses the Met-AP2 gene of the microsporidia of Bombyx mori and its application in the detection of microsporidia.
  • a large number of experimental results show that the design of suitable primers according to the gene can not only detect a plurality of microsporidia, but also have a very Good detection sensitivity is a good target for universal detection of microsporidia, and it has practical significance for the detection of microscopic pathogens of other insect sources.
  • the invention also provides a set of universal detection primers for microsporidia, which is a primer designed according to the Met-AP2 gene sequence of the microsporidia of the silkworm, and the primer is used for PCR detection of various microsporidia diseases, and has good versatility and Sensitivity, universal detection of a variety of microsporidia, especially for a variety of mulberry main microsporidia, such as silkworm microsporidia, mulberry microsporidia, mulberry microsporidia, Spodoptera litura microsporidia, Plutella xylostella, microsporidia, or microsporidia, have broad application value and significance in the practical detection of microsporidia.
  • the primers and related reagents of the invention can be assembled into a kit, which is convenient to use and saves time.
  • the sample is microsporidia, microsporidia, mulberry microsporidia, sporozoite, microsporidium, diamondback moth.
  • the larvae can detect positive results, and the screening of the samples is more accurate, and no missed detection occurs.
  • Figure 1 is a schematic diagram of the design of four pairs of primers, 2047-F/2047-R, MC-F/MC-R, M6-F/M6-R, and M7-F/M7-R.
  • Figure 2 shows the results of homology alignment of the protein encoded by the Met-AP2 gene.
  • Figure 3 shows the specific detection results of primer MC-F/MC-R, 1. Microspores, microspores, 2. Microsporidia, 3. Microsporidia, 4. Microsporidia, 5. Plutella xylostella, 6. Sporozoites, Sporozoites, 7. Microsporidium, 8. Microsporidia, 9. Microsporidia, 10. Silkworm midgut, 11.ddH 2 O , M.DL2000Maker.
  • Figure 4 shows the specific detection results of primer 2047-F/2047-R, 1. M. mulberry microsporidia, 2. Maize microsporidia, 3. S. cerevisiae microsporidia, 4. Spodoptera litura microsporidia, 5. Microsporidia, Plutella xylostella, 7. Microsporidia, 7. Microsporidia, 8. Microsporidia, 9. Microsporidia, 10. Microsporidia, 11. Silkworm midgut, 12.ddH 2 O, M.DL1000Maker.
  • Figure 5 shows the specific detection results of the primers M5-F/M5-R and M6-F/M6-R, 1-12 is the specific detection result of the primer M5-F/M5-R, and 13-24 is the primer M6- Specific detection results of F/M6-R; 1/13. Microsporidia mulberry, 2/14. Microsporidia, 3/15. Microsporidia, 4/16. Sporozoite microspore Insect, 5/17. Microsporidia, Plutella xylostella, 6/18. Microsporidia, 7/19. Microspores, Microspores, 8/20. Microsporidia, 9/21. Sporozoites, 10/22. Bombyx mori, 11/23. Bombyx midgut, 12/24.ddH 2 O, M.DL1000Maker.
  • Figure 6 shows the specific detection results of the primers M7-F/M7-R, 1. M. mulberry microsporidia, 2. Maize microsporidia, 3. S. cerevisiae, 3. Spodoptera litura microsporidia, 5. Microsporidia, Plutella xylostella, 7. Microsporidia, 7. Microsporidia, 8. Microsporidia, 9. Microsporidia, 10. Microsporidia, 11. Silkworm midgut, 12.ddH 2 O, M.DL1000Maker.
  • Figure 7 shows the results of detection of the sensitivity of the primer MC-F/MC-R to the microsporidia of the silkworm, 1 to 8 being 1.85 ⁇ 10 1 , 1.85 ⁇ 10 0 , 1.85 ⁇ 10 -1 , 1.85 ⁇ 10 -2 , 1.85 , respectively . ⁇ 10 -3 , 1.85 ⁇ 10 -4 , 1.85 ⁇ 10 -5 , 1.85 ⁇ 10 -6 ⁇ g / mL of Bombyx mori microsporidia DNA, M is DL1000Maker.
  • Figure 8 shows the results of sensitivity detection of primer MC-F/MC-R against Spodoptera litura microsporidia
  • 1 to 8 are 1.21 ⁇ 10 1 , 1.21 ⁇ 10 0 , 1.21 ⁇ 10 -1 , 1.21 ⁇ 10 -2 , respectively .
  • M is DL1000Maker.
  • Figure 9 shows the results of detection of the sensitivity of primer MC-F/MC-R to Microsporidia, which are 1.64 ⁇ 10 1 , 1.64 ⁇ 10 0 , 1.64 ⁇ 10 -1 , 1.64 ⁇ 10 -2 , respectively. 1.64 ⁇ 10 -3 , 1.64 ⁇ 10 -4 , 1.64 ⁇ 10 -5 , 1.64 ⁇ 10 -6 ⁇ g/mL of Microsporidium DNA of M. serrata, and M is DL1000Maker.
  • Figure 10 shows the results of detection of the sensitivity of the primer MC-F/MC-R to the microsporidia of the tussah, which are 2.87 ⁇ 10 1 , 2.87 ⁇ 10 0 , 2.87 ⁇ 10 -1 , 2.87 ⁇ 10 -2 , 2.87 , respectively . ⁇ 10 -3 , 2.87 ⁇ 10 -4 , 2.87 ⁇ 10 -5 , 2.87 ⁇ 10 -6 ⁇ g / mL of the microsporidia DNA of M. sinensis, and M is DL1000Maker.
  • Figure 11 shows the results of detection of the sensitivity of the primer MC-F/MC-R to Microsporidia, which are 1.21 ⁇ 10 1 , 1.21 ⁇ 10 0 , 1.21 ⁇ 10 -1 , and 1.21 ⁇ 10 -2 , respectively. 1.21 ⁇ 10 -3 , 1.21 ⁇ 10 -4 , 1.21 ⁇ 10 -5 , 1.21 ⁇ 10 -6 ⁇ g/mL of microsporidia DNA, and M is DL1000Maker.
  • Figure 12 shows the results of detection of the sensitivity of primer MC-F/MC-R to Microsporidia of Plutella xylostella, which are 1.21 ⁇ 10 1 , 1.21 ⁇ 10 0 , 1.21 ⁇ 10 -1 , and 1.21 ⁇ 10 -2 , respectively. 1.21 ⁇ 10 -3 , 1.21 ⁇ 10 -4 , 1.21 ⁇ 10 -5 , 1.21 ⁇ 10 -6 ⁇ g/mL of microsporidia DNA of Plutella xylostella, and M is DL1000Maker.
  • Figure 13 shows the results of detection of the sensitivity of the primer MC-F/MC-R to M. mulberry, 1-8 are 1.8 ⁇ 10 1 , 1.8 ⁇ 10 0 , 1.8 ⁇ 10 -1 , 1.8 ⁇ 10 -2 , respectively. 1.8 ⁇ 10 -3 , 1.8 ⁇ 10 -4 , 1.8 ⁇ 10 -5 , 1.8 ⁇ 10 -6 ⁇ g/mL of M. sinensis Microsporidia DNA, M is DL1000Maker.
  • Figure 14 shows the results of detection of the sensitivity of the primer 2047-F/2047-R to the microsporidia of the silkworm, which are 1.85 ⁇ 10 1 , 1.85 ⁇ 10 0 , 1.85 ⁇ 10 -1 , 1.85 ⁇ 10 -2 , 1.85 , respectively . ⁇ 10 -3 , 1.85 ⁇ 10 -4 , 1.85 ⁇ 10 -5 , 1.85 ⁇ 10 -6 ⁇ g / mL of Bombyx mori microsporidia DNA, M is DL1000Maker.
  • Figure 15 shows the results of detection of the sensitivity of the primer 2047-F/2047-R against Microsporidia, which are 1.8 ⁇ 10 1 , 1.8 ⁇ 10 0 , 1.8 ⁇ 10 -1 , 1.8 ⁇ 10 -2 , respectively .
  • M is DL1000Maker.
  • Figure 16 shows the results of detection of the sensitivity of primer 2047-F/2047-R against Microsporidia of Plutella xylostella
  • 1 to 8 are 1.21 ⁇ 10 1 , 1.21 ⁇ 10 0 , 1.21 ⁇ 10 -2 , 1.21 ⁇ 10 -3 , respectively .
  • M is DL1000Maker.
  • Figure 17 shows the results of detection of primer 2047-F/2047-R for microsporidia and microsporidia of Spodoptera litura; 1 to 8 are 1.64 ⁇ 10 1 , 1.64 ⁇ 10 0 , 1.64 ⁇ 10 -1 , respectively 1,64 ⁇ 10 -2 , 1.64 ⁇ 10 -3 , 1.64 ⁇ 10 -4 , 1.64 ⁇ 10 -5 , 1.64 ⁇ 10 -6 ⁇ g / mL of microsporidia DNA of S.
  • serrata, 9 to 16 were 1.21 ⁇ 10 1 , 1.21 ⁇ 10 0 , 1.21 ⁇ 10 -1 , 1.21 ⁇ 10 -2 , 1.21 ⁇ 10 -3 , 1.21 ⁇ 10 -4 , 1.21 ⁇ 10 -5 , 1.21 ⁇ 10 -6 ⁇ g / mL of Spodoptera litura microspores Insect DNA, M is DL1000Maker.
  • Figure 18 shows the results of detection of the sensitivity of the primer 2047-F/2047-R to M. mulberry, 1 to 8 being 1.8 ⁇ 10 1 , 1.8 ⁇ 10 0 , 1.8 ⁇ 10 -1 , 1.8 ⁇ 10 -2 , respectively.
  • M is DL1000Maker.
  • Upstream primer MC-F (SEQ ID NO. 4): ATGAGGCCTATTGTTTTATCAGAAG;
  • Downstream primer MC-R (SEQ ID NO. 5): TTAAAAATCATCTCCTTTTGTAAGA.
  • the DNA and cDNA of Bombyx mori were used as templates to carry out PCR amplification with primer MC-F/MC-R.
  • the reaction system and reaction procedure were as follows:
  • the PCR reaction system (total volume 20 ⁇ L):
  • reaction buffer 2 ⁇ Taq Master Mix (reaction buffer)
  • the components of 2 ⁇ Taq Master Mix were Taq DNA polymerase, 160 mM Tris-HCl, 40 mM (NH 4 ) 2 SO 4 , 3.0 mM MgCl 2 , 400 ⁇ M dNTP.
  • the PCR reaction procedure was: 94 ° C for 5 min; 94 ° C for 30 s, 53 ° C for 45 s, 72 ° C for 90 s, 32 cycles; 72 ° C for 10 min.
  • the PCR cloned fragment was ligated to the PMD18-T vector, and then sent to the company (Shanghai Shenggong) for sequencing to obtain the full-length DNA and cDNA sequences of the Met-AP2 gene of Bombyx mori.
  • the DNA sequence of the Met-AP2 gene is shown in SEQ ID NO. 1, and the cDNA sequence of the Met-AP2 gene is shown in SEQ ID NO.
  • the MEGA5 software was used to analyze the homology of other microsporidia genomic genes, and the Met-AP2 gene coding region of Bombyx mori was obtained, which was consistent with the sequence shown in SEQ ID NO.
  • the protein sequence encoded by the Met-AP2 gene is shown in SEQ ID NO. Homology alignment was performed in the microsporidia database, and the alignment results are shown in Fig. 2. The results of BLAST analysis indicated that the Met-AP2 gene of Bombyx mori was not highly homologous to the protein in the library, and it was extremely important for microsporidia, so this protein is a newly discovered gene protein.
  • primer sequences of each group are as follows:
  • Upstream primer MC-F (SEQ ID NO. 4): ATGAGGCCTATTGTTTTATCAGAAG;
  • Downstream primer MC-R (SEQ ID NO. 5): TTAAAAATCATCTCCTTTTGTAAGA.
  • Upstream primer 2047-F (SEQ ID NO. 6): GGAGAAGGGTGATGGAATAG;
  • Downstream primer 2047-R (SEQ ID NO. 7): CGACGGTAGATAACCACATA.
  • Upstream primer M6-F (SEQ ID NO. 8): CCCCTAAATGAGGCC;
  • Downstream primer M6-R (SEQ ID NO. 9): CCTATTCCATCACCCT.
  • Upstream primer M7-F (SEQ ID NO. 10): CCCCTAAATGAGGCC;
  • Downstream primer M7-R (SEQ ID NO. 11): TGGAAGCACGGTAAA.
  • Upstream primer M8-F (SEQ ID NO. 12): TTTTCGCTCCCCTAAA;
  • Downstream primer M8-R (SEQ ID NO. 13): TTCCATCACCCTTCTC.
  • Upstream primer M9-F (SEQ ID NO. 14): TTTTCGCTCCCCTAAA;
  • Downstream primer M9-R (SEQ ID NO. 15): CCTATTCCATCACCCT.
  • Upstream primer M10-F (SEQ ID NO. 16): TTTTCGCTCCCCTAAA;
  • Downstream primer M10-R (SEQ ID NO. 17): TCCATGATTCTCCCAT.
  • Upstream primer M11-F (SEQ ID NO. 18): AAACTTCCCTCTTACAC;
  • Downstream primer M11-R (SEQ ID NO. 19): TCGACGGTAGATAACC.
  • Upstream primer M12-F (SEQ ID NO. 20): GCTTACTATGATGGGTCT;
  • Downstream primer M12-R (SEQ ID NO. 21): TGAACACGGATACTTTA.
  • Upstream primer M13-F (SEQ ID NO. 22): ATCGCCTTGTACTCAT;
  • Downstream primer M13-R (SEQ ID NO. 23): TCGACGGTAGATAACC.
  • Upstream primer M14-F (SEQ ID NO. 24): AAATCGCCTTGTACTCA;
  • Downstream primer M14-R (SEQ ID NO. 25): CGACGGTAGATAACCACAT.
  • Upstream primer M15-F (SEQ ID NO. 26): AGGAACTCTACCCTTTA;
  • Downstream primer M15-R (SEQ ID NO. 27): TGAACACGGATACTTTA.
  • Upstream primer M16-F (SEQ ID NO. 28): AAATCGCCTTGTACTCA;
  • Downstream primer M16-R (SEQ ID NO. 29): TCGACGGTAGATAACC.
  • Upstream primer M17-F (SEQ ID NO. 30): TCTGATAAATCGCCTTGT;
  • Downstream primer M17-R (SEQ ID NO. 31): CGACGGTAGATAACCACAT.
  • the sample DNA was extracted using the Plant DNA Rapid Kit (Qeasy) mini kit (Qeasy), as follows (according to the instructions):
  • a 20 g sample was placed in a mortar, the liquid nitrogen was thoroughly ground, and the ground powder was collected into a 1.5 mL centrifuge tube.
  • the supernatant was transferred to a 1.5 mL or 2 mL centrifuge tube; 40 ⁇ L of buffer AE was added and allowed to stand at room temperature for 5 min; centrifuged at 4200 rpm for 1 min; the previous step was repeated (ie, 40 ⁇ L of buffer AE was added and allowed to stand at room temperature for 5 min, Centrifuge at 4200rpm for 1min ); the extracted total DNA was stored in a refrigerator at -20 ° C for use.
  • PCR amplification was carried out using the above primers.
  • the PCR reaction system (total volume 20 ⁇ L):
  • reaction buffer 2 ⁇ Taq Master Mix (reaction buffer)
  • the components of 2 ⁇ Taq Master Mix were Taq DNA polymerase, 160 mM Tris-HCl, 40 mM (NH 4 ) 2 SO 4 , 3.0 mM MgCl 2 , 400 ⁇ M dNTP.
  • the PCR reaction procedure was: 94 ° C for 5 min; 94 ° C for 30 s, 53 ° C for 45 s, 72 ° C for 90 s, 32 cycles; 72 ° C for 10 min.
  • the product after the end of the PCR reaction was subjected to agarose gel electrophoresis, and it was judged whether or not the sample was infected with the microsporidium, depending on whether or not the target band (DNA fragment) was amplified.
  • the target band can be specifically amplified, it can be judged that the sample is infected with the microsporidia.
  • Example 2 The detection specificity of the 7 pairs of primers screened in Example 2 was studied by the PCR method of Example 2 using the DNA of the larva, the corn borer microsporidium or the megasporozoite of Shandong.
  • MC-F and MC-R detected the most microsporidia species, and simultaneously detected microsporidia, microsporidia, microsporidia, microsporidia, spores, and microspore of Plutella xylostella.
  • the main microsporidia larvae of the larvae, the microsporidia, and the microsporidia are the best versatility, and have a good application prospect in the microsporidia detection of actual samples.
  • Example 2 The detection sensitivity of the 7 pairs of primers screened in Example 2 was studied by the PCR method of Example 2 using the DNA of the larva, the corn borer microsporidium or the megasporozoite of Shandong.
  • the detection sensitivity results of the primer MC-F/MC-R for the seven species of mulberry main microsporidia are shown in Figs. 7 to 13, respectively. Among them, the detection sensitivity of Microsporidia, Sporozoites and Microsporidia was the highest, which could reach 1.64 ⁇ 10 -4 , 1.21 ⁇ 10 -4 and 2.87 ⁇ 10 -4 ⁇ g/mL respectively. The detection sensitivity of Bombyx mori microsporidia can also reach 1.85 ⁇ 10 -2 ⁇ g/mL.
  • Primer 2047-F/2047-R had the best sensitivity to the detection of microsporidia and microsporidia, and reached 1.85 ⁇ 10 -2 and 1.8 ⁇ 10 -1 ⁇ g/mL, respectively.
  • the detection sensitivities of sporozoans and Plutella xylostella were 1.21 ⁇ g/mL, and the detection sensitivities to M. mulberry and M. sinensis were 18 and 16.4 ⁇ g/mL, respectively, as shown in Figures 14-18. Shown.
  • the detection sensitivities of the primers M6-F/M6-R for microsporidia, microsporidia, microsporidia, microsporidia, and microsporidia of Plutella xylostella were 1.21 ⁇ 10 -2 and 1.85, respectively. ⁇ 10 -1, 1.21 ⁇ 10 -1 , 1.64 ⁇ 10 - 1, 12.1 ⁇ g / mL.
  • the detection sensitivities of the primers M7-F/M7-R for Microsporidia, Microsporidium, Microsporidium, and Microsporidia were 2.87 ⁇ 10 -2 , 1.85, 1.21 and 16.4 ⁇ g/mL, respectively. .
  • primers M13-F/M13-R, M16-F/M16-R, M17-F/M17-R, etc. not only have poor detection versatility, but also have poor detection sensitivity, and can detect microsporidia DNA.
  • the concentration is basically greater than 1.0 ⁇ 10 -1 ⁇ g / mL.
  • MC-F and MC-R detected the most microsporidia species, and simultaneously detected microsporidia, microsporidia, microsporidia, sporozoites, spores, and small vegetables.
  • the main microsporidia larvae of the microsporidia, microsporidia and microsporidia are the most versatile, and the detection sensitivity is also the best. In the actual sample microsporidia detection Very good application prospects.

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Abstract

提供了家蚕微孢子虫Met-AP2基因及其在微孢子虫检测方面的应用。还提供了Met-AP2基因为靶基因的微孢子虫通用检测引物对及试剂盒。

Description

家蚕微孢子虫Met-AP2基因及其应用 技术领域
本发明属于生物技术领域。更具体地，涉及家蚕微孢子虫Met-AP2基因及其应用。
背景技术
家蚕微孢子虫病的病原家蚕微孢子虫是一类细胞内专性寄生的真核生物，之前一直属于原虫，近些年被划分到真菌类。因其具有垂直传播的特点使之成为了家蚕病原微生物中最受人关注的一种，也是家蚕病原微生物中唯一法定检疫的疫病。从19世纪发现到现在，家蚕微孢子虫病对各国的养蚕业造成了巨大的损失。除此之外，其他不同的微孢子虫也可侵害其它昆虫如蜜蜂、蝗虫，水产如鱼，哺乳动物如兔、狗，甚至也可以感染到人类，造成人角膜炎、脑炎、肠炎等疾病。不论是在哪种微孢子虫病的治疗上都未取得很好的结果，而准确检测指示微孢子虫是治疗的前提。
最初人们判别微孢子虫主要根据微粒子病的发病特点进行肉眼观察。显微镜发明以后，人们根据微孢子虫的形态和大小进行显微镜镜检，一定程度上提高了检测的灵敏度和效率，并因此遏制了法国等欧洲蚕桑生产国流行的家蚕微粒子病。但是显微镜镜检有明显的缺点，如对操作人员的技术及经验要求较高，而且微孢子虫个体及其微小，常用的显微镜镜检检测方法特异性和灵敏度较低，难以区分微孢子虫类似物。
随着PCR技术的普及，人们开始使用PCR方法来检测微孢子虫并且达到了较高的灵敏度。“分子钟”是分子水平分析生物系统进化中的有效手段，SSU rRNA(16S rDNA)是微生物进化研究中常用的“分子钟”(Pei A Y,et al.,2010)。家蚕微粒子病PCR检测技术研究中所设计的引物针对的靶基因多数也是SSU rRNA，针对其他微孢子虫基因设计的引物较少或灵敏度较差，因而很少被报道。Baker et al(1995)和Terry et al(1999)根据相近种属微孢子虫SSU rRNA高度保守区设计的PCR引物V1f/530r，可鉴别多种种属的微孢子虫的DNA模板扩增约450bp的特异目的条带，但是经常出现假阳性等问题。
微孢子虫寄主域广泛，不仅可侵害各种昆虫，也可侵染水产生物、陆生的哺乳动物。微孢子虫感染人后，造成人角膜炎、脑炎、肠炎以及腹泻等疾病。而同一种物种也可能会被不同种属微孢子虫同时感染。因此，在实际工作中，尤其是检验检疫工作，对于样品中存在多种微孢子虫病原的同时检测是很重要的，且不能出现漏检的情况，因此寻求一种既能够通用的检测多种微孢子虫，又具有很好的检测灵敏性的引物组，在微孢子虫及微孢子虫病的实际检测应用中具有广泛的应用价值和意义。
发明内容
本发明要解决的技术问题是克服现有昆虫微孢子虫通用检测技术的缺陷和不足，提供一种家蚕微孢子虫Met-AP2基因及其在微孢子虫检测方面的应用。
本发明另一目的是提供一组微孢子虫通用检测引物及其应用。
本发明上述目的通过以下技术方案实现：
本发明获得了家蚕微孢子虫Met-AP2基因，其DNA的核苷酸序列如SEQ ID NO.1所示。其cDNA的核苷酸序列如SEQ ID NO.2所示。所述家蚕微孢子虫Met-AP2基因编码的具有生物活性的蛋白的氨基酸序列如SEQ ID NO.3所示。
本发明还提供了所述家蚕微孢子虫Met-AP2基因在微孢子虫检测方面的应用。在获得家蚕微孢子虫Met-AP2基因的基础上，以Met-AP2基因为靶基因设计引物进行PCR扩增，根据扩增DNA片段的结果判定样品中是否含有微孢子虫。
另外，本发明还提供了一组微孢子虫通用检测引物，包括上游引物MC-F和下游引物MC-R，上游引物MC-F的核苷酸序列如SEQ ID NO.4所示，下游引物MC-R的核苷酸序列如SEQ ID NO.5所示。
上述引物是以Met-AP2基因为靶基因设计，经过大量的实验和研究获得的，该引物既能够通用的检测多种微孢子虫，又具有很好的检测灵敏性，在微孢子虫的实际检测应用中具有广泛的应用价值和意义。所述引物尤其适用于同时检测多种桑园及野外常见的昆虫微孢子虫，如家蚕微孢子虫、桑螟微孢子虫、桑尺蠖微孢子虫、斜纹夜蛾微孢子虫、小菜蛾微孢子虫、菜粉蝶微孢子虫或柞蚕微孢子虫等。
本发明还提供了一种微孢子虫通用检测试剂盒，包括上游引物MC-F和下游引物MC-R，上游引物MC-F的核苷酸序列如SEQ ID NO.4所示，下游引物MC-R的核苷酸序列如SEQ ID NO.5所示。
所述试剂盒的使用方法如下：
以样品DNA或cDNA为模板，利用引物MC-R和MC-F进行PCR反应，反应结束后凝胶电泳检测扩增产物，根据扩增产物判定结果，当琼脂糖凝胶上出现特异性地1077bp的DNA片段产物，即该样品感染了微孢子虫。
所述PCR反应体系(总体积20μL)：
2×Taq Master Mix(反应缓冲液)   10μL
10μM上游引物   0.5μL
10μM下游引物   0.5μL
模板DNA           1μL；
ddH2O           补至20μL。
其中，2×Taq Master Mix(反应缓冲液)的组分为Taq DNA聚合酶，160mM Tris-HCl，40mM(NH4)2SO4，3.0mM MgCl2，400μM dNTP。
PCR反应程序为：94℃5min；94℃30s，53℃45s，72℃90s，32个循环；72℃10min。
本发明具有以下有益效果：
本发明公开了家蚕微孢子虫Met-AP2基因及其在微孢子虫检测方面的应用，大量实验结果显示，根据该基因设计合适的引物，既能够通用的检测多种微孢子虫，又具有很好的检测灵敏性，是一个微孢子虫通用检测的良好靶点，对于其他昆虫来源的微粒子病病原检测有现实意义。
本发明还提供了一组微孢子虫通用检测引物，是根据家蚕微孢子虫Met-AP2基因序列设计的引物，该引物用于多种微孢子虫病的PCR检测，具有很好的通用性和灵敏性，能够通用的检测多种微孢子虫，尤其是对多种桑园主要微孢子虫，如家蚕微孢子虫、桑螟微孢子虫、桑尺蠖微孢子虫、斜纹夜蛾微孢子虫、小菜蛾微孢子虫、菜粉蝶微孢子虫或柞蚕微孢子虫等，在微孢子虫的实际检测应用中具有广泛的应用价值和意义。
另外，本发明引物及相关试剂可组装成试剂盒，使用方便，节省时间，当样品被家蚕微孢子虫、桑螟微孢子虫、桑尺蠖微孢子虫、斜纹夜蛾微孢子虫、小菜蛾微孢子虫、菜粉蝶微孢子虫或柞蚕微孢子虫等中的一种或几种感染时，都能够检测出阳性结果，对样品的筛选更加准确，不会出现漏检。
附图说明
图1为2047-F/2047-R、MC-F/MC-R、M6-F/M6-R、M7-F/M7-R四对引物的设计简图。
图2为Met-AP2基因编码蛋白的同源性比对结果。
图3为引物MC-F/MC-R的特异性检测结果，1.浙江小孢子微孢子虫、2.玉米螟微孢子虫、3.桑螟微孢子虫、4.菜粉蝶微孢子虫、5.小菜蛾微孢子虫、6.斜纹夜蛾微孢子虫、7.桑尺蠖微孢子虫、8.柞蚕微孢子虫、9.家蚕微孢子虫、10.家蚕中肠、11.ddH2O、M.DL2000Maker。
图4为引物2047-F/2047-R的特异性检测结果，1.桑螟微孢子虫、2.玉米螟微孢子虫、3.桑尺蠖微孢子虫、4.斜纹夜蛾微孢子虫、5.小菜蛾微孢子虫、6.菜粉蝶微孢子虫、7.浙江小孢子微孢子虫、8.山东大孢子微孢子虫、9.柞蚕微孢子虫、10.家蚕微孢子虫、11.家蚕中肠、 12.ddH2O、M.DL1000Maker。
图5为引物M5-F/M5-R和M6-F/M6-R的特异性检测结果，1～12为引物M5-F/M5-R的特异性检测结果，13～24为引物M6-F/M6-R的特异性检测结果；1/13.桑螟微孢子虫、2/14.玉米螟微孢子虫、3/15.桑尺蠖微孢子虫、4/16.斜纹夜蛾微孢子虫、5/17.小菜蛾微孢子虫、6/18.菜粉蝶微孢子虫、7/19.浙江小孢子微孢子虫、8/20.山东大孢子微孢子虫、9/21.柞蚕微孢子虫、10/22.家蚕微孢子虫、11/23.家蚕中肠、12/24.ddH2O、M.DL1000Maker。
图6为引物M7-F/M7-R的特异性检测结果，1.桑螟微孢子虫、2.玉米螟微孢子虫、3.桑尺蠖微孢子虫、4.斜纹夜蛾微孢子虫、5.小菜蛾微孢子虫、6.菜粉蝶微孢子虫、7.浙江小孢子微孢子虫、8.山东大孢子微孢子虫、9.柞蚕微孢子虫、10.家蚕微孢子虫、11.家蚕中肠、12.ddH2O、M.DL1000Maker。
图7为引物MC-F/MC-R对家蚕微孢子虫的灵敏性检测结果，1～8分别为1.85×101、1.85×100、1.85×10-1、1.85×10-2、1.85×10-3、1.85×10-4、1.85×10-5、1.85×10-6μg/mL的家蚕微孢子虫DNA，M为DL1000Maker。
图8为引物MC-F/MC-R对斜纹夜蛾微孢子虫的灵敏性检测结果，1～8分别为1.21×101、1.21×100、1.21×10-1、1.21×10-2、1.21×10-3、1.21×10-4、1.21×10-5、1.21×10-6μg/mL的斜纹夜蛾微孢子虫DNA，M为DL1000Maker。
图9为引物MC-F/MC-R对桑尺蠖微孢子虫的灵敏性检测结果，1～8分别为1.64×101、1.64×100、1.64×10-1、1.64×10-2、1.64×10-3、1.64×10-4、1.64×10-5、1.64×10-6μg/mL的桑尺蠖微孢子虫DNA，M为DL1000Maker。
图10为引物MC-F/MC-R对柞蚕微孢子虫的灵敏性检测结果，1～8分别为2.87×101、2.87×100、2.87×10-1、2.87×10-2、2.87×10-3、2.87×10-4、2.87×10-5、2.87×10-6μg/mL的柞蚕微孢子虫DNA，M为DL1000Maker。
图11为引物MC-F/MC-R对菜粉蝶微孢子虫的灵敏性检测结果，1～8分别为1.21×101、1.21×100、1.21×10-1、1.21×10-2、1.21×10-3、1.21×10-4、1.21×10-5、1.21×10-6μg/mL的菜粉蝶微孢子虫DNA，M为DL1000Maker。
图12为引物MC-F/MC-R对小菜蛾微孢子虫的灵敏性检测结果，1～8分别为1.21×101、1.21×100、1.21×10-1、1.21×10-2、1.21×10-3、1.21×10-4、1.21×10-5、1.21×10-6μg/mL的小菜蛾微孢子虫DNA，M为DL1000Maker。
图13为引物MC-F/MC-R对桑螟微孢子虫的灵敏性检测结果，1～8分别为1.8×101、1.8×100、1.8×10-1、1.8×10-2、1.8×10-3、1.8×10-4、1.8×10-5、1.8×10-6μg/mL的桑螟 微孢子虫DNA，M为DL1000Maker。
图14为引物2047-F/2047-R对家蚕微孢子虫的灵敏性检测结果，1～8分别为1.85×101、1.85×100、1.85×10-1、1.85×10-2、1.85×10-3、1.85×10-4、1.85×10-5、1.85×10-6μg/mL的家蚕微孢子虫DNA，M为DL1000Maker。
图15为引物2047-F/2047-R对菜粉蝶微孢子虫的灵敏性检测结果，1～8分别为1.8×101、1.8×100、1.8×10-1、1.8×10-2、1.8×10-3、1.8×10-4、1.8×10-5、1.8×10-6μg/mL的菜粉蝶微孢子虫DNA，M为DL1000Maker。
图16为引物2047-F/2047-R对小菜蛾微孢子虫的灵敏性检测结果，1～8分别为1.21×101、1.21×100、1.21×10-2、1.21×10-3、1.21×10-4、1.21×10-5、1.21×10-6、1.21×10-1μg/mL的小菜蛾微孢子虫DNA，M为DL1000Maker。
图17为引物2047-F/2047-R对桑尺蠖微孢子虫和斜纹夜蛾微孢子虫的灵敏性检测结果；1～8分别为1.64×101、1.64×100、1.64×10-1、1.64×10-2、1.64×10-3、1.64×10-4、1.64×10-5、1.64×10-6μg/mL的桑尺蠖微孢子虫DNA，9～16分别为1.21×101、1.21×100、1.21×10-1、1.21×10-2、1.21×10-3、1.21×10-4、1.21×10-5、1.21×10-6μg/mL的斜纹夜蛾微孢子虫DNA，M为DL1000Maker。
图18为引物2047-F/2047-R对桑螟微孢子虫的灵敏性检测结果，1～8分别为1.8×101、1.8×100、1.8×10-1、1.8×10-2、1.8×10-3、1.8×10-4、1.8×10-5、1.8×10-6μg/mL的桑螟微孢子虫DNA，M为DL1000Maker。
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家蚕微孢子虫Met-AP2基因的克隆
1、引物设计
基于基因组分析数据，对其他物种的Met-AP2基因进行分析，并结合各类微孢子虫基因组进行同源性分析，通过引物设计软件Primer5.0设计了一对引物MC-F/MC-R，引物序列如下所示：
上游引物MC-F(SEQ ID NO.4)：ATGAGGCCTATTGTTTTATCAGAAG；
下游引物MC-R(SEQ ID NO.5)：TTAAAAATCATCTCCTTTTGTAAGA。
2、PCR扩增
分别以家蚕微孢子虫的DNA和cDNA为模板，以引物MC-F/MC-R进行PCR扩增，反应体系和反应程序如下：
所述PCR反应体系(总体积20μL)：
2×Taq Master Mix(反应缓冲液)   10μL
10μM上游引物        0.5μL
10μM下游引物          0.5μL
模板DNA            1μL；
ddH2O              补至20μL。
其中，2×Taq Master Mix(反应缓冲液)的组分为Taq DNA聚合酶，160mM Tris-HCl，40mM(NH4)2SO4，3.0mM MgCl2，400μM dNTP。
PCR反应程序为：94℃5min；94℃30s，53℃45s，72℃90s，32个循环；72℃10min。
3、基因克隆
将PCR克隆的片段连接到PMD18-T载体上，然后送去公司(上海生工)进行测序，获得家蚕微孢子虫Met-AP2基因的DNA和cDNA全长序列。Met-AP2基因DNA序列如SEQ ID NO.1所示，Met-AP2基因cDNA序列如SEQ ID NO.2所示。
另外，应用MEGA5软件通过与其他种微孢子虫基因组基因进行同源性分析，得到了家蚕微孢子虫Met-AP2基因序列编码区，与SEQ ID NO.2所示序列一致。
4、蛋白分析
Met-AP2基因编码的蛋白序列如SEQ ID NO.3所示。将其在微孢子虫数据库进行同源性比对，比对结果如图2所示。BLAST分析结果表明，家蚕微孢子虫Met-AP2基因与库中蛋白的同源性并不高，而其对于微孢子虫又极为重要，因此该蛋白是一个新发现的基因蛋白。
实施例2检测引物设计及PCR扩增方法的建立
1、引物设计
(1)在获得家蚕微孢子虫Met-AP2基因的基础上，应用Primer premier 5.0软件设计，设计了14对引物，各组引物序列如下：
上游引物MC-F(SEQ ID NO.4)：ATGAGGCCTATTGTTTTATCAGAAG；
下游引物MC-R(SEQ ID NO.5)：TTAAAAATCATCTCCTTTTGTAAGA。
上游引物2047-F(SEQ ID NO.6)：GGAGAAGGGTGATGGAATAG；
下游引物2047-R(SEQ ID NO.7)：CGACGGTAGATAACCACATA。
上游引物M6-F(SEQ ID NO.8)：CCCCTAAATGAGGCC；
下游引物M6-R(SEQ ID NO.9)：CCTATTCCATCACCCT。
上游引物M7-F(SEQ ID NO.10)：CCCCTAAATGAGGCC；
下游引物M7-R(SEQ ID NO.11)：TGGAAGCACGGTAAA。
上游引物M8-F(SEQ ID NO.12)：TTTCTGCTCCCCTAAA；
下游引物M8-R(SEQ ID NO.13)：TTCCATCACCCTTCTC。
上游引物M9-F(SEQ ID NO.14)：TTTCTGCTCCCCTAAA；
下游引物M9-R(SEQ ID NO.15)：CCTATTCCATCACCCT。
上游引物M10-F(SEQ ID NO.16)：TTTCTGCTCCCCTAAA；
下游引物M10-R(SEQ ID NO.17)：TCCATGATTCTCCCAT。
上游引物M11-F(SEQ ID NO.18)：AAACTTCCCTCTTACAC；
下游引物M11-R(SEQ ID NO.19)：TCGACGGTAGATAACC。
上游引物M12-F(SEQ ID NO.20)：GCTTACTATGATGGGTCT；
下游引物M12-R(SEQ ID NO.21)：TGAACACGGATACTTTA。
上游引物M13-F(SEQ ID NO.22)：ATCGCCTTGTACTCAT；
下游引物M13-R(SEQ ID NO.23)：TCGACGGTAGATAACC。
上游引物M14-F(SEQ ID NO.24)：AAATCGCCTTGTACTCA；
下游引物M14-R(SEQ ID NO.25)：CGACGGTAGATAACCACAT。
上游引物M15-F(SEQ ID NO.26)：AGGAACTCTACCCTTTA；
下游引物M15-R(SEQ ID NO.27)：TGAACACGGATACTTTA。
上游引物M16-F(SEQ ID NO.28)：AAATCGCCTTGTACTCA；
下游引物M16-R(SEQ ID NO.29)：TCGACGGTAGATAACC。
上游引物M17-F(SEQ ID NO.30)：TCTGATAAATCGCCTTGT；
下游引物M17-R(SEQ ID NO.31)：CGACGGTAGATAACCACAT。
(2)通过大量的特异性和灵敏性检测，最终选取了相对较好的7对引物，即MC-F和MC-R、2047-F和2047-R、M6-F和M6-R、M7-F和M7-R、M13-F和M13-R、M16-F和M16-R、M17-F和M17-R。
2、PCR扩增方法的建立
(1)样品总DNA的提取
采用QIAGEN公司生产的植物DNA快捷迷你型抽提试剂盒(DNeasy Plant mini kit试剂盒)抽提样品DNA，步骤如下(按照说明书进行)：
取20g样品放入研钵中，液氮充分研磨，将研磨后的粉末收集至1.5mL的离心管中。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AP2，混合后冰浴5min；然后14,000rpm，离心5min；吸取上清于过滤柱(QIAshredder spin column)中的收集管，14,000rpm，离心2min；将离心管中上清液移至新管(勿搅动出现的残渣)，加入1.5倍体积的AP3/E，移液器混合；将650μL混合液移至吸附柱(DNeasy Mini spin column)中，4200rpm，离心1min；剩下的液体重复此步骤；将吸附柱放入新收集管中，加入500μL缓冲液AW，4200rpm，离心1min；弃上清；再加入500μL缓冲液AW，14000rpm，离心2min(保证收集管不接触到底部上清)；移收集管至1.5mL或2mL离心管中；加入40μL缓冲液AE洗脱，室温放置5min；4200rpm离心1min；重复上一步(即加入40μL缓冲液AE洗脱，室温放置5min，4200rpm离心1min)；将提取好的总DNA置于-20℃冰箱保存备用。
(2)PCR扩增方法
以样品总DNA为模板，用上述引物进行PCR扩增。
所述PCR反应体系(总体积20μL)：
2×Taq Master Mix(反应缓冲液)   10μL
10μM上游引物         0.5μL
10μM下游引物        0.5μL
模板DNA           1μL；
ddH2O               补至20μL。
其中，2×Taq Master Mix(反应缓冲液)的组分为Taq DNA聚合酶，160mM Tris-HCl，40mM(NH4)2SO4，3.0mM MgCl2，400μM dNTP。
PCR反应程序为：94℃5min；94℃30s，53℃45s，72℃90s，32个循环；72℃10min。
(3)结果判断
将PCR反应结束后的产物进行琼脂糖凝胶电泳，根据是否扩增到目的条带(DNA片段)判定样品是否感染了微孢子虫。当能特异性地扩增出目的条带，即可判断该样品感染了微孢子虫。
实施例3引物特异性检测
1、分别以家蚕微孢子虫、桑螟微孢子虫、桑尺蠖微孢子虫、斜纹夜蛾微孢子虫、小菜蛾微孢子虫、菜粉蝶微孢子虫、柞蚕微孢子虫、浙江小孢子微孢子虫、玉米螟微孢子虫或山东大孢子微孢子虫的DNA为模板，利用实施例2的PCR方法，对实施例2筛选的7对引物的检测特异性进行研究。
2、结果显示，只有MC-F和MC-R、2047-F和2047-R、M5-F和M5-R、M6-F和M6-R、M7-F和M7-R这五对引物能够检测到的微孢子虫种类较多，分别能检测到7种、6种、5种、5种、4种微孢子虫。具体如附图3～6所示。
其中，MC-F和MC-R所检测的微孢子虫种类最多，能同时检测到家蚕微孢子虫、桑螟微孢子虫、桑尺蠖微孢子虫、斜纹夜蛾微孢子虫、小菜蛾微孢子虫、菜粉蝶微孢子虫和柞蚕微孢子虫7种常见的桑园主要微孢子虫，检测通用性最好，在实际样品的微孢子虫检测中有很好的应用前景。
实施例4引物灵敏性检测
1、分别以家蚕微孢子虫、桑螟微孢子虫、桑尺蠖微孢子虫、斜纹夜蛾微孢子虫、小菜蛾微孢子虫、菜粉蝶微孢子虫、柞蚕微孢子虫、浙江小孢子微孢子虫、玉米螟微孢子虫或山东大孢子微孢子虫的DNA为模板，利用实施例2的PCR方法，对实施例2筛选的7对引物的检测灵敏性进行研究。
2、检测结果
(1)引物MC-F/MC-R对7种桑园主要微孢子虫的检测灵敏度结果分别如附图7～13所示。其中，对桑尺蠖微孢子虫、斜纹夜蛾微孢子虫和柞蚕微孢子虫的检测灵敏度最高，分别可达到1.64×10-4、1.21×10-4、2.87×10-4μg/mL，对家蚕微孢子虫的检测灵敏度亦可达到1.85×10-2μg/mL。
(2)引物2047-F/2047-R对家蚕微孢子虫和菜粉蝶微孢子虫的检测灵敏度最好，分别达到1.85×10-2、1.8×10-1μg/mL，对斜纹夜蛾微孢子虫和小菜蛾微孢子虫的检测灵敏度均为1.21μg/mL，对桑螟微孢子虫和桑尺蠖微孢子虫的检测灵敏度分别为18、16.4μg/mL，具体分别如附图14～18所示。
(3)引物M5-F/M5-R对家蚕微孢子虫的检测灵敏度最好，为18.5μg/mL。
引物M6-F/M6-R对菜粉蝶微孢子虫、家蚕微孢子虫、斜纹夜蛾微孢子虫、桑尺蠖微孢子虫和小菜蛾微孢子虫的检测灵敏度分别为1.21×10-2、1.85×10-1、1.21×10-1、1.64×10- 1、12.1μg/mL。
引物M7-F/M7-R对柞蚕微孢子虫、家蚕微孢子虫、斜纹夜蛾微孢子虫、桑尺蠖微孢子虫的检测灵敏度分别为2.87×10-2、1.85、1.21、16.4μg/mL。
(4)另外，引物M13-F/M13-R、M16-F/M16-R、M17-F/M17-R等不仅检测通用性差，检测灵敏性也很差，能检测到的微孢子虫DNA的浓度基本都大于1.0×10-1μg/mL。
综上所述，MC-F和MC-R所检测的微孢子虫种类最多，能同时检测到家蚕微孢子虫、桑螟微孢子虫、桑尺蠖微孢子虫、斜纹夜蛾微孢子虫、小菜蛾微孢子虫、菜粉蝶微孢子虫和柞蚕微孢子虫7种常见的桑园主要微孢子虫，检测通用性最好，而且检测灵敏性也最好，在实际样品的微孢子虫检测中有很好的应用前景。

Claims (10)

  1. 家蚕微孢子虫Met-AP2基因，其特征在于，其DNA核苷酸序列如SEQ ID NO.1所示。
  2. 根据权利要求1所述家蚕微孢子虫Met-AP2基因，其特征在于，其cDNA核苷酸序列如SEQ ID NO.2所示。
  3. 权利要求1所述家蚕微孢子虫Met-AP2基因编码的具有生物活性的蛋白，其特征在于，其氨基酸序列如SEQ ID NO.3所示。
  4. 权利要求1所述家蚕微孢子虫Met-AP2基因在微孢子虫检测方面的应用。
  5. 根据权利要求4所述应用，其特征在于，以Met-AP2基因为靶基因设计引物进行PCR扩增，根据扩增DNA片段的结果判定样品中是否含有微孢子虫。
  6. 一组微孢子虫通用检测引物，其特征在于，包括上游引物MC-F和下游引物MC-R，上游引物MC-F的核苷酸序列如SEQ ID NO.4所示，下游引物MC-R的核苷酸序列如SEQ ID NO.5所示。
  7. 权利要求6所述微孢子虫通用检测引物在微孢子虫检测方面的应用。
  8. 根据权利要求4、5或7所述应用，其特征在于，所述微孢子虫为家蚕微孢子虫、桑螟微孢子虫、桑尺蠖微孢子虫、斜纹夜蛾微孢子虫、小菜蛾微孢子虫、菜粉蝶微孢子虫或柞蚕微孢子虫中的一种或几种。
  9. 一种微孢子虫通用检测试剂盒，其特征在于，包括上游引物MC-F和下游引物MC-R，上游引物MC-F的核苷酸序列如SEQ ID NO.4所示，下游引物MC-R的核苷酸序列如SEQ ID NO.5所示。
  10. 根据权利要求9所述试剂盒，其特征在于，所述试剂盒的使用方法如下：
    以样品DNA或cDNA为模板，利用引物MC-F和MC-R进行PCR反应，反应结束后凝胶电泳检测扩增产物，根据扩增产物判定结果，当琼脂糖凝胶上出现特异性1077bp的DNA片段产物，即该样品感染了微孢子虫。
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