WO2016090965A1 - Hmg1基因及其在微孢子虫分子检测中的应用 - Google Patents

Hmg1基因及其在微孢子虫分子检测中的应用 Download PDF

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WO2016090965A1
WO2016090965A1 PCT/CN2015/088443 CN2015088443W WO2016090965A1 WO 2016090965 A1 WO2016090965 A1 WO 2016090965A1 CN 2015088443 W CN2015088443 W CN 2015088443W WO 2016090965 A1 WO2016090965 A1 WO 2016090965A1
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microsporidia
hmg1
primer
silkworm
seq
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French (fr)
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刘吉平
宋小景
程伟
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华南农业大学
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Priority claimed from CN201410755669.1A external-priority patent/CN104498599B/zh
Priority claimed from CN201410755670.4A external-priority patent/CN104498509B/zh
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Priority to US15/310,783 priority Critical patent/US10435759B2/en
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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  • the invention belongs to the technical field of molecular detection of entomopathogenic microorganisms. More specifically, it relates to the HMG1 gene and its use in the detection of microsporidia molecules.
  • silkworm microsomal disease also known as silkworm microsporidia
  • Pasteur determined that the "microparticles" he observed were the cause of microparticle disease, and later Balbiani determined it to be Bombyx mori, Nosema bombycis (JVMaddox et al., 2000).
  • the microsporidia of Bombyx mori has two transmission pathways: horizontal transmission and vertical transmission. Vertical transmission has caused great harm to silkworm production in silkworm production, and has a great negative impact on the yield and quality of silkworm cocoon. Affect the development of the downstream economy of the silk industry chain (Cai Shunfeng et al., 2011).
  • 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 AY, et al., 2010).
  • Most of the target genes targeted by primers designed for PCR detection of silkworm microparticle disease are also SSUrRNA, and the primers designed for other microsporidia genes are less or less sensitive, so they are rarely Report.
  • Microsporidia is 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 at the same time, and the DNA of silkworm eggs has serious interference to the detection. Therefore, If you want to directly use the silkworm egg DNA as a template for the detection of microsporidia, higher requirements are imposed on the detection.
  • the worm has a good detection sensitivity, and it has a very sensitive primer set when the silkworm egg DNA is used as a template. It has wide application value and significance in the practical detection application of microsporidia.
  • the technical problem to be solved by the present invention is to overcome the shortcomings of the existing microsporidia detection technology, and to design a specific detection primer for the microsporidia of the silkworm, and a universal detection primer for the microsporidium, using the HMG1 gene of the silkworm, Bombyx mori.
  • the specific detection primer can be used as a template for silkworm DNA/cDNA, silkworm egg DNA/cDNA or silkworm midgut DNA/cDNA, and the detection result is reliable, easy to operate (simple and rapid), specific and sensitive, and can be used for Rapid detection of microsporidia in Bombyx mori, especially early detection of infection and rapid detection of microsporidia in silkworm eggs.
  • the universal detection primer can simultaneously detect various microsporidia such as the microsporidia, the microsporidia, and the microsporidia of the corn borer, and the detection result is reliable and sensitive.
  • the object of the present invention is to provide a Bombyx mori HMG1 gene.
  • Another object of the present invention is to provide a set of primers for rapidly and specifically detecting Bombyx mori microsporidia using the HMG1 gene of Bombyx mori, and a preparation kit for the microsporidia of Bombyx mori.
  • a further object of the present invention is to provide a set of universal detection primers for microsporidia molecules and a universal detection kit for microsporidia.
  • a Bombyx mori microsporidium HMG1 gene having a full-length DNA sequence as shown in SEQ ID NO. 1 and a full-length cDNA sequence of the cDNA as shown in SEQ ID NO.
  • HMG1 gene sequence As the target gene for detection, and designed specific detection primers and microsporidia of Bombyx mori.
  • General detection primers are as follows:
  • the primer set includes the upstream primer HMG1-sF and the downstream primer HMG1-sR, and the nucleotide sequence of the upstream primer HMG1-sF is SEQ. As shown in ID No. 5, the nucleotide sequence of the downstream primer HMG1-sR is shown in SEQ ID NO.
  • the primers are sensitive, rapid and specific, and the primers can effectively and specifically detect the microsporidia of the silkworm, especially for the early detection of infection and the rapid detection of the microsporidia of the silkworm eggs, which is of great significance.
  • the invention also provides the application of the primer set for rapidly detecting the microsporidia of the silkworm, in the preparation of a microsporidia detection kit for silkworm.
  • a kit for detecting microsporidia of the silkworm including the upstream primer HMG1-sF and the downstream primer HMG1-sR, the nucleotide sequence of the upstream primer HMG1-sF is shown in SEQ ID NO. 5, and the downstream primer HMG1-sR is provided. The nucleotide sequence is shown in SEQ ID NO.
  • the kit is used as follows:
  • the PCR reactions were carried out by using primers HMG1-sF and HMG1-sR. After the reaction, the amplified products were detected by gel electrophoresis. The size of the segment is judged. The criterion for the determination result is that a specific 684 bp DNA fragment product appears on the agarose gel, that is, the silkworm or silkworm egg is infected with the silkworm microsporidia.
  • the reaction system of the PCR reaction is:
  • the components of the 2 ⁇ reaction buffer were Taq DNA polymerase, 160 mM Tris-HCl, 40 mM (NH 4 ) 2 SO 4 , 3.0 mM MgCl 2 , and 400 ⁇ M dNTP.
  • the procedure of the PCR reaction is: 94 ° C for 5 min; 94 ° C for 30 s, 50 ° C for 45 s, 72 ° C for 45 s, 32 cycles; 72 ° C for 10 min.
  • a set of microsporidia universal detection primers including the upstream primer HMG1F And the downstream primer HMG1R, the nucleotide sequence of the upstream primer HMG1F is shown in SEQ ID NO. 3, and the nucleotide sequence of the downstream primer HMG1R is shown in SEQ ID NO.
  • the invention obtains the HMG1 gene of Bombyx mori, the full-length nucleotide sequence of the HMG1 gene is shown in SEQ ID NO. 1, and the full-length cDNA sequence of the cDNA is as shown in SEQ ID NO.
  • the HMG1 gene was used as the target gene, and the above-mentioned microsporidia universal detection primer was designed.
  • the primer can not only detect a variety of microsporidia, but also has good detection sensitivity, and has practical detection application in microsporidia. Wide range of application values and meanings.
  • the primers are particularly suitable for simultaneous detection of Bombyx mori, Microsporidium and Microsporidia.
  • the invention also provides the application of the universal detection primer of the microsporidium in preparing a universal detection kit for microsporidia.
  • a universal detection kit for microsporidia is provided, which comprises an upstream primer HMG1F and a downstream primer HMG1R, a nucleotide sequence of the upstream primer HMG1F is shown in SEQ ID NO. 3, and a nucleotide sequence of the downstream primer HMG1R is SEQ ID. Shown in NO.4.
  • the kit is used as follows:
  • the PCR reaction was carried out by using the primers HMG1F and HMG1R. After the reaction, the amplified product was detected by gel electrophoresis, and the result was determined according to the size of the amplified DNA fragment; the criterion of the determination result was: agarose gel A specific 561 bp DNA fragment product appeared on the sample, i.e., the sample was infected with Bombyx mori.
  • the reaction system of the PCR reaction is:
  • the components of the 2 ⁇ reaction buffer were Taq DNA polymerase, 160 mM Tris-HCl, 40 mM (NH 4 ) 2 SO 4 , 3.0 mM MgCl 2 , and 400 ⁇ M dNTP.
  • the reaction procedure of the PCR reaction is: 94 ° C for 5 min; 94 ° C for 30 s, 58.5 ° C for 45 s, 72 ° C for 45 s, 32 cycles; 72 ° C for 10 min.
  • the present invention clones the full-length DNA and cDNA sequences of the MMG1 gene of Bombyx mori, and on the basis of this, a set of specificity and sensitivity is rapidly obtained to rapidly detect the microsporidia of Bombyx mori.
  • the primer set can be used for PCR detection of microsporidia of silkworm, and can accurately determine whether the sample contains the microsporidia of the silkworm, and can be a "toxic" silkworm egg (ie, a silkworm egg infected with microsporidia). The detection and safety of the seed provides assurance.
  • the invention also designs a set of universal detection primers for microsporidia according to the HMG1 gene, which can be used for PCR detection of microsporosis, has good versatility and sensitivity, and can comprehensively detect various microsporidia. It has good detection sensitivity and has wide application value and significance in the practical detection application of microsporidia.
  • the primers and related reagents of the present invention can be assembled into a kit, which is convenient to use.
  • the applicable PCR amplification template is very diverse, and can be applied to a wide range of samples, such as DNA or cDNA of a sample, and can directly use silkworm egg DNA as a template; wherein the specific detection primer of the microsporidia of the silkworm can also be used for silkworm DNA/cDNA, silkworm egg DNA. /cDNA, silkworm midgut DNA/cDNA, etc. as templates, greatly increasing the range of detection objects.
  • both detection primers and kits of the present invention can be specifically detected in the early stage of microsporidia infection, providing a simple and rapid method for early detection of microsporidia.
  • Figure 1 shows the results of HMG1F/HMG1R primers; lanes: M: DL1000; 1: toxic silkworm egg DNA, 2: purified silkworm microsporidia (Nb) DNA, 3: (normal) non-toxic silkworm egg DNA, 4: ddH 2 O.
  • Figure 2 shows the results of HMG1-sF/HMG1-sR primers; lanes: M: DL1000; 1: venomous silkworm DNA, 2: purified silkworm microsporidia (Nb) DNA, 3: (normal) non-toxic silkworm eggs DNA, 4: ddH 2 O.
  • Figure 3 shows the results of HMG1-xF/HMG1-xR primers; lanes: M: DL1000; 1: toxic silkworm egg DNA, 2: purified silkworm microsporidia (Nb) DNA, 3: (normal) non-toxic silkworm eggs DNA, 4: ddH 2 O.
  • Figure 4 shows the specific detection results of HMG1F/HMG1R primers; lanes: M: DL1000; 1: Nb (Microsporidia) DNA; 2: Na (Microsporidia) DNA; 3: Nf (Maize ⁇ Microsporidium) ) DNA; 4: water control.
  • Figure 5 shows the specific detection results of HMG1-sF/HMG1-sR primers; lanes: M: DL1000; 1: Nb (microsporidia) DNA; 2: Na (microsporidium) DNA; 3: Nf (corn) Microsporidia) DNA; 4: water control.
  • Figure 6 shows the specific detection results of the HMG1-xF/HMG1-xR primers; lane: M: DL1000; 1: N.b (microsporidia) DNA; 2: N.a (microsporidia) DNA; 3: N.f (maize microsporidia) DNA; 4: water control.
  • Figure 7 shows the results of sensitivity detection of HMG1F/HMG1R primers; lanes: M: DL1000; 1 to 7 are 5.0 ⁇ 10 0 , 5.0 ⁇ 10 -1 , 5.0 ⁇ 10 -2 , 5.0 ⁇ 10 -3 , 5.0 ⁇ 10 , respectively -4 , 5.0 x 10 -5 , 5.0 x 10 -6 ng/ ⁇ L of Nb DNA, and 8 is a water control.
  • Figure 8 shows the results of sensitivity detection of HMG1-sF/HMG1-sR primers; lanes: M: DL1000; 1 to 7 are 5.0 ⁇ 10 0 , 5.0 ⁇ 10 -1 , 5.0 ⁇ 10 -2 , 5.0 ⁇ 10 -3 , respectively , 5.0 ⁇ 10 -4 , 5.0 ⁇ 10 -5 , 5.0 ⁇ 10 -6 ng/ ⁇ L of Nb DNA, and 8 is a water control.
  • Figure 9 shows the results of sensitivity detection of HMG1-xF/HMG1-xR primers; lanes: M: DL1000; 1 to 7 are 5.0 ⁇ 10 0 , 5.0 ⁇ 10 -1 , 5.0 ⁇ 10 -2 , 5.0 ⁇ 10 -3 , respectively , 5.0 ⁇ 10 -4 , 5.0 ⁇ 10 -5 , 5.0 ⁇ 10 -6 ng/ ⁇ L of Nb DNA, and 8 is a water control.
  • Figure 10 shows the PCR results of HMG1F/HMG1R primers for Nb-infected 4th instar silkworm cDNA samples at different times; Lanes: M is DL1000; 1:6h; 2:12h; 3:18h; 4:24h; 5:36h;6:48h 7:60h; 8:72h; 9:84h; 10:96h; 11:108h; 12: purified Nb cDNA; 13: normal silkworm midgut cDNA; 14: ddH 2 O.
  • Figure 11 shows the PCR results of HMG1F/HMG1R primers for cDNA templates of Nb-infected silkworm eggs (before pickling) at different times; Lanes: M is DL1000; 1:2h; 2:8h; 3:10h; 4:12h; 5:17h ; 6: cDNA of purified Nb spores; 7: normal silkworm eggs cDNA; 8: ddH 2 O.
  • Figure 12 shows the PCR results of HMG1F/HMG1R primers on cDNA templates of Nb-infected silkworm eggs (not pickled) at different times; Lanes: M is DL1000; 1:24h; 2:48h; 3:72h; 4:96h; 5:120h ; 6: 144h; 7: 168h; 8: 192h; 9: 216h; 10: 240h; 11: purified Nb cDNA; 12: normal silkworm egg cDNA; 13: ddH 2 O.
  • Figure 13 shows the PCR results of HMG1F/HMG1R primers for cDNA templates of Nb-infected silkworm eggs (implanted acid) at different times; Lanes: M is DL1000; 1:24h; 2:48h; 3:72h; 4:96h; 5:120h; 6: 144h; 7: 168h; 8: 192h; 9: 216h; 10: 240h; 11: purified Nb cDNA; 12: normal silkworm egg cDNA; 13: ddH 2 O.
  • the primer primers HMG1F/HMG1R were designed using Primer premier 5.0 software combined with comprehensive analysis, and the sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
  • Upstream primer HMG1F (SEQ ID NO. 3):
  • Downstream primer HMG1R (SEQ ID NO. 4):
  • the full-length DNA sequence of the MMG 1 gene of Bombyx mori was obtained by genomic high-throughput sequencing and after a large number of gene prediction and comparison analysis, and finally confirmed by PCR sequencing, as shown in SEQ ID NO.
  • Upstream primer HMG1F (SEQ ID NO. 3):
  • Downstream primer HMG1R (SEQ ID NO. 4):
  • Upstream primer HMG1-sF (SEQ ID NO. 5):
  • Upstream primer HMG1-xF (SEQ ID NO. 7):
  • Downstream primer HMG1-xR (SEQ ID NO. 8):
  • the genomic DNA of silkworm eggs was extracted using the DNeasy Plant mini kit from QIAGEN. The procedure is as follows (according to the instructions):
  • 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 total DNA extracted is placed at -20 ° C The refrigerator is kept for use.
  • 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, 50 ° C for 45 s, 72 ° C for 45 s, 32 cycles; 72 ° C for 10 min.
  • the first pair of primers HMG1F/HMG1R After the end of the PCR reaction, agarose gel electrophoresis was carried out, and it was determined whether or not the silkworm egg sample was infected with the microsporidia of the silkworm according to whether or not the 561 bp DNA fragment was amplified. When the 561 bp DNA fragment product can be specifically amplified, it can be judged that the silkworm or silkworm egg is infected with the silkworm microsporidia.
  • the second pair of primers HMG1-sF/HMG1-sR After the end of the PCR reaction, agarose gel electrophoresis was carried out, and whether the silkworm egg sample was infected with the microsporidia was determined according to whether or not the 684 bp DNA fragment was amplified. When the 684 bp DNA fragment product can be specifically amplified, it can be judged that the silkworm or silkworm egg is infected with the silkworm microsporidia.
  • the third pair of primers HMG1-xF/HMG1-xR After the end of the PCR reaction, agarose gel electrophoresis was carried out, and whether the silkworm egg sample was infected with the microsporidia was determined according to whether or not the 251 bp DNA fragment was amplified. When the 251 bp DNA fragment product can be specifically amplified, it can be judged that the silkworm or silkworm egg is infected with the silkworm microsporidia.
  • the DNA of the microsporidia (Nb), the microsporidia (Na), and the microsporidia (Nf) of maize were used as templates, with primers HMG1F/HMG1R, HMG1-sF/HMG1-sR, HMG1-xF. /HMG1-xR, PCR amplification was carried out by the method of Example 2, and the results of agarose gel electrophoresis were measured after the amplification.
  • the above Nb DNA was diluted with ddH 2 O, and diluted 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , and 10 6 times, respectively. That is, the concentration gradients were 5.0 ⁇ 10 0 , 5.0 ⁇ 10 -1 , 5.0 ⁇ 10 -2 , 5.0 ⁇ 10 -3 , 5.0 ⁇ 10 -4 , 5.0 ⁇ 10 -5 , and 5.0 ⁇ 10 -6 ng / ⁇ L.
  • the amplification results of the three pairs of primers are shown in Figures 7-9, respectively.
  • the results showed that the primers HMG1F/HMG1R and the primers HMG1-sF/HMG1-sR were able to detect Nb DNA at a concentration of 5.0 ⁇ 10 -4 ng/ ⁇ L, which has good detection sensitivity.
  • the sensitivity of the primers HMG1-xF/HMG1-xR was one order of magnitude, and Nb DNA at a concentration of 5.0 ⁇ 10 -3 ng/ ⁇ L was detected.
  • the primers HMG1-sF/HMG1-sR can specifically detect the microsporidia of Bombyx mori, and have good detection sensitivity, especially for detecting silkworms.
  • the microsporidia of the silkworm silkworm provides a guarantee for the detection and safe seeding of the "toxic" silkworm species in the silkworm production.
  • the experimental results also show that the HMG1 gene is a good test for silkworm micro Molecular targets of sporozoans.
  • primers HMG1F/HMG1R can not only detect a variety of microsporidia, but also have good detection sensitivity, and have wide application value and significance in the practical detection applications of various microsporidia.
  • Example 5 Detection of silkworm eggs in the midgut of four-year-old silkworm infected with N.b at different time intervals and infected with N.b
  • RNA is reverse transcribed into cDNA
  • Genomic DNA reaction conditions were removed: 42 ° C, 2 min (or room temperature 5 min for up to 30 min).
  • Reverse transcription reaction conditions 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C / -20 ° C for storage.
  • the RT-PCR reaction was carried out using the specific primer HMG1F/HMGR.
  • the results of the reaction are shown in Fig. 10.
  • the HMG1 gene was not detected in the first 12 hours of Nb infection in the midgut of the silkworm, indicating that the microsporidia did not start to multiply, and the HMG1 gene was transcriptionally active from 18 h, at this time, the microspore The worm may have begun to divide and multiply. This conclusion is consistent with the life history of the silkworm microsporidia infecting the silkworm in the prior art, and it is also confirmed from the side that the detection primer of the present invention has good specificity and sensitivity.
  • Nb-infected silkworm eggs and normal silkworm eggs were used as test objects, respectively.
  • the cDNA of silkworm eggs at different times after Nb infection without acid pickling, before pickling, and after pickling were used as templates to normal silkworms.
  • the silkworm egg DNA was used as a control, and the HMG1F/HMG1R primer was used for PCR detection.
  • pickling treatment divide an egg ring into two parts on average, about 20h after spawning, pickling acid (specific gravity of 1.075), pickling conditions: picking acid for 5min at 46°C, rinsing with water for 20min, and placing at temperature Incubate in an artificial climate incubator at 25 ° C and 85% humidity to the ant silkworm.
  • Figure 11 shows the PCR results of HMG1F/HMG1R primers for cDNA templates of Nb-infected silkworm eggs (before pickling) at different times; Lanes: M is DL1000; 1:2h; 2:8h; 3:10h; 4:12h; 5:17h ; 6: cDNA of purified Nb spores; 7: normal silkworm eggs cDNA; 8: ddH 2 O.
  • Figure 12 shows the PCR results of HMG1F/HMG1R primers on cDNA templates of Nb-infected silkworm eggs (not pickled) at different times; Lanes: M is DL1000; 1:24h; 2:48h; 3:72h; 4:96h; 5:120h ; 6: 144h; 7: 168h; 8: 192h; 9: 216h; 10: 240h; 11: purified Nb cDNA; 12: normal silkworm egg cDNA; 13: ddH 2 O.
  • Figure 13 shows the PCR results of HMG1F/HMG1R primers for cDNA templates of Nb-infected silkworm eggs (implanted acid) at different times; Lanes: M is DL1000; 1:24h; 2:48h; 3:72h; 4:96h; 5:120h; 6: 144h; 7: 168h; 8: 192h; 9: 216h; 10: 240h; 11: purified Nb cDNA; 12: normal silkworm egg cDNA; 13: ddH 2 O.
  • the results of the tests in Figures 11 to 13 indicate that the HMG1 gene is transcribed at 2 h after silkworm eggs are produced, and the HMG1 gene is transcribed throughout the development of silkworm eggs (acid picking and non-leaching). Activity, so the HMG1 gene can be used as a molecular target to detect whether early silkworm eggs are infected with Bombyx mori.
  • the detection primers and kits of the present invention can accurately determine whether the sample contains the microsporidia of the silkworm, especially the microsporidia of the silkworm, and detect and safely produce the "toxic" silkworm species in the silkworm production.
  • the experimental results also show that the HMG1 gene is a good molecular target for the detection of microsporidia.

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Abstract

本发明公开了家蚕微孢子虫HMG1基因，以及一对特异性的快速检测家蚕微孢子虫的引物组和一组微孢子虫通用检测引物及其应用。所述引物组为引物HMG1-sF和HMG1-sR，核苷酸序列分别如SEQ ID NO.5～6所示。所述通用检测引物包括引物HMG1F和HMG1R，核苷酸序列分别如SEQ ID NO.3～4所示。

Description

HMG1基因及其在微孢子虫分子检测中的应用 技术领域
本发明属于昆虫病原微生物分子检测技术领域。更具体地，涉及HMG1基因及其在微孢子虫分子检测中的应用。
背景技术
家蚕微粒子病(又名家蚕微孢子虫病)的研究始于1845年发生在法国的全国流行微粒子病，巴斯德确定他观察到的“微粒”是微粒子病的成因因素，后来Balbiani确定其为家蚕微孢子虫Nosema bombycis(J.V.Maddox等，2000)。家蚕微孢子虫有水平传播和垂直传播两种传染途径，其中垂直传播对蚕业生产中的蚕种生产造成巨大的危害，同时对丝茧育蚕茧产量和品质带来很大的负面影响，严重影响蚕丝产业链下游经济的发展(蔡顺风等，2011)。自法国全国微粒子病爆发以来，家蚕微孢子虫始终是各国蚕种生产和进出口贸易的重要检测对象。19世纪末，在日本养蚕业最发达的时期，《母蛾镜检法》、《蚕病预防法》曾被写入宪法(Takeshi Kawarabata，2003)，我国也将其列为进出口动物检疫二类疫病[《中华人民共和国进境动物一、二类传染病、寄生虫病名录》，(1992)农(检疫)字第12号]。
最初人们判别家蚕微孢子虫主要根据家蚕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，并因此遏制了法国全国流行的微粒子病，拯救了世界的养蚕业。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，而且家蚕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，特别是蚕卵中的家蚕微孢子虫一般都是未成熟的孢子，普通光学显微镜根本无法鉴别判定等。
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。“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 AY,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SSUrRNA，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，一方面其对家蚕微孢子虫不具有特异性，另一方面，本发明人研究发现，使用该引物检测蚕卵微孢子虫，检测的灵敏度极低，暗示蚕卵抽提物中可能存在某种抑制因素干扰了对家蚕微孢子虫(N.b)DNA的PCR扩增。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。
另外，在实际工作中，尤其是检疫工作，对于样品的各种微孢子虫病原的检测是很重要的，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，且以蚕卵DNA为模板时也具有很好的灵敏性的引物组，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。
发明内容
本发明要解决的技术问题是克服现有微孢子虫检测技术的不足，以家蚕微孢子虫HMG1基因为靶基因，设计家蚕微孢子虫的特异检测引物以及多种微孢子虫的通用检测引物。所述特异检测引物可以以家蚕DNA/cDNA、家蚕蚕卵DNA/cDNA或家蚕中肠DNA/cDNA等为模板，并且检测结果可靠、易于操作(简单快速)、特异性强、灵敏度高，可用于家蚕微孢子虫的快速检测，尤其是侵染早期检测和蚕卵中家蚕微孢子虫的快速检测。所述通用检测引物可以同时检测出样品中存在的家蚕微孢子虫、柞蚕微孢子虫和玉米螟微孢子虫等多种微孢子虫，而且检测结果可靠、灵敏度高。
本发明的目的是提供一种家蚕微孢子虫HMG1基因。
本发明另一目的是提供一组以家蚕微孢子虫HMG1基因为靶基因、快速特异检测家蚕微孢子虫的引物组以及制备的家蚕微孢子虫检测试剂盒。
本发明的再一目的是提供一组微孢子虫分子通用检测引物以及微孢子虫通用检测试剂盒。
本发明上述目的通过以下技术方案实现：
一种家蚕微孢子虫HMG1基因，其DNA全长核苷酸序列如SEQ ID NO.1所示，其cDNA全长核苷酸序列如SEQ ID NO.2所示。
本发明人基于对家蚕微孢子虫HMG1基因的研究实验和针对性分析总结，选择该HMG1基因序列作为检测的靶基因，分别设计出了家蚕微孢子虫的特异检测引物和多种微孢子虫的通用检测引物，具体如下：
首先，一组以HMG1基因为靶基因的快速检测家蚕微孢子虫的引物组，所述引物组包括上游引物HMG1-sF和下游引物HMG1-sR，上游引物HMG1-sF的核苷酸序列如SEQ ID NO.5所示，下游引物HMG1-sR的核苷酸序列如SEQ ID NO.6所示。所述引物灵敏、快速、特异性高，依据本引物可有效地特异地检测家蚕微孢子虫，尤其是对侵染早期的检测和蚕卵中家蚕微孢子虫的快速检测，具有重要的意义。
本发明还提供所述快速检测家蚕微孢子虫的引物组在制备家蚕微孢子虫检测试剂盒方面的应用。并提供了一种家蚕微孢子虫检测试剂盒，包括上游引物HMG1-sF和下游引物HMG1-sR，上游引物HMG1-sF的核苷酸序列如SEQ IDNO.5所示，下游引物HMG1-sR的核苷酸序列如SEQ ID NO.6所示。
优选地，所述试剂盒的使用方法如下：
以家蚕DNA/cDNA、家蚕蚕卵DNA/cDNA或家蚕中肠DNA/cDNA为模板，利用引物HMG1-sF和HMG1-sR进行PCR反应，反应结束后凝胶电泳检测扩增产物，根据扩增DNA片段的大小判定结果。所述判定结果的标准为：琼脂糖凝胶上出现特异性地684bp的DNA片段产物，即该家蚕或蚕卵感染了家蚕微孢子虫。
优选地，所述PCR反应的反应体系为：
Figure PCTCN2015088443-appb-000001
其中，2×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。
优选地，所述PCR反应的程序为：94℃5min；94℃30s，50℃45s，72℃45s，32个循环；72℃10min。
其次，一组微孢子虫通用检测引物，所述通用检测引物包括上游引物HMG1F 和下游引物HMG1R，上游引物HMG1F的核苷酸序列如SEQ ID NO.3所示，下游引物HMG1R的核苷酸序列如SEQ ID NO.4所示。
本发明在获得了家蚕微孢子虫HMG1基因的基础上(HMG1基因DNA全长核苷酸序列如SEQ ID NO.1所示，其cDNA全长核苷酸序列如SEQ ID NO.2所示)，以HMG1基因为靶基因，设计了上述微孢子虫通用检测引物，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家蚕微孢子虫、柞蚕微孢子虫和玉米螟微孢子虫。
本发明还提供了所述微孢子虫通用检测引物在制备微孢子虫通用检测试剂盒方面的应用。并提供了一种微孢子虫通用检测试剂盒，包括上游引物HMG1F和下游引物HMG1R，上游引物HMG1F的核苷酸序列如SEQ ID NO.3所示，下游引物HMG1R的核苷酸序列如SEQ ID NO.4所示。
优选地，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HMG1F和HMG1R进行PCR反应，反应结束后凝胶电泳检测扩增产物，根据扩增DNA片段的大小判定结果；所述判定结果的标准为：琼脂糖凝胶上出现特异性地561bp的DNA片段产物，即该样品感染了家蚕微孢子虫。
优选地，所述PCR反应的反应体系为：
Figure PCTCN2015088443-appb-000002
其中，2×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。
优选地，所述PCR反应的反应程序为：94℃5min；94℃30s，58.5℃45s，72℃45s，32个循环；72℃10min。
本发明具有以下有益效果：
本发明首次克隆了家蚕微孢子虫HMG1基因的DNA和cDNA全长序列，并在此基础上，设计获得了一组特异性、灵敏性非常好的快速检测家蚕微孢子虫的 引物组，该引物可用于家蚕微孢子虫病的PCR检测，能够准确地判断样品是否含有家蚕微孢子虫，且可为蚕种生产中“有毒”蚕卵(即感染微孢子虫的蚕卵)的检测及安全发种提供保证。
本发明还根据HMG1基因设计出了一组微孢子虫通用检测引物，该引物可用于微孢子虫病的PCR检测，具有很好的通用性和灵敏性，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。
另外，本发明引物及相关试剂可组装成试剂盒，使用方便。而且适用的PCR扩增模板非常多样，适用范围广，可以是样品的DNA或cDNA，可以直接以蚕卵DNA为模板；其中家蚕微孢子虫特异检测引物还可以家蚕DNA/cDNA、家蚕蚕卵DNA/cDNA、家蚕中肠DNA/cDNA等为模板，大大增加了检测对象的范围。
更重要的是，本发明的两种检测引物和试剂盒均能够在微孢子虫侵染早期就能够特异性的检测出来，为微孢子虫病的早期检测提供了一种简单快速的方法。
附图说明
图1为HMG1F/HMG1R引物的检测结果；泳道：M:DL1000；1：有毒蚕卵DNA，2：纯化的家蚕微孢子虫(N.b)DNA，3：(正常)无毒蚕卵DNA，4：ddH2O。
图2为HMG1-sF/HMG1-sR引物的检测结果；泳道：M:DL1000；1：有毒蚕卵DNA，2：纯化的家蚕微孢子虫(N.b)DNA，3：(正常)无毒蚕卵DNA，4：ddH2O。
图3为HMG1-xF/HMG1-xR引物的检测结果；泳道：M:DL1000；1：有毒蚕卵DNA，2：纯化的家蚕微孢子虫(N.b)DNA，3：(正常)无毒蚕卵DNA，4：ddH2O。
图4为HMG1F/HMG1R引物的特异性检测结果；泳道：M：DL1000；1：N.b(家蚕微孢子虫)DNA；2：N.a(柞蚕微孢子虫)DNA；3：N.f(玉米螟微孢子虫)DNA；4：水对照。
图5为HMG1-sF/HMG1-sR引物的特异性检测结果；泳道：M：DL1000；1：N.b(家蚕微孢子虫)DNA；2：N.a(柞蚕微孢子虫)DNA；3：N.f(玉米螟微孢子虫)DNA；4：水对照。
图6为HMG1-xF/HMG1-xR引物的特异性检测结果；泳道：M：DL1000； 1：N.b(家蚕微孢子虫)DNA；2：N.a(柞蚕微孢子虫)DNA；3：N.f(玉米螟微孢子虫)DNA；4：水对照。
图7为HMG1F/HMG1R引物的灵敏性检测结果；泳道：M：DL1000；1～7分别为5.0×100、5.0×10-1、5.0×10-2、5.0×10-3、5.0×10-4、5.0×10-5、5.0×10-6ng/μL的N.b DNA，8为水对照。
图8为HMG1-sF/HMG1-sR引物的灵敏性检测结果；泳道：M：DL1000；1～7分别为5.0×100、5.0×10-1、5.0×10-2、5.0×10-3、5.0×10-4、5.0×10-5、5.0×10-6ng/μL的N.b DNA，8为水对照。
图9为HMG1-xF/HMG1-xR引物的灵敏性检测结果；泳道：M：DL1000；1～7分别为5.0×100、5.0×10-1、5.0×10-2、5.0×10-3、5.0×10-4、5.0×10-5、5.0×10-6ng/μL的N.b DNA，8为水对照。
图10为HMG1F/HMG1R引物对N.b感染四龄蚕不同时间cDNA模板的PCR结果；泳道：M为DL1000；1：6h；2：12h；3：18h；4：24h；5：36h；6：48h；7：60h；8：72h；9：84h；10：96h；11：108h；12：纯化的N.b cDNA；13：正常家蚕中肠cDNA；14：ddH2O。
图11为HMG1F/HMG1R引物对N.b感染蚕卵(浸酸前)不同时间cDNA模板的PCR结果；泳道：M为DL1000；1：2h；2：8h；3：10h；4：12h；5：17h；6：纯化的N.b孢子的cDNA；7：正常家蚕蚕卵cDNA；8：ddH2O。
图12为HMG1F/HMG1R引物对N.b感染蚕卵(不浸酸)不同时间cDNA模板的PCR结果；泳道：M为DL1000；1：24h；2：48h；3：72h；4：96h；5：120h；6：144h；7：168h；8：192h；9：216h；10：240h；11：纯化的N.b的cDNA；12：正常家蚕蚕卵cDNA；13：ddH2O。
图13为HMG1F/HMG1R引物对N.b感染蚕卵(浸酸)不同时间cDNA模板的PCR结果；泳道：M为DL1000；1：24h；2：48h；3：72h；4：96h；5：120h；6：144h；7：168h；8：192h；9：216h；10：240h；11：纯化的N.b的cDNA；12：正常家蚕蚕卵cDNA；13：ddH2O。
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，以下实施例所用试剂和材料均为市购。
实施例1家蚕微孢子虫HMG1基因
1、根据分子生物学的基因克隆技术中基因同源克隆的方法，克隆获得了家蚕微孢子虫HMG 1基因的cDNA和DNA全长序列。
2、cDNA全长的获得，具体方法如下：
(1)运用Primer premier 5.0软件，结合综合分析，设计了引物引物HMG1F/HMG1R，序列分别如SEQ ID NO.3和SEQ ID NO.4所示。
上游引物HMG1F(SEQ ID NO.3)：
5’ATGACTGCTCAAAAAGACGATAC 3’
下游引物HMG1R(SEQ ID NO.4)：
5’TTATTCATCACTATCTCCTACTTCT 3’。
(2)以纯化的家蚕微孢子虫(N.b)孢子DNA为模板，以引物HMG1F/HMG1R进行PCR扩增。
(3)PCR产物经过纯化，连接到pMD19T中，并转化到E,coli DH-5α中培养。
(4)提取重组质粒，并测序，获得HMG1基因的cDNA全长，如SEQ ID NO.2所示。
3、DNA全长的获得
利用基因组高通量测序的方法，并经过大量的基因预测对比分析，最后经PCR测序验证，获得了家蚕微孢子虫HMG 1基因的DNA全长序列，如SEQ ID NO.1所示。
4、根据测序结果的多次确认，得出家蚕微孢子虫HMG 1基因的DNA全长序列，如SEQ ID NO.1所示，其cDNA全长核苷酸序列如SEQ ID NO.2所示。
实施例2检测引物设计及PCR扩增方法的建立
1、引物设计
在获得家蚕微孢子虫HMG 1基因的基础上，应用Primer premier 5.0软件设计，设计了多对引物，通过大量的抗药性、特异性和灵敏性检测，最终选取了3对引物有代表性的引物组，各组引物序列如下：
(1)第一对：
上游引物HMG1F(SEQ ID NO.3)：
5’ATGACTGCTCAAAAAGACGATAC 3’
下游引物HMG1R(SEQ ID NO.4)：
5’TTATTCATCACTATCTCCTACTTCT 3’。
(2)第二对：
上游引物HMG1-sF(SEQ ID NO.5)：
TTCCGAAATAATCTTCTTTTAATTG
下游引物HMG1-sR(SEQ ID NO.6)：
TTGTGCACCGAATCGTAAATAG
(3)第三对：
上游引物HMG1-xF(SEQ ID NO.7)：
TCCCTAGGAACTTTTAAAGAGAAG
下游引物HMG1-xR(SEQ ID NO.8)：
TCCTTTTATTCATCACTATCTCCT
2、PCR扩增方法的建立
(1)家蚕或家蚕蚕卵的总DNA的提取
采用QIAGEN公司生产的植物DNA小提试剂盒(DNeasy Plant mini kit试剂盒)抽提蚕卵基因组DNA，步骤如下(按照说明书进行)：
取20粒蚕卵放入研钵中，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
以家蚕或家蚕蚕卵的总DNA为模板，用实施例1所述三对引物进行PCR扩增。
所述PCR反应体系(总体积20μL)：
Figure PCTCN2015088443-appb-000003
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反应的程序为：94℃5min；94℃30s，50℃45s，72℃45s，32个循环；72℃10min。
(3)结果判断
第一对引物HMG1F/HMG1R：PCR反应结束后进行琼脂糖凝胶电泳，根据是否扩增到561bp的DNA片段判定蚕卵样品是否感染了家蚕微孢子虫。当能特异性地扩增出561bp的DNA片段产物，即可判断该家蚕或蚕卵感染了家蚕微孢子虫。
第二对引物HMG1-sF/HMG1-sR：PCR反应结束后进行琼脂糖凝胶电泳，根据是否扩增到684bp的DNA片段判定蚕卵样品是否感染了家蚕微孢子虫。当能特异性地扩增出684bp的DNA片段产物，即可判断该家蚕或蚕卵感染了家蚕微孢子虫。
第三对引物HMG1-xF/HMG1-xR：PCR反应结束后进行琼脂糖凝胶电泳，根据是否扩增到251bp的DNA片段判定蚕卵样品是否感染了家蚕微孢子虫。当能特异性地扩增出251bp的DNA片段产物，即可判断该家蚕或蚕卵感染了家蚕微孢子虫。
(4)分别取50粒“有毒”蚕卵(即感染家蚕微孢子虫的家蚕所产的蚕卵)、健康蚕卵和纯化的家蚕微孢子虫样本，分别提取DNA，按照上述的PCR方法进行PCR扩增。
琼脂糖凝胶电泳检测结果分别如附图1～3所示，三对引物均可在“有毒”蚕卵和纯化的家蚕微孢子虫中检出特异性的DNA片段，而健康蚕卵中未检测出特异性片段。表明三对引物和建立的PCR方法均可以很好的用于家蚕微孢子虫的快速检测。
实施例3引物特异性检测
1、分别以家蚕微孢子虫(N.b)、柞蚕微孢子虫(N.a)、玉米螟微孢子虫(N.f)的DNA为模板，以引物HMG1F/HMG1R，HMG1-sF/HMG1-sR，HMG1-xF/HMG1-xR，以实施例2的方法进行PCR扩增，扩增结束后琼脂糖凝胶电泳检测结果。
2、三对引物的扩增结果分别如附图4～6所示。结果显示，引物HMG1-sF/HMG1-sR能特异的检测家蚕微孢子虫，而引物HMG1F/HMG1R和引物HMG1-xF/HMG1-xR均能够检测到所有的微孢子虫，具有很好的通用检测性，但是不具有对家蚕微孢子虫的特异性。
实施例4引物灵敏性检测
1、提取家蚕微孢子虫(N.b)的DNA，原始浓度为5.0ng/μL。
将上述N.b DNA用ddH2O进行稀释，分别稀释100、101、102、103、104、105、106倍。即得到浓度梯度为5.0×100、5.0×10-1、5.0×10-2、5.0×10-3、5.0×10-4、5.0×10-5、5.0×10-6ng/μL。
2、以上述各浓度的N.b DNA为模板，以引物HMG1F/HMG1R，HMG1-sF/HMG1-sR，HMG1-xF/HMG1-xR，以实施例2的方法进行PCR扩增，扩增结束后琼脂糖凝胶电泳检测结果。
3、三对引物的扩增结果分别如附图7～9所示。结果显示，引物HMG1F/HMG1R和引物HMG1-sF/HMG1-sR均能检测到5.0×10-4ng/μL浓度的N.b DNA，具有很好的检测灵敏性。而引物HMG1-xF/HMG1-xR的灵敏性差了1个数量级，能检测到5.0×10-3ng/μL浓度的N.b DNA。
因此，综上所述，从特异性和灵敏性的角度考虑，只有引物HMG1-sF/HMG1-sR既能够特异性的检测家蚕微孢子虫，又具有很好的检测灵敏性，尤其是检测蚕卵家蚕微孢子虫，为蚕种生产中“有毒”蚕种的检测及安全发种提供了保证，在家蚕微孢子虫的实际检测以及早期蚕卵是否感染了家蚕微孢子虫的检测应用中具有重要的意义。实验结果也表明，HMG1基因是一个很好的检测家蚕微 孢子虫的分子靶标。
另外，只有引物HMG1F/HMG1R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，在多种微孢子虫的实际检测应用中具有广泛的应用价值和意义。
以下实施例进一步对引物HMG1F/HMG1R的检测适用性和灵敏性进行测试。
实施例5感染N.b不同时间段的四龄家蚕中肠和感染N.b的蚕卵检测
1、总RNA的提取
使用按照艾德莱生物公司的EASYspin植物RNA快速提取试剂盒说明书进行操作，分别提取感染N.b不同时间段的四龄家蚕中肠和感染N.b的蚕卵的RNA，具体步骤如下：
(1)取500μL裂解液RLT，转入1.5mL离心管中，加入50μL植物RNA助提剂(PLANTaid，结合有多糖多酚)混匀备用；
(2)液氮中研磨家蚕组织成细粉后，取50mg细粉转入上述装有裂解液RLT和PLANTaid的离心管，立即用手剧烈振荡20s，充分裂解；
(3)将裂解物13000rpm离心5～10min，沉淀不能裂解的碎片和结合有多糖多酚的PLANTaid；
(4)取裂解物上清转到一个新的离心管。加入上清体积一半的无水乙醇，此时可能出现沉淀，但是不影响提取过程，立即吹打混匀，不要离心；
(5)将混合物(每次小于720μL，多可以分两次加入)加入一个吸附柱RA中，(吸附柱放入收集管中)13000rpm离心2min，弃掉废液；
(6)加700μL去蛋白液RW1，室温放置1min，13000rpm离心30s，弃掉废液；
(7)加入500μL漂洗液RW，13000rpm离心30s，弃掉废液。加入500μL漂洗液Rw，重复一遍；
(8)将吸附柱RA放回空收集管中，13000rpm离心2min，尽量除去漂洗液，以免漂洗液中残留乙醇抑制下游反应；
(9)取出吸附柱RA，放入一个RNase离心管中，根据预期RNA产量在吸附膜的中间部位加30～50μL去除RNA酶的无菌水(RNase free water，事先在70～90℃水浴中加热，可提高产量)，室温放置1min，12000rpm离心1min。
2、RNA反转录成cDNA
将提取到的RNA进行反转录反应，使用TAKARA公司生产的PrimeScriptTM RT reagent Kit with gDNAEraser(Perfect Real Time)试剂盒，步骤如下：
(1)去除基因组DNA反应
Figure PCTCN2015088443-appb-000004
去除基因组DNA反应条件：42℃，2min(或室温5min最多可处理30min)。
(2)反转录反应
Figure PCTCN2015088443-appb-000005
反转录反应条件：37℃，15min；85℃，5s；4℃/-20℃保存备用。
3、PCR检测
以步骤2得到的各cDNA为模板，用特异性引物HMG1F/HMGR进行RT-PCR反应。
反应结果如附图10所示，在N.b感染家蚕中肠的前12h都没有检测到HMG1基因，说明此时微孢子虫还没有开始繁殖，而从18h开始HMG1基因有转录活性，此时微孢子虫可能已经开始进行分裂繁殖。而这一结论与现有技术中家蚕微孢子虫侵染家蚕的生活史是一致的，也从侧面证实了本发明所述检测引物具有很好的特异性和敏感性。
实施例6N.b感染蚕卵不同时间cDNA模板的检测
1、本实施例分别以N.b感染蚕卵和正常家蚕蚕卵为检测对象，分别以不浸酸、浸酸前、浸酸后的N.b感染后不同时间的蚕卵的cDNA为模板，以正常家蚕蚕卵DNA为对照，以HMG1F/HMG1R引物进行PCR检测。
2、实验方法
(1)浸酸前取样：家蚕微孢子虫感染家蚕后，在蚕蛾交配2h、8h、10h、12h、17h时分别取蚕卵，置于-80℃冰箱保存备用。
(2)浸酸处理：将一个卵圈平均分成两份，产卵后20h左右，浸酸(盐酸比重为1.075)，浸酸条件为：46℃浸酸5min，清水冲洗20min，置于温度为25℃，湿度为85％的人工气候培养箱中培养，至蚁蚕。
(3)将浸酸与不浸酸的蚕卵按产卵后24h、48h、72h、96h、120h、144h、168h、192h、216h、240h(蚁蚕)取样，置于-80℃冰箱保存备用。
3、结果如附图11～13所示。
图11为HMG1F/HMG1R引物对N.b感染蚕卵(浸酸前)不同时间cDNA模板的PCR结果；泳道：M为DL1000；1：2h；2：8h；3：10h；4：12h；5：17h；6：纯化的N.b孢子的cDNA；7：正常家蚕蚕卵cDNA；8：ddH2O。
图12为HMG1F/HMG1R引物对N.b感染蚕卵(不浸酸)不同时间cDNA模板的PCR结果；泳道：M为DL1000；1：24h；2：48h；3：72h；4：96h；5：120h；6：144h；7：168h；8：192h；9：216h；10：240h；11：纯化的N.b的cDNA；12：正常家蚕蚕卵cDNA；13：ddH2O。
图13为HMG1F/HMG1R引物对N.b感染蚕卵(浸酸)不同时间cDNA模板的PCR结果；泳道：M为DL1000；1：24h；2：48h；3：72h；4：96h；5：120h；6：144h；7：168h；8：192h；9：216h；10：240h；11：纯化的N.b的cDNA；12：正常家蚕蚕卵cDNA；13：ddH2O。
附图11～13的检测结果表明，在蚕卵产下2h时，即可检测到HMG1基因有转录活性，而且在此后蚕卵(浸酸与不浸酸)的整个发育过程HMG1基因都有转录活性，因此可以将HMG1基因作为一个分子靶标，来检测早期蚕卵是否感染了家蚕微孢子虫。
综上所述，本发明的检测引物和试剂盒能够准确地判断样品是否含有家蚕微孢子虫，尤其是检测蚕卵家蚕微孢子虫，为蚕种生产中“有毒”蚕种的检测及安全发种提供了保证。实验结果也表明，HMG1基因是一个很好的检测微孢子虫的分子靶标。

Claims (10)

  1. 一种家蚕微孢子虫HMG1基因，其特征在于，其DNA全长核苷酸序列如SEQ ID NO.1所示，其cDNA全长核苷酸序列如SEQ ID NO.2所示。
  2. 一组快速检测家蚕微孢子虫的引物组，其特征在于，所述引物组包括上游引物HMG1-sF和下游引物HMG1-sR，上游引物HMG1-sF的核苷酸序列如SEQ ID NO.5所示，下游引物HMG1-sR的核苷酸序列如SEQ ID NO.6所示。
  3. 权利要求2所述快速检测家蚕微孢子虫的引物组在制备家蚕微孢子虫检测试剂盒方面的应用。
  4. 一种家蚕微孢子虫检测试剂盒，其特征在于，包括上游引物HMG1-sF和下游引物HMG1-sR，上游引物HMG1-sF的核苷酸序列如SEQ ID NO.5所示，下游引物HMG1-sR的核苷酸序列如SEQ ID NO.6所示。
  5. 根据权利要求4所述试剂盒，其特征在于，所述试剂盒的使用方法如下：以家蚕DNA/cDNA或家蚕蚕卵DNA/cDNA为模板，利用上游引物HMG1-sF和下游引物HMG1-sR进行PCR反应，反应结束后凝胶电泳检测扩增产物，根据扩增DNA片段的大小判定结果；
    所述PCR反应的反应体系为：
    Figure PCTCN2015088443-appb-100001
    其中，2×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；
    所述PCR反应的程序为：94℃5min；94℃30s，50℃45s，72℃45s，32个循环；72℃10min。
  6. 一组微孢子虫通用检测引物，其特征在于，所述通用检测引物包括上游引物HMG1F和下游引物HMG1R，上游引物HMG1F的核苷酸序列如SEQ ID NO.3所示，下游引物HMG1R的核苷酸序列如SEQ ID NO.4所示。
  7. 权利要求6所述微孢子虫分子通用检测引物在制备微孢子虫通用检测试剂盒方面的应用。
  8. 根据权利要求7所述应用，其特征在于，所述微孢子虫为家蚕微孢子虫、柞蚕微孢子虫和/或玉米螟微孢子虫。
  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HMG1F和下游引物HMG1R，上游引物HMG1F的核苷酸序列如SEQ ID NO.3所示，下游引物HMG1R的核苷酸序列如SEQ ID NO.4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，利用上游引物HMG1F和下游引物HMG1R进行PCR反应，反应结束后凝胶电泳检测扩增产物，根据扩增DNA片段的大小判定结果；
    所述PCR反应的反应体系为：
    Figure PCTCN2015088443-appb-100002
    其中，2×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；
    所述PCR反应的反应程序为：94℃5min；94℃30s，58.5℃45s，72℃45s，32个循环；72℃10min。
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CN110656186A (zh) * 2019-10-31 2020-01-07 新疆农业科学院植物保护研究所 一种快速鉴定核桃黑斑蚜的试剂盒及方法
CN110656186B (zh) * 2019-10-31 2023-07-14 新疆农业科学院植物保护研究所 一种快速鉴定核桃黑斑蚜的试剂盒及方法

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