CN107400719B - Tussah microsporidian detection primers and application thereof - Google Patents
Tussah microsporidian detection primers and application thereof Download PDFInfo
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Abstract
The invention discloses a group of tussah microsporidian detection primers and application thereof, wherein the base sequence of the primers is shown as SEQ ID No. 1-3, the template extraction method is characterized in that the release, enrichment, separation and purification of DNA are carried out by combining alkaline cracking with magnetic beads, the primers have good specificity and high sensitivity, and the tussah microsporidian detection method established by using the primers is simple and rapid in operation, low in cost, high in flux, capable of sampling live pupae, strong in anti-interference capability, low in pollution risk, high in extracted DNA quality and capable of being automated. The method can be widely applied to molecular detection, can obviously improve the detection flux and the operation convenience, obviously reduces the false positive rate of detection, and improves the detection sensitivity, the accuracy and the detection efficiency; has important significance for centralized quarantine of tussah disease and germplasm resource protection in tussah industry.
Description
Technical Field
The invention belongs to the technical field of detection of pathogenic microorganisms, and particularly relates to a group of primers for specifically detecting nosema bombycis and application thereof.
Background
Tussah (Antheraea pernyi) originates from China and is one of the most valuable biological resources of human beings. China is the first major country of the tussah industry in the world, and the cocoon yield accounts for 90% of the world total cocoon yield. At present, the annual output of China oak silkworm cocoons is more than 8 million tons, the direct output value is 25 hundred million yuan, and the output value of processing and comprehensive utilization of the oak silkworm cocoons is more than 180 hundred million yuan. Tussah has become a dominant industry difficult to replace in mountain countryside in China. However, since the tussah industry is scaled up, the development of the tussah industry is always troubled by the tussah micro-particle disease, the disease is mainly characterized by long harm time, serious resource waste, wide distribution and complex infection route, and once the disease is outbreak, the disease causes great economic loss, so the disease is classified as an important epidemic disease in tussah production.
At present, the female moths are screened in the seed production process by using an optical Microscopy method (microscopic examination), and the transmission of microsporidia is cut off by eliminating disease moths and eggs thereof. The method is simple and easy to learn and has strong practicability. However, the method has higher experience dependence on seed production personnel, and the difference between the sensitivity and the accuracy of a detection result is larger. The development of microsporidia can be roughly divided into four stages, namely, sporogenous stage (sporoplast), merozoite stage (Merogony), sporogenous stage (Sporogony) and mature Spore stage (spoore), and microscopic examination mainly aims at mature microsporidia, so that the examination is inevitably missed, and the effect of effectively controlling the microparticulate diseases is limited. Meanwhile, microscopic examination is time-consuming and labor-consuming, and easily induces eye diseases of detection personnel.
In the prior art, Wangbangyang et al disclose a method for obtaining a polyclonal antibody against microsporidian of tussah by directly immunizing a rabbit with microsporidian spore liquid of tussah, and a colloidal gold immunochromatography detection method is established, but the method has low sensitivity, and when the concentration of microsporidian of tussah is lower than 8000/mu L, the detection test strip has light color development or no color development, and the result is unstable, so that accurate judgment cannot be carried out. Subsequently, the tussah microsporidian indirect competitive enzyme-linked immunosorbent assay (ELISA) method is established by Jiangyang and the like, the detection sensitivity is improved to 160/mu L, but the operation is very complicated and the matching production difficulty is high.
The molecular diagnosis technology achieves the diagnosis purpose through specific amplification target genes, has high sensitivity and good specificity, can be operated in batches, can obviously improve the detection accuracy and the detection flux, and is widely applied to the detection of pathogenic microorganisms. In 2010, Dengzhenhua et al disclose a PCR diagnosis technique for tussah microsporidian, which has high sensitivity, good specificity and low detection lower limit (each reaction system contains 0.94ng of tussah microsporidian genome DNA). However, this technique has the problems of complicated DNA extraction process, time consumption, low throughput, etc. Considering the characteristics of tussah industry, a large number of samples are generally required to be diagnosed intensively in a short time, individuals infected with microsporidia are eliminated, the aim of cutting off the propagation of microparticle disease parents is achieved, and the production application of the microsporidia is limited due to low flux. Meanwhile, PCR equipment is required in the technology, and the use of the technology in the field is restricted.
The Recombinase Polymerase Amplification (RPA) is a constant-temperature in-vitro rapid nucleic acid Amplification technology developed on the basis of the existing in-vitro nucleic acid Amplification principle, and is firstly proposed by Piepenburg et al in 2006, and has the obvious advantages of capability of realizing Amplification of a specific nucleic acid sequence at a constant temperature of 25-43 ℃, capability of observing results within 5-20 minutes, low requirement on RPA technical equipment, wide carrier in an Amplification process, capability of realizing quantitative analysis and visual result judgment by combining methods such as a probe or a transverse flow test strip and the like, capability of meeting the requirement of field sample measurement, and wide application in the fields of in-vitro diagnosis, veterinary medicine, food safety, biological safety and the like. However, based on the limitations of key technologies such as expression and purification of key enzymes, design of primer probes and the like, no report on the related technology for detecting the nosema bombycis by applying the RPA technology is found.
Disclosure of Invention
The invention provides an antheraea pernyi microsporidian detection primer and application thereof, aiming at various problems listed in the background technology of complicated operation, low flux, low detection sensitivity and the like of the existing antheraea pernyi microsporidian detection technology, in particular to a template DNA acquisition method and a kit thereof, and discloses application of the primer in the aspect of detection.
The invention discloses a group of tussah microsporidian detection primers, wherein the base sequence of the primers is shown as SEQ ID No. 1-3. Meanwhile, a method for detecting the nosema bombycis by using the primer, namely a recombinase polymerization amplification technology is disclosed.
Specifically, in the above-described detection method, the reaction configuration system in the method for amplifying the template DNA is as follows:
further preferred concentrations for the above agents are:
| dithiothreitol | 4mM |
| ATP | 3mM |
| Creatine phosphate | 50mM |
| gp32 | 250ng/μL |
| UvsX | 120ng/μL |
| UvsY | 22ng/μL |
| Magnesium acetate | 10mM |
| Forward primer |
480nM |
| Reverse guideSubstance |
480nM |
| Methoxylated primer |
480nM |
The reaction system can be prepared immediately or can be freeze-dried into powder in a freeze dryer for later use. After the reaction system is freeze-dried, deionized water is needed to dissolve the system into a solution again.
The recombinase polymerization amplification reaction is preferably carried out under the following reaction conditions: the amplification temperature is 37-40 ℃, and the amplification time is 20-60 minutes.
When the recombinase polymerization amplification reaction adopts a fluorescent quantitative PCR instrument, the preferable reaction conditions are as follows: 60 minutes at 39 ℃, followed by a melting curve reaction conditions: 95 ℃ for 15s, 60 ℃ for 60s, 95 ℃ for 15s, and 60 ℃ for 15 s.
And (4) judging a result:
after the recombinase polymerization amplification reaction is finished, the PCR product is boiled for 2 minutes and then subjected to PAGE gel electrophoresis. If the specific band is present at 72bp, the result is judged to be positive, namely, the tussah microsporidian is infected. Otherwise, the result is judged to be negative.
When the recombinase polymerization amplification reaction adopts a fluorescent quantitative PCR instrument, the Ct value of the sample can be compared with the Ct value of water, and whether the melting curve and the positive reference peak at the same Tm value is judged. Of course, the result determination can also be combined with a lateral flow test strip to realize visual result determination.
For the above technical solution, particularly preferably, the method for obtaining the microsporidian template DNA of tussah is different from the conventional method, and the specifically adopted technical solution is as follows:
carrying out cracking treatment on a sample to be detected by using an alkali solution, and carrying out enrichment, separation and purification on DNA by using magnetic beads;
the source and the collection method of the sample to be detected are obviously different from the prior art, and specifically comprise the following steps: the tussah microsporidian sample can be derived from any body part of tussah at any period or obtained by artificial purification and culture. The period of collecting the sample from tussah can be egg period, silkworm period, pupal period and moth period, and the part of collecting the sample can be tissue, epidermis and blood, etc. The artificially purified and cultured Antheraea pernyi Microsporum can be cell culture solution, suspension with buffer solution, etc. And in vivo sampling can be achieved.
Specifically, in the above technical scheme, the base is an inorganic base, for example: sodium hydroxide, potassium hydroxide, sodium carbonate and the like can be selected, the concentration of the alkali solution can be 0.05-1M, the treatment process is usually accompanied by shaking and uniformly mixing operations, and the treatment time is 1 minute-40 days.
Specifically, in the above technical scheme, Sodium Dodecyl Sulfate (SDS) may be added to the alkali solution during the cracking treatment, and the concentration is less than 10%. The treatment time is 5 minutes to 48 hours, and the treatment temperature is 0 to 40 ℃.
In a preferable case, in the technical scheme described above, the lysis treatment process may be freeze thawing at-20 to-16 ℃ after adding the alkali liquor, and the number of freeze thawing may be 1 or more. More preferably, the action time of adding the alkali liquor is 1 minute to 24 hours, and then freeze thawing is carried out; the treatment time of the alkali liquor can be 5 minutes to 40 days.
Specifically, the magnetic beads in the above technical solutions are widely used in bio-separation as separation carriers, and in the magnetic separation process, the magnetic microspheres are directly put into a mixed solution containing a target substance, the target substance and the magnetic microspheres are tightly combined, and then an external magnetic field is used for separation. The magnetic beads specifically applicable to the present invention are magnetic microspheres containing silica and a ferroferric oxide component, such as SiO2@Fe3O4，C@SiO2@Fe3O4And the like.
In a preferred case, for the above technical solution, the process of DNA enrichment is to add magnetic beads or a suspension of magnetic beads to the lysis solution, mix them uniformly, and stand for a while. Meanwhile, absolute ethyl alcohol is added into the lysate to improve the enrichment efficiency, and the volume ratio of the absolute ethyl alcohol to the lysate is usually 0.7-1: 1.
Specifically, in the above technical solution, the DNA separation process refers to separating magnetic beads from a liquid phase by an external magnetic field and discarding the liquid phase. The externally applied magnetic field is typically a magnet.
Specifically, in the DNA purification process described in the above technical scheme, the magnetic beads with enriched DNA are usually washed with 70-75% alcohol, and the washing is usually accompanied by drying and elution steps, and the elution is usually performed by ddH2O, TE buffer solution or PCR reaction solution.
In addition, in the DNA purification process described in the above technical scheme, when the sample size is very low, the PCR reaction system can be directly used for elution of the template DNA.
It is to be noted that, when the sample is derived from tussah (larva, pupa, moth, egg), the template DNA obtained by the above method is a total genomic DNA including tussah genomic DNA, tussah microsporidian genomic DNA, and genomic DNA of other microorganisms parasitizing in tussah.
Furthermore, the invention also provides a tussah microsporidian molecular detection kit.
Specifically, the kit comprises primers with base sequences shown in SEQ ID No. 1-3 and the following reagents: the reagent combination used for detection in the method for acquiring the Antheraea pernyi microsporidian template DNA; the main reagents include alkali solution and Sodium Dodecyl Sulfate (SDS); wherein the alkali solution is selected from sodium hydroxide, potassium hydroxide, sodium carbonate, etc.; the concentration of the alkali solution can be 0.05-1M or high concentration convenient for storage; the SDS concentration may be 0.1 to 10% or a high concentration convenient for storage.
Has the advantages that:
compared with the prior art, the invention has the following advantages and remarkable effects:
1. compared with the existing molecular detection technology, the recombinase polymerization amplification technology can realize the amplification of a specific nucleic acid sequence at the constant temperature of 25-43 ℃, the result can be observed within 5-60 minutes, quantitative analysis and visual result judgment can be realized by combining a probe or a transverse flow test strip and other methods, the equipment requirement is low, a simple constant temperature device with a power supply can meet the requirement of field sample measurement, and the method is particularly suitable for the industrial characteristics of wild breeding of tussah, but the related report of detecting the tussah microsporidia by adopting the recombinase polymerization amplification technology is not seen so far. The invention provides a group of primers for detecting the microsporidian of tussah by a recombinase polymerization amplification technology, can realize quick and high-sensitivity detection on the microsporidian of tussah, and is one of the breakthroughs of the invention.
2. The invention provides a group of primers for detecting the nosema oak silkworm by a recombinase polymerization amplification technology, which comprises a methoxylated probe (PNA-IO), can obviously avoid nonspecific amplification, reduce the influence of a miscellaneous band on result judgment, solve the technical limitation that the recombinase polymerization amplification technology is often accompanied by nonspecific amplification due to smaller amplified fragments, and is another breakthrough of the invention.
3. The invention provides a rapid extraction method of a DNA template, which comprises the steps of carrying out cracking treatment on a sample by using mixed solution of alkali solution and SDS (sodium dodecyl sulfate), and carrying out enrichment, separation and purification of DNA by combining magnetic beads. Compared with the prior art, the method has the advantages that: (a) the sample is cracked by using the alkali solution, so that the cost is lower than that of the conventional technology in which proteinase K is generally used for digestion, the technical defect that the conventional and generally adopted proteinase K digestion method cannot effectively digest protein and release DNA after tissue blackening is overcome, and the result uncertainty caused by the instability of DNA quality is reduced; (b) the method can realize the cracking of the sample within 5 minutes at the fastest speed, the timeliness is far beyond the 3-4 hours required by the conventional protease K digestion, and the method is more suitable for the requirement of field rapid detection; (c) the whole extraction process does not need heating, so that the risk of steam pollution is greatly reduced; (d) the magnetic beads are adopted for enrichment, separation and purification, the operation is simple, centrifugation is not needed, multiple samples can be simultaneously extracted on a 96-well plate or even a 384-well plate, the speed is high, and the flux is high; (e) the magnetic beads can be recycled, so that the energy is saved and the environment is protected; (f) the sampling quantity requirement is low, the loss is less, and the in vivo sampling can be realized. This is one of the important breakthroughs of the invention.
Drawings
FIG. 1 shows an agarose gel electrophoresis chart of example 1 of the present invention;
FIG. 2 shows the agarose gel electrophoresis of example 2 of the present invention.
FIG. 3 shows an agarose gel electrophoresis image of example 3 of the present invention.
FIG. 4 shows an agarose gel electrophoresis image of example 4 of the present invention.
FIG. 5 shows an agarose gel electrophoresis image of example 5 of the present invention.
Detailed Description
The present invention is further described below with reference to examples. It should be noted that the following description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Materials, actual equipment and experimental equipment related to the embodiment of the invention are consistent with commercially available products in the field of pathogenic microorganism molecule detection if no special description is provided.
The primers related to the embodiment of the invention are synthesized by entrusted bioengineering company, and the specific primer information is as follows:
SEQ ID NO:1:
5’-TTGACGGAAGAATACCACAAG-3’
SEQ ID NO:2：
5’-TACCTGGTAAATTACCCCGCG-3’
SEQ ID NO:3：
5’-TCGTGCATGCACTTCCACAAGGAGTGGATTGTGCGGCTTAATTTGACmUmCmA mAmCmGmCmGmGmGmGmUmA/InvdT/-3’
the underlined content in sequence SEQ ID NO 3 is a methoxylated primer used as a probe.
SEQ ID NO:4：
5’-GTGCGGCTTAATTTGACTCAACG-3’
SEQ ID NO:5：
5’-CTATATGAGGGTCTCACATCTTG-3’SEQ ID NO:6：
5’-GACGGAAGAATACCACAAGGAGT-3’
SEQ ID NO:7：
5’-CTATATGAGGGTCTCACATCTTGT-3’
SEQ ID NO:8:
5’-GTGATCTCGGTCTACGGTTCTC-3’
SEQ ID NO:9:
5’-TAGTTCACCATCTTTCGGGTCC-3’
Example 1
Preparation of plasmid standard:
taking the tussah microsporidian suspension, centrifuging for 5 minutes at 5000r/min, removing the supernatant, retaining spore precipitate, adding an extraction solution (10mmol/L Tris-HCL pH 8.0; 10mmol/L EDTA pH 8.0; 100mmol/L NaCl; 2% SDS; 0.039mol/L DTT), adding protease K (final concentration of 100 mu g/mL), digesting for 4 hours at 55 ℃, extracting with phenol/chloroform, washing twice with 70% ethanol, and eluting with TE after drying to obtain the tussah microsporidian genomic DNA. Carrying out conventional PCR by using the genomic DNA of the microsporidian of the tussah, wherein the PCR system is as follows: 2.5 μ L10 XPCR buffer, 2 μ L10 mmol dNTPs, 22 μ L MgCl, 0.5 μ L each of 10 μmol/L upstream and downstream primers, 1U Taq enzyme, 2 μ L template DNA, and 25 μ L sterile deionized water. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 65 ℃ and 60s at 72 ℃ for 35 cycles; extension at 72 ℃ for 10 min. And after the reaction is finished, performing electrophoresis detection on 10 mu L of PCR amplification product by using 2% agarose gel, recovering and purifying a specific PCR product of 72bp, connecting the specific PCR product to a pMD-18T vector, transforming the specific PCR product to DH5 alpha competent cells, coating the competent cells on a plate culture medium for culture, selecting positive bacterial colonies to extract plasmid DNA, performing enzyme digestion and sequencing for identification, and determining the correct sequence as a plasmid standard product.
Extracting the genome DNA of the healthy tussah pupa:
taking a little of healthy tussah pupa tissue, grinding the healthy tussah pupa tissue by liquid nitrogen, adding the ground healthy tussah pupa tissue into tissue extract (10mmol/L Tris-HCL pH 8.0; 10mmol/L EDTA pH 8.0; 100mmol/L NaCl; 2% SDS; 0.039mol/L DTT), adding proteinase K (final concentration is 100 mu g/mL), digesting the mixture at 55 ℃ for 4 hours, extracting the mixture by phenol/chloroform, washing the mixture twice by 70% ethanol, and eluting the mixture by TE after drying to obtain the healthy tussah pupa genomic DNA.
Extracting the DNA of the microsporidian template of the tussah:
selecting one pupa infected with the later period of pupae of the nosema bombycis, cutting a knife edge of about 5mm on the back of the pupae by using an operating blade, quickly picking a small amount of tissue by using a toothpick, placing the picked tissue in a 1.5mL EP tube, disinfecting the knife edge on the back of the pupae by using 75% alcohol cotton, and carrying out wax sealing by using melted section wax (Hualingbrand). Adding 200 mu L of lysis solution (0.2M NaOH, 1% SDS), shaking and mixing uniformly, standing for 20 minutes at room temperature, adding 10 mu L (sigma) of magnetic beads and 150mL of absolute ethyl alcohol, shaking and mixing uniformly gently, placing an EP tube on a magnetic frame for separation, pouring out the liquid phase after the magnetic beads are completely separated from the liquid phase, removing the magnetic frame, adding 150 mu L of 70% ethanol, shaking and mixing uniformly, placing on the magnetic frame for separation, discarding the liquid, removing the magnetic frame, adding 150 mu L of 70% ethanol again, shaking and mixing uniformly, placing on the magnetic frame for separation, sucking the liquid as much as possible by a liquid-moving gun, removing the magnetic frame, placing the EP tube in a 37 ℃ incubator for drying for 30 minutes, and eluting by 20 mu L of TE. And (5) freezing and storing the DNA at the temperature of-20 ℃ for later use. And putting the injured pupa into a cocoon shell, and placing at normal temperature for hatching condition observation.
The configuration of a recombinase polymerization amplification reaction system:
conditions for recombinase polymerase amplification reaction: the amplification temperature was 37 ℃ and the amplification time was 20 minutes. Boiling in water for 2 min, subjecting 10 μ L to PAGE gel electrophoresis, and observing under ultraviolet lamp after electrophoresis, to obtain the result shown in FIG. 1. In addition, the injured pupae hatched into moths after 5 days.
The results are illustrated below: m represents nucleic acid electrophoresis marker, T-Na represents standard plasmid, Na represents sample DNA, Gm represents healthy tussah pupa genome DNA, and H2O represents deionized water.
This example shows that the method has the advantages of rapid extraction of DNA, good technical effect of DNA extraction, and high sensitivity for detecting the microparticulate disease by combining with late molecular detection. Meanwhile, the DNA extraction technology of the invention has low sampling amount and can realize sampling of live pupa.
Example 2
Preparation of plasmid standard:
taking the tussah microsporidian suspension, centrifuging for 5 minutes at 5000r/min, removing the supernatant, retaining spore precipitate, adding an extraction solution (10mmol/L Tris-HCL pH 8.0; 10mmol/L EDTA pH 8.0; 100mmol/L NaCl; 2% SDS; 0.039mol/L DTT), adding protease K (final concentration of 100 mu g/mL), digesting for 4 hours at 55 ℃, extracting with phenol/chloroform, washing twice with 70% ethanol, and eluting with TE after drying to obtain the tussah microsporidian genomic DNA. Carrying out conventional PCR by using the genomic DNA of the microsporidian of the tussah, wherein the PCR system is as follows: 2.5 μ L10 XPCR buffer, 2 μ L10 mmol dNTPs, 22 μ L MgCl, 0.5 μ L each of 10 μmol/L upstream and downstream primers, 1U Taq enzyme, 2 μ L template DNA, and 25 μ L sterile deionized water. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 65 ℃ and 60s at 72 ℃ for 35 cycles; extension at 72 ℃ for 10 min. And after the reaction is finished, performing electrophoresis detection on 10 mu L of PCR amplification product by using 2% agarose gel, recovering and purifying a specific PCR product of 72bp, connecting the specific PCR product to a pMD-18T vector, transforming the specific PCR product to DH5 alpha competent cells, coating the competent cells on a plate culture medium for culture, selecting positive bacterial colonies to extract plasmid DNA, performing enzyme digestion and sequencing for identification, and determining the correct sequence as a plasmid standard product.
Extracting the genome DNA of the healthy tussah pupa:
taking a little of healthy tussah pupa tissue, grinding the healthy tussah pupa tissue by liquid nitrogen, adding the ground healthy tussah pupa tissue into tissue extract (10mmol/L Tris-HCL pH 8.0; 10mmol/L EDTA pH 8.0; 100mmol/L NaCl; 2% SDS; 0.039mol/L DTT), adding proteinase K (final concentration is 100 mu g/mL), digesting the mixture at 55 ℃ for 4 hours, extracting the mixture by phenol/chloroform, washing the mixture twice by 70% ethanol, and eluting the mixture by TE after drying to obtain the healthy tussah pupa genomic DNA.
Extracting the DNA of the microsporidian template of the tussah:
selecting one diseased pupa infected with microsporidian of tussah, placing a small amount of tissues in 1.5mL of EP tubes respectively, adding 200 μ L of lysis solution (0.2M NaOH and 1% SDS), shaking and mixing uniformly, placing at room temperature for 5 minutes, adding 10 μ L (sigma) of magnetic beads and 150mL of absolute ethyl alcohol, shaking and mixing uniformly gently, placing the EP tubes on a magnetic frame for separation, pouring out the liquid phase after the magnetic beads are completely separated from the liquid phase, removing the magnetic frame, adding 150 μ L of 70% ethanol, shaking and mixing uniformly, placing on the magnetic frame for separation, discarding the liquid, removing the magnetic frame, adding 150 μ L of 70% ethanol again, shaking and mixing uniformly, placing on the magnetic frame for separation, sucking the liquid as much as possible by a pipette gun, removing the magnetic frame, placing the EP tubes in a 37 ℃ incubator for drying for 30 minutes, and eluting by 20 μ L of TE. And (5) freezing and storing the DNA at the temperature of-20 ℃ for later use.
The configuration of a recombinase polymerization amplification reaction system:
conditions for recombinase polymerase amplification reaction: the amplification temperature was 38 ℃ and the amplification time was 60 minutes. After the amplification, the PCR product was boiled in water for 2 minutes, 10. mu.L of the product was subjected to PAGE gel electrophoresis, and the result was observed under an ultraviolet lamp after the electrophoresis was completed, as shown in FIG. 2.
The results are illustrated below: m represents nucleic acid electrophoresis Marker DL2000, T-Na represents standard plasmid, Na represents sample DNA, Gm represents healthy tussah pupa genome DNA, and H2O represents deionized water.
This example shows that the method has the advantages of rapid extraction of DNA, good technical effect of DNA extraction, and high sensitivity for detecting the microparticulate disease by combining with late molecular detection. Meanwhile, compared with the embodiment 1, the embodiment can effectively avoid non-specific amplification along with the addition of the methoxylated probe primer, reduce the influence of the hybrid band on result judgment, and has good specificity.
Example 3
The DNA of the nosema Antheraea pernyi is automatically extracted by adopting a Thermo nucleic acid automatic extractor 706. A small amount of 12 cases of the tissue of the diseased pupa with the corpuscles was soaked in 210. mu.L of lysis solution (0.1M NaOH, 1% SDS) for 2 hours, and 200. mu.L of supernatant was removed for further use. The solvent amount in the DW wells in column A was slightly adjusted using the KF _ tissue DNA _ Duo program preset by the instrument: take 200. mu.L of lysate, 20. mu.L of magnetic bead suspension (sigma), and 140. mu.L of absolute ethanol, wherein the sample has been fully lysed. 2 mu L of the extracted DNA is taken as a PCR template to configure a reaction system: each 25. mu.L system contained 0.5. mu. L, rTaq 0.25. mu.L, 10 XBuffer 2.5. mu.L, 2. mu.L dNTPs, 17.25. mu.L deionized water and 2. mu.L DNA template of the primers (10pM) shown in SEQ ID NO. 4 and SEQ ID NO. 5. The PCR reaction condition is 95 ℃ for 4 minutes; 40 cycles of 95 ℃ 30 seconds, 65 ℃ 30 seconds, 72 ℃ 1 minute, extension at 72 ℃ for 5 minutes, adding a nucleic acid staining agent to the obtained PCR product, performing gel electrophoresis with 2% agarose, and observing the result under ultraviolet light after the electrophoresis is finished, as shown in FIG. 3.
The results are illustrated below: m represents the nucleic acid electrophoresis Marker DL2000, and the sample holes of No. 1-12 all have specific bands of about 144 bp. Therefore, the DNA extracted automatically has better quality, which shows that the DNA extraction technology provided by the method can completely realize automatic extraction and greatly save manpower.
Example 4
Recycling of magnetic beads: selecting one diseased pupa infected with microsporidian of tussah, placing a small amount of tissue in a 1.5mL EP tube, and numbering Pos. Adding 200 mu L of lysis solution S (0.2M NaOH, 1% SDS) into a tube, shaking and uniformly mixing, standing at room temperature for 60 minutes, adding 20 mu L (sigma) of magnetic bead suspension and 150mL of absolute ethyl alcohol, shaking and uniformly mixing lightly, placing an EP tube on a magnetic frame for separation, pouring out the liquid phase when the magnetic beads are completely separated from the liquid phase, removing the magnetic frame, adding 150 mu L of 70% ethanol, shaking and uniformly mixing, placing on the magnetic frame for separation, removing the liquid, removing the magnetic frame, adding 150 mu L of 70% ethanol again, shaking and uniformly mixing, placing on the magnetic frame for separation, absorbing the liquid as far as possible by a liquid-transferring gun, removing the magnetic frame, placing the EP tube in a 37 ℃ incubator for drying for 30 minutes, eluting by 50 mu L of TE, transferring eluent, and freezing and storing DNA at-20 ℃ for later use.
Adding 1mL of water into the EP tube with transferred eluent, blowing by a pipette gun to uniformly disperse the magnetic beads in the water, placing on a magnetic frame, removing the water phase after the water phase is clarified, repeating the operation for 5 times, adding 50 mu L of TE to elute the magnetic beads, transferring the eluent, numbering Washed, storing at 4 ℃ for standby, recovering the magnetic beads, and placing the EP tube containing the magnetic beads in a 37 ℃ incubator to dry for 20 minutes for standby.
Selecting one healthy pupa which is determined not to be infected with the microsporidian of tussah, taking a small amount of tissues and placing the tissues in an EP tube with the volume of 1.5mL, and numbering Neg. Adding 200 mu L of lysis solution S (0.2M NaOH, 1% SDS) into a tube, shaking and mixing uniformly, standing at room temperature for 60 minutes, transferring the lysis solution into the recovered magnetic bead EP tube, adding 150mL of absolute ethyl alcohol, shaking and mixing uniformly slightly, placing the EP tube on a magnetic frame for separation, pouring the liquid phase when the magnetic beads are completely separated from the liquid phase, removing the magnetic frame, adding 150 mu L of 70% ethanol, shaking and mixing uniformly, placing on the magnetic frame for separation, discarding the liquid, removing the magnetic frame, adding 150 mu L of 70% ethanol again, shaking and mixing uniformly, placing on the magnetic frame for separation, absorbing the liquid as far as possible by a liquid-transferring gun, removing the magnetic frame, placing the EP tube in a 37 ℃ incubator for drying for 30 minutes, eluting by 50 mu L of TE, transferring the eluent, and freezing and storing DNA at-20 ℃ for later use.
The Pos, Washed, Neg samples were subjected to conventional PCR using 0.5. mu.L each of the primers shown in SEQ ID NO:6 and SEQ ID NO:7 (10pM), 2. mu. L, rTaq 0.25.25. mu.L of template DNA, 2.5. mu.L of 10 XBuffer, 2. mu.L of dNTPs, and 17.25. mu.L of deionized water per 25. mu.L system. PCR conditions were 95 ℃ for 4 minutes; 40 cycles at 95 ℃ for 30 seconds, 60 ℃ for 30 seconds, 72 ℃ for 1 minute, and extension at 72 ℃ for 5 minutes.
Another 2. mu.L Neg template DNA is taken, and 0.5. mu. L, rTaq 0.25, 10 XBuffer 2.5. mu.L each, dNTPs 2. mu.L and deionized water 17.25. mu.L tussah 28SrRNA specific primers (10pM) shown as SEQ ID NO:8 and SEQ ID NO:9 are added. Carrying out conventional PCR on the prepared system, wherein the PCR condition is 95 ℃ for 4 minutes; 40 cycles at 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, 72 ℃ for 1 minute, and extension at 72 ℃ for 5 minutes.
Adding nucleic acid staining agent into the two PCR products, performing gel electrophoresis with 2% agarose, and observing the result under ultraviolet light after electrophoresis, as shown in FIG. 4.
The results are explained as follows, the sample holes are from left to right, the number 1-3 are the specific primer amplification results of the nosema bombycis of Pos, Washed and Neg, the number 4 is the specific primer amplification result of the Neg tussah of the sample: pos samples extracted by fresh magnetic beads are specifically taken out at about 173bp, and the DNA containing the tussah microsporidian genome is determined; the Washed sample and the Neg sample do not have specific bands of the nosema pernyi, which shows that the magnetic beads for extracting the sample do not contain the eluted nosema pernyi genome DNA after being cleaned, and meanwhile, the cleaned magnetic beads do not have false positive interference on the sample when being reused; the Neg sample has tussah specific bands at 106bp, which shows that the recovered magnetic beads still have the capability of extracting DNA with high quality. This example illustrates the recyclability of the magnetic beads well and also the economy and greenness of the process.
Example 5
Selecting 1 pupa infected with microsporidian of tussah, selecting a small amount of tissue in a sterile mortar, adding a proper amount of PBS, slightly grinding and dispersing, fully and uniformly blowing, diluting by 10 times to obtain two gradients, numbering sample-1# and sample-2# respectively, and standing at room temperature for 20 minutes for blackening. Each of the suspension after blackening No.1 and No. 2 was taken and 100. mu.L of each of the suspension was added to 6 EP tubes (1.5 mL), numbered FT-1, FT-2, S-1, S-2, Pk-1 and Pk-2, respectively, 0.4M KOH was added to the tubes FT-1 and FT-2, respectively, 2X lysate S (final concentration: NaOH 0.2M, SDS 1%) was added to the tubes S-1 and S-2, and 2X plaque extract (protease K-containing 10. mu.L) was added to the tubes Pk-1 and Pk-2. Placing Pk-1 and Pk-2 in a thermostatic water bath at 56 ℃, and placing FT-1, FT-2, S-1 and S-2 at room temperature.
Pk-1 and Pk-2 are placed in a 56 ℃ constant temperature water bath for 3 hours, then taken out, 10 mu L of magnetic bead suspension (Sigma) and 150mL of absolute ethyl alcohol are added, the mixture is gently shaken and uniformly mixed, an EP tube is placed on a magnetic frame for separation, the liquid phase is poured out after the magnetic beads are completely separated from the liquid phase, the magnetic frame is removed, 150 mu L of 70% ethyl alcohol is added, the mixture is shaken and uniformly mixed and then placed on the magnetic frame for separation, the liquid is discarded, the magnetic frame is removed, 150 mu L of 70% ethyl alcohol is added again, the mixture is shaken and uniformly mixed and then placed on the magnetic frame for separation, a liquid transferring gun sucks the liquid as much as possible, the magnetic frame is removed, the EP tube is placed in a 37 ℃ incubator for drying for 30 minutes, and 20 mu L of.
Placing FT-1 and FT-2 and S-1 and S-2 at room temperature for 40 minutes, then placing FT-1 and FT-2 at-20 ℃ for freezing, adding 10 mu L of magnetic beads (Sigma) and 150mL of absolute ethyl alcohol into tubes S-1 and S-2, shaking gently and mixing uniformly, placing the tubes EP on a magnetic frame for separation, pouring out the liquid phase after the magnetic beads are completely separated from the liquid phase, removing the magnetic frame, adding 150 mu L of 70% ethyl alcohol, shaking and mixing uniformly, then placing on the magnetic frame for separation, discarding the liquid, removing the magnetic frame, adding 150 mu L of 70% ethyl alcohol again, shaking and mixing uniformly, placing on the magnetic frame for separation, sucking the liquid as far as possible by a liquid-moving gun, removing the magnetic frame, placing the tubes in an incubator at 37 ℃ for drying for 30 minutes, and eluting by 20 mu L of TE. And (5) freezing and storing the DNA at the temperature of-20 ℃ for later use.
Taking out FT-1 and FT-2, melting at room temperature, adding 10 μ L of magnetic beads (Sigma) and 150mL of absolute ethanol, shaking gently, mixing, separating the EP tube on a magnetic frame, pouring out the liquid phase after the magnetic beads are completely separated from the liquid phase, removing the magnetic frame, adding 150 μ L of 70% ethanol, shaking, mixing, separating on the magnetic frame, discarding the liquid, removing the magnetic frame, adding 150 μ L of 70% ethanol again, shaking, mixing, separating on the magnetic frame, removing the liquid as much as possible by a liquid transfer gun, removing the magnetic frame, drying the EP tube in an incubator at 37 ℃ for 30 minutes, and eluting with 20 μ L of TE.
Taking purified tussah microsporidian cell culture solution, diluting two gradients by 10 times, taking 100 mu L of each gradient in each 2-1.5 mL EP tube, adding 2 Xspot extractive solution (containing protease K10 μ L), placing in 56 deg.C constant temperature water bath for 3 hr, taking out, adding 150mL of 10 mu L of magnetic beads (Sigma) and anhydrous ethanol, slightly shaking and mixing uniformly, placing an EP tube on a magnetic frame for separation, pouring out the liquid phase after the magnetic beads are completely separated from the liquid phase, removing the magnetic frame, adding 150 mu L of 70% ethanol, shaking and mixing uniformly, placing on the magnetic frame for separation, discarding the liquid, removing the magnetic frame, adding 150 mu L of 70% ethanol again, shaking and mixing uniformly, placing on the magnetic frame for separation, sucking the liquid as much as possible by a liquid transfer gun, removing the magnetic frame, placing the EP tube in an incubator at 37 ℃ for drying for 30 minutes, eluting by 20 mu L of TE, and numbering Pkna-1 and Pkna-2.
As a reference, the DNA sample also included a positive reference, healthy pupae (negative reference), and deionized water.
Carrying out tussah microsporidian detection by using a PCR (polymerase chain reaction) technology:
each 25. mu.L system contained 0.5. mu. L, rTaq 0.25. mu.L, 10 XBuffer 2.5. mu.L, 2. mu.L dNTPs, 17.25. mu.L deionized water and 2. mu.L DNA template of the primers (10pM) shown in SEQ ID NO:6 and SEQ ID NO:7, respectively. The PCR reaction condition is 95 ℃ for 4 minutes; 40 cycles of 95 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 1 minute, extension at 72 ℃ for 5 minutes, adding a nucleic acid staining agent to the obtained PCR product, performing gel electrophoresis with 2% agarose, and observing the result under ultraviolet light after the electrophoresis is finished, as shown in FIG. 5.
Description of the experimental results: the number of gel electrophoresis pore canals 1-11 is FT-1, FT-2, S-1, S-2, Pk-1, Pk-2, Pkna-1, Pkna-2, deionized water, healthy pupa reference and positive reference respectively. As shown in FIG. 4, FT-1, FT-2, S-1, S-2, Pk-1, Pkna-1 and Pkna-2 gave a positive result, and Pk-2 gave a negative result. The practical Pk-2 is a positive result, but a false negative result occurs due to poor digestion effect of the proteinase K caused by blackening, so that compared with the traditional method, the method disclosed by the invention has the advantages of strong anti-interference capability in the DNA extraction process, higher quality of extracted DNA, lower false positive rate of detection on microsporidia, better sensitivity and higher accuracy.
Example 6
For 94 cases of tussah female moths which have been laid with eggs and are negative by microscopic examination, fat bodies in the belly of the tussah female moths are respectively placed in an EP tube filled with 200 mu L of lysate for soaking overnight, the next day, the lysate is transferred to a 96-well plate by 100 mu L, 10 mu L of magnetic beads are respectively added, 70 mu L of absolute ethyl alcohol is added, the mixture is slightly shaken and evenly mixed, the mixture is placed on a magnetic frame for separation, the liquid phase is poured out, the mixture is washed for 3 times by 70 percent of ethyl alcohol, a blast oven is dried for 30 minutes at 37 ℃, and each 30 mu LTE is eluted at minus 20 ℃ for standby.
The quality of the extraction was checked using conventional PCR. Each 25 mu L system contains 0.5 mu L of tussah 28SrRNA specific primers (10pM) shown as SEQ ID NO. 8 and SEQ ID NO. 9, 2 mu L, rTaq 0.25.25 mu L of template DNA, 2.5 mu L of 10 XBuffer, 2 mu L of dNTPs and 17.25 mu L of deionized water. The DNA template included 94 samples, 1 water negative reference and 1 positive plasmid reference as described above. The PCR reaction condition is 95 ℃ for 4 minutes; 40 cycles at 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, 72 ℃ for 1 minute, and extension at 72 ℃ for 5 minutes. Adding nucleic acid staining agent into the two PCR products, performing gel electrophoresis with 2% agarose, observing the result under ultraviolet light after electrophoresis, and obtaining specific bands of about 106bp except water.
And (3) detecting the microsporidian of tussah by using conventional PCR. Each 25. mu.L system contained 0.5. mu. L, rTaq 0.25. mu.L, 10 XBuffer 2.5. mu.L, 2. mu.L dNTPs, 17.25. mu.L deionized water and 2. mu.L DNA template of the primers (10pM) shown in SEQ ID NO. 4 and SEQ ID NO. 5. The PCR reaction condition is 95 ℃ for 4 minutes; carrying out 40 cycles at 95 ℃ for 30 seconds, 65 ℃ for 30 seconds and 72 ℃ for 1 minute, extending at 72 ℃ for 5 minutes, adding a nucleic acid staining agent into the obtained PCR product, carrying out gel electrophoresis by using 2% agarose for result judgment, wherein 4 samples in 94 samples have microsporidian specific bands as positive references and are positive; water and the remaining 90 samples showed no specific bands and were negative.
The embodiment shows that the DNA extraction method has the advantages of simplicity, rapidness and high flux, and meanwhile, the extracted DNA has high quality, and the detection sensitivity, the specificity and the detection rate are high by combining a molecular detection technology.
While the molecular detection methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the inventive techniques can be practiced with modification, or with appropriate modification and combination, of the methods described herein without departing from the spirit and scope of the invention. It is specifically noted that all similar substitutions and alterations: such as similar primer substitutions and modifications, such as changes in PCR cycling temperature, such as reasonable substitutions and modifications of the components of the PCR reaction system used, such as reasonable variations in base concentration, treatment time, and the like, which are apparent to those skilled in the art, are to be considered as included within the spirit, scope, and content of the present invention.
Sequence listing
<110> institute of biotechnology of the university of academy of agricultural sciences of Liaoning province
<120> a group of tussah microsporidian detection primers and application thereof
<141> 2017-07-27
<160> 9
<170> SIPOSequenceListing 1.0
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<213> Artificial Synthesis of primer
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<210> 2
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<212> DNA
<213> Artificial Synthesis of primer
<400> 2
tacctggtaa attaccccgc g 21
<210> 3
<211> 47
<212> DNA
<213> Artificial Synthesis of a methoxylated primer
<400> 3
tcgtgcatgc acttccacaa ggagtggatt gtgcggctta atttgac 47
<210> 4
<211> 23
<212> DNA
<213> Artificial Synthesis of primer
<400> 4
gtgcggctta atttgactca acg 23
<210> 5
<211> 23
<212> DNA
<213> Artificial Synthesis of primer
<400> 5
ctatatgagg gtctcacatc ttg 23
<210> 6
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<212> DNA
<213> Artificial Synthesis of primer
<400> 6
gacggaagaa taccacaagg agt 23
<210> 7
<211> 24
<212> DNA
<213> Artificial Synthesis of primer
<400> 7
ctatatgagg gtctcacatc ttgt 24
<210> 8
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<212> DNA
<213> Artificial Synthesis of primer
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<213> Artificial Synthesis of primer
<400> 9
tagttcacca tctttcgggt cc 22
Claims (9)
1. The base sequence of the tussah microsporidian detection primers is shown in SEQ ID No. 1-3.
2. The use of the primer of claim 1 for the detection of microsporidian of tussah silkworm, wherein the detection is carried out by a recombinase polymerization amplification technique; the method comprises the step of carrying out recombinase polymerization amplification reaction on template DNA, wherein the configuration reaction system is as follows:
the reaction conditions are 37-40 ℃, and the amplification time is 5-60 minutes.
3. Use according to claim 2, characterized in that: the method for obtaining the template DNA is as follows:
cracking the microsporidian sample of tussah with alkali solution, and enriching, separating and purifying DNA with magnetic beads.
4. Use according to claim 3, characterized in that: the alkali is inorganic alkali; the concentration of the alkali solution is 0.05-1M; the alkali solution also contains sodium dodecyl sulfate with the concentration of less than 10 percent.
5. Use according to claim 3, characterized in that: the treatment time in the cracking treatment process is 1 minute to 40 days; the treatment temperature is-20 to 40 ℃; the treatment process also comprises zero to multiple freezing and thawing processes, and the single freezing time is 10 minutes to 40 days.
6. Use according to claim 3, characterized in that: in the process of enriching, separating and purifying DNA by using magnetic beads, directly putting magnetic microspheres into a mixed solution containing a sample to be detected, then separating by using an external magnetic field, and adopting ddH (ddH) after separation2O, TE buffer solution or PCR reaction solution.
7. Use according to claim 3, characterized in that: the magnetic beads are magnetic microspheres containing silicon dioxide and ferroferric oxide components.
8. Use according to claim 3, characterized in that: the process of DNA enrichment by using the magnetic beads comprises the steps of adding the magnetic beads or the suspension of the magnetic beads into the alkali solution, uniformly mixing, and standing for a moment; and absolute ethyl alcohol is also added into the aqueous alkali, and the volume ratio of the absolute ethyl alcohol to the aqueous alkali is 0.7-1: 1.
9. A tussah microsporidian molecular detection kit, characterized by comprising the primer of claim 1 and a reagent combination used in the method for the application of any one of claims 2 to 8.
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