CN109055573B - Method for rapidly identifying common storage spider mites based on multiple PCR technology - Google Patents
Method for rapidly identifying common storage spider mites based on multiple PCR technology Download PDFInfo
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Abstract
The invention discloses a method for rapidly identifying common storage spider mites based on a multiple PCR technology, which comprises the steps of firstly extracting genome DNA of a single spider mite, taking the extracted DNA as a PCR template, designing a specific primer of the spider mite on ribosome DNA, establishing a multiple PCR identification method under a common PCR system and a common PCR program, and determining the species of the spider mites through agarose gel electrophoresis detection; various experimental verifications show that the method is rapid, efficient and high in specificity, and compared with a powdery mite morphological identification method, the method does not need complete specimens, eggs, larvae, nymphs, dormancy bodies and other juvenile stages, hidden seeds and damaged samples can also be identified; compared with an enzyme digestion method and a DNA sequencing method, the method is simple, convenient and economic, only one PCR reaction is needed, the complex steps of multiple PCR identifications are avoided, the workload is reduced, the detection time is shortened, and the method can be operated by general technicians.
Description
Technical Field
The invention belongs to the technical field of multiplex PCR, and particularly relates to a method for rapidly identifying common storage spider mites based on the multiplex PCR technology.
Background
The flour mites belong to Acarina (Acari), Eudermans (Acariformes) and Astigmata, are small in size, various in variety and wide in habitat, can be bred in a large amount in the stored substances, mainly eat organic residues of plants or animals, but mainly take stored grains, dried fruits, fur and the like in the stored substances. The aleyrodids not only cause great loss of quality and quantity of stored materials, but also can cause mite allergic diseases, even lung acariasis, intestinal acariasis, uroacariasis and the like. Under the global economy, the frequency and the convenience of international trade and tourism provide convenience for the international spreading and diffusion of the pink mites, and 98 kinds of the pink mites which are known to be widely harmful worldwide are provided, wherein the common pink mites comprise: spider mites (Acarus sroro), Tyrophagus putrescentiae (Tyrophagus putrescentiae), Dermatophagoides naesbioti (Suidasia neostiti), Dermatophagoides pteronyssinus (Dermatophagoides pteronyssinus), Dermatophagoides farinae (Dermatophagoides farina), and Lepidoglyphus insect (Lepidoglyphus destructor). In recent years, the flour mites are pests which are often captured by entry and exit, and the inspection of the flour mites has become an important aspect of the inspection of the entry and exit of stored grains. In the real-time detection of the stored grain mites entering and exiting a large number of agricultural product trades, the demand for a simple, quick, economic and accurate identification technology is urgent.
At present, the identification of the imported quarantine flour mite is mainly carried out according to the morphological characteristics. Due to the tiny size and various varieties of the flour mites, the rapid and accurate identification is quite difficult when the flour mites frequently encounter diseases such as young stages (including eggs, larvae, nymphs and dormant bodies), hidden species and damaged samples. In recent years, molecular marker-based techniques have been widely used in the field of species identification. Among them, the transcribed internal spacer 2(ITS2) of ribosomal gene has a high rate of evolution, and thus has a large difference in sequence length and base composition even in closely related species, and is widely used as an effective molecular marker. The method for identifying the acarid based on the molecular marker technology comprises the following steps: polymerase chain reaction and PCR-RFLP, but the above method steps are complicated and the identification time is long.
Disclosure of Invention
According to the defects of the prior art, the technical problem to be solved by the invention is to provide a method for rapidly identifying common storage spider mites based on a multiplex PCR technology, the method overcomes the problems in the existing technology for identifying common storage spider mites, firstly, the genomic DNA of a single spider mite is extracted, the extracted DNA is used as a PCR template to perform clone sequencing and analysis of ribosome ITS sequences, and 6 specific downstream primers are designed based on the sequence length and the difference of base composition of ITS2 and are respectively named as Acas-R (Dermatophagoides farinae), Tyrp-R (Tyrophagus putrescentiae), Suin-R (Suin-crinophagus naeslunensis), Derp-R (house dust mite), Derf-R (dust mite) and Lepd-R (lepidoptera pest). As 5.8S is well conserved among different species of aleyrodids, an Asti-F primer is designed as a universal upstream primer of 6 specific primers. Multiple PCR amplification is carried out on the basis of a reaction system and a program of common PCR, and the identification of 6 common storage spider mites is realized simultaneously through agarose gel electrophoresis. And subsequent experiments prove that the primers have no obvious difference in amplification results no matter whether the primers carry out common PCR specific amplification or multiple PCR specific amplification, and prove that the multiple PCR primers designed by the invention have high specificity. Only one PCR reaction is needed, 6 different product lengths can be amplified simultaneously, and 6 aleyrodids can be directly identified by simply judging the sizes of the products.
In order to solve the technical problems, the invention adopts the technical scheme that:
a method for rapidly identifying common storage spider mites based on a multiplex PCR technology comprises the following steps:
(1) extracting the genome DNA of a single acarid;
(2) amplifying ribosome rDNA ITS sequences by using the extracted gene group DNA of the aleyrotes dermatatus as a template, designing 7 primers to be mixed as primers of PCR based on the differential sites of ITS regions, wherein the primers are as follows:
Asti-F：5’-GTGGTGGATCACTCGGC-3’；
Acas-R：5’-GGCAAACATACATTGGCTCCC-3’；
Tyrp-R：5’-AGCCCCCTACATTAGACTACCA-3’；
Suin-R：5’-AGAGTCGACACGACCAACTG-3’；
Derp-R：5’-TGGAAATGACCTGACGACGTT-3’；
Derf-R：5’-TGGAAATGACCTGACGACGTT-3’；
Lepd-R：5’-ACCCGATCGATGCAATATGCT-3’；
(3) on the basis of a reaction system and a program of common PCR, adding 7 primers to carry out multiple PCR amplification reaction;
(4) performing electrophoresis detection on the multiple PCR reaction product, using 100+50bp DNAlader DNAMark J as reference, performing PCR product size judgment, and determining the variety of the aleyrotes tetranychii: the tetranychus urticae koch is 429bp, Tyrophagus putrescentiae 355bp, Dermatophagoides naeslundii 192bp, house dust mite 215bp, Dermatophagoides farinae 280bp, and Dermatophagoides pteronyssinus 306 bp.
Preferably, in the 7 primers in step (2), Asti-F is an upstream primer, and the remaining 6 primers are specific downstream primers.
Preferably, the conditions of the ordinary PCR reaction and the multiplex PCR amplification reaction described in step (3) are both: pre-denaturation at 94 deg.C for 3 min; 26 cycles: denaturation at 94 deg.C, 30sec, annealing at 60 deg.C, 40sec, and extension at 72 deg.C for 1 min; the extension was stopped at 72 ℃ for 10min and finally at 4 ℃ for 59 min.
Preferably, the electrophoresis in the step (4) is agarose gel electrophoresis, and the type of the acarid is judged according to the size of the PCR product as follows: the tetranychus urticae koch is 429bp, Tyrophagus putrescentiae 355bp, Dermatophagoides naeslundii 192bp, house dust mite 215bp, Dermatophagoides farinae 280bp, and Dermatophagoides pteronyssinus 306 bp.
The invention has the beneficial effects that:
1. the invention utilizes the multiple PCR technology to simultaneously amplify the specific genes of the spider mites, the Tyrophagus putrescentiae, the Dermatophagoides naeslungsiensis, the dermatophagoides pteronyssinus, the dust mites and the lepidoptera pest at one time, and obtains the identification result through simple judgment of the agarose gel electrophoresis result.
2. Compared with the traditional morphological identification, the method does not need a complete specimen, can also identify the residue and the young stage (eggs, larvae and nymphs), and has higher accuracy and specificity of the identification result. The experimental result shows that for the same aleyrodids, the primer has specificity no matter whether the primer carries out common PCR specific amplification or multiplex PCR specific amplification, and the amplification results have no obvious difference.
3. Compared with an enzyme digestion method and a DNA sequencing method, the method is simple, convenient and economic, only one PCR reaction is needed, the complex steps of multiple PCR identifications are avoided, the workload is reduced, the detection time is shortened, and the method can be operated by general technicians.
4. The requirement for the DNA template is not high, and even the DNA template stored for a long time or the fragment of the template from the polypide has good amplification result.
5. The experimental operation is simple, the reaction system and the procedure are similar to those of common PCR, and only one PCR reaction is needed, so that 6 common pink mites can be identified simultaneously. The method is suitable for entry and exit ports, and accurate, rapid and high-throughput port quarantine is realized.
Drawings
The contents of the drawings and the reference numerals in the drawings are briefly described as follows:
FIG. 1 is a schematic diagram showing the PCR electrophoresis result of 6 kinds of flour mites of the present invention.
I: common PCR electrophoresis results of 6 kinds of aleyrotes dermatatus, II: multiple PCR electrophoresis result of 6 kinds of flour mites
1. Dermatophagoides pteronyssinus, 2. Tyrophagus putrescentiae, 3. Dermatophagoides Naphschnei, 4. dermatophagoides pteronyssinus, 5. dermatophagoides pteronyssinus, 6. Lepidoptera
Detailed Description
The following embodiments are described in further detail to help those skilled in the art to more fully, accurately and deeply understand the inventive concept and technical solutions of the present invention.
Example 1
Extracting 6 powder mite monomer genome DNAs by STE method
(1) In a centrifuge tube containing a single female mite, 20 μ L of STE buffer (10mM Tris-HCl, 1mM EDTA, 100mM NaCl, PH 8.0) was added, and then the centrifuge tube was ground onto ice, respectively;
(2) adding 0.5 mu L of proteinase K (25mg/mL) into the centrifugal tubes respectively, and fully and uniformly mixing;
(3) centrifuging 1000g for 1min at room temperature, and subjecting the mixture to 56 deg.C water bath for 60 min;
(4) after the water bath is finished, initially denaturing at 95 ℃ for 5min, and inactivating the protease K;
(5) at room temperature, centrifugation was carried out for 1min at 2000g, and 2. mu.L of the supernatant was used as a template for PCR reaction, and individual DNA samples of the powdery mites were numbered and stored in a refrigerator at-20 ℃ for further use.
Example 2
2 groups of PCR amplification are carried out, the group I carries out common PCR amplification on 6 aleurites mite monomer genome DNA, and the group II carries out multiplex PCR amplification on 6 aleurites mite monomer genome. Both templates are the same monomer aleyrodids genome DNA, one aleyrodids corresponds to one PCR reaction, the volume of the reaction system is 25 mu L, and the group I specific reaction reagents and the using amount are shown in Table 2; group ii specific reagents and amounts are shown in table 3:
common PCR amplification system for 26 kinds of aleyrotes tetranychii in table
Table 36 multiple PCR amplification system for flour mites
The 6 kinds of aleyrodids are aleyrotes destructor, Tyrophagus putrescentiae, Dermatophagoides naeslundii, house dust mite, aleyrodids dermatitidis and Dermatophagoides pteronyssinus respectively; the 7 pairs of primers are Asti-F, Acas-R, Tyrp-R, Suin-R, Derp-R, Derf-R, Lepd-R respectively; the reaction procedure was either normal PCR or multiplex PCR: 94 ℃ for 3min, 26 × (94 ℃ for 30sec, 60 ℃ for 40sec, 72 ℃ for 1min), 72 ℃ for 10min, and 4 ℃ for 59 min.
Example 3
And detecting the multiple PCR reaction products by 2% agarose gel electrophoresis, and judging the size of the PCR products by using 100+50bp DNA Ladder DNA Mark J as reference to determine the species of the aleyrotes tetranychii. Judging the variety of the aleyrotes tetranychii according to the size of the PCR product as follows: the tetranychus urticae koch is 429bp, Tyrophagus putrescentiae 355bp, Dermatophagoides naeslundii 192bp, house dust mite 215bp, Dermatophagoides farinae 280bp, and Dermatophagoides pteronyssinus 306 bp.
The invention has been described in an illustrative manner, and it is to be understood that the invention is not limited to the precise form disclosed, and that various insubstantial modifications of the inventive concepts and solutions, or their direct application to other applications without such modifications, are intended to be covered by the scope of the invention. The protection scope of the present invention shall be subject to the protection scope defined by the claims.
Sequence listing
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Claims (3)
1. A method for rapidly identifying common storage spider mites based on a multiplex PCR technology is characterized by comprising the following steps:
(1) extracting the genome DNA of a single acarid;
(2) amplifying ribosome rDNA ITS sequences by using the extracted gene group DNA of the aleyrotes dermatatus as a template, designing 7 primers to be mixed as primers of PCR based on the differential sites of ITS regions, wherein the primers are as follows:
Asti-F：5’-GTGGTGGATCACTCGGC-3’；
Acas-R：5’-GGCAAACATACATTGGCTCCC-3’；
Tyrp-R：5’-AGCCCCCTACATTAGACTACCA-3’；
Suin-R：5’-AGAGTCGACACGACCAACTG-3’；
Derp-R：5’-TGGAAATGACCTGACGACGTT-3’；
Derf-R：5’-TGGAAATGACCTGACGACGTT-3’；
Lepd-R：5’-ACCCGATCGATGCAATATGCT-3’；
(3) on the basis of a reaction system and a program of common PCR, adding 7 primers to carry out multiple PCR amplification reaction;
(4) carrying out electrophoresis detection on the multiple PCR reaction products, using 100+50bp DNA Ladder DNA Mark J as reference, carrying out PCR product size judgment, and determining the species of the aleyrotes tetranychii;
wherein, the electrophoresis in the step (4) is agarose gel electrophoresis, and the variety of the aleyrotes tetranychii is judged to be: the tetranychidae is 429bp, the Tyrophagus putrescentiae is 355bp, the Dermatophagoides naeslundii is 192bp, the dermatophagoides pteronyssinus is 215bp, the dermatophagoides farinae is 280bp, and the Hemochagoides pteronyssinus is 306 bp;
the test results are defined for non-disease diagnostic purposes.
2. The method for rapidly identifying common storage varroa mites on the basis of the multiplex PCR technology as claimed in claim 1, wherein in the 7 primers in step (2), Asti-F is an upstream primer, and the other 6 primers are specific downstream primers.
3. The method for rapidly identifying common storage spider mites on the basis of the multiplex PCR technology as claimed in claim 1, wherein the conditions of the common PCR reaction and the multiplex PCR amplification reaction in step (3) are as follows: pre-denaturation at 94 deg.C for 3 min; 26 cycles: denaturation at 94 deg.C, 30sec, annealing at 60 deg.C, 40sec, and extension at 72 deg.C for 1 min; the extension was stopped at 72 ℃ for 10min and finally at 4 ℃ for 59 min.
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| WO2015007787A1 (en) * | 2013-07-16 | 2015-01-22 | Alk-Abelló A/S | Molecular identification of allergy causing mites by pcr |
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| CN103805708A (en) * | 2014-02-20 | 2014-05-21 | 南京农业大学 | PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) identification method for Chinese tetranychus common species |
| CN104521828A (en) * | 2014-12-18 | 2015-04-22 | 中国科学院水生生物研究所 | Method for building gyrodactylus purebred artificial infection system |
| ES2674128A1 (en) * | 2016-12-27 | 2018-06-27 | Universidad De Salamanca | Method for diagnosing allergic sensitization in a subject (Machine-translation by Google Translate, not legally binding) |
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