CN112921106A - Real-time fluorescent PCR detection method for bark beetle of yellow fir, and primer and probe for detection - Google Patents
Real-time fluorescent PCR detection method for bark beetle of yellow fir, and primer and probe for detection Download PDFInfo
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Abstract
The invention provides a method for detecting bark beetle of yellow fir, and the combination of specific primers (HS-F and HS-R) and a probe (HS-P) which are screened by tests can achieve the aim of identifying the bark beetle in a Taq-Man real-time fluorescent quantitative PCR method. The invention is suitable for the quarantine identification of bark beetles in different states, makes up the defects of the conventional morphological identification method, and compared with the DNA bar code identification technology applied in the prior art, the primer and the MGB probe have strong specificity, high sensitivity, simple experimental operation and short time; the identification result is visual, and the result can be obtained by observing the amplification curve. The research can meet the original requirement of rapid clearance in and out of the border, improve the detection rate, effectively prevent the invasion and diffusion of the dangerous bark beetle and have important significance for protecting the agriculture and forestry production and ecological safety of China.
Description
Technical Field
The invention belongs to the technical field of molecular identification of biological species, and relates to a real-time fluorescence PCR detection method for bark beetles, in particular to a detection method for bark beetles of yellow fir and application thereof; in addition, the invention also relates to a primer and a probe for detecting the bark beetle of the yellow fir.
Background
Dendroctonus of bark beetle belongs to Coleoptera of Coleoptera, Curculionidae of elephant family and Scolyytina of bark beetle family, and is an important pest for trunk borer of forest ecosystem, which mainly causes harm to trees of Pinus, spruce, yellow cedar and the like, and the genus (non-Chinese species) is listed in the name of imported plant quarantine pest in China. The bark beetle genus is known in 20 species all over the world, 18 species are distributed in central and north america, and 3 species are distributed in europe and asia. The yellow fir bark beetle Dendroctonusjudaotsugae belongs to a non-Chinese species of bark beetle, is distributed in countries such as Canada, America, Mexico and the like, and hosts of the yellow fir bark beetle Dendroctonusjudaotsugae, P.
With the increasing frequency of foreign trade and the increasing quantity of imported wood in China, the variety and the quantity of pests intercepted and captured by the port are continuously increased, so that the rapid and accurate quarantine identification of the pests has important significance for preventing quarantine species from invading.
At present, identification based on external morphological characteristics aiming at the species of bark beetles is still the most common method of quarantine systems. However, the morphological identification mainly takes adults as a research object, and a considerable part of the ips caught by the port is larvae, pupae or adults which are partially broken, so that the morphological identification is difficult to realize accurate identification. Moreover, the species of the large and small moth pests are similar in shape, small in difference, difficult to identify and high in requirements on experience and classification bases of identification personnel. Therefore, it is important and urgent to search for new methods to make up for the deficiencies of morphological identification. In recent years, with the development of molecular biology techniques, various techniques such as DNA barcode technique, conventional PCR detection technique, real-time fluorescence quantitative PCR detection technique, Restriction Fragment Length Polymorphism (RFLP) technique, and microsatellite technique are gradually applied to molecular identification methods of species. However, the research reports on the molecular identification technology of the bark beetle in China are still few, and the feasibility and the accuracy of the rapid identification of 6 bark beetle insects which are frequently intercepted by the port of China based on the mitochondrial COI gene of Yingyusheng and the like are researched (see the details: Yingyusheng, Zhang Sai, Anyulin, the identification of the bark beetle insects based on the mitochondrial COI gene [ J ]. Fujian forestry technology, 2014, 41 (01): 63-67.); sequence analysis of 17 species of the large and small silverfish through the 867bp fragment sequence of the mitochondrial COI gene by the Jing and the like shows that the COI gene can be used as a basis for classifying the large and small silverfish insects (see in detail: Jing, Zhengsi bamboo, Anyulin, Yang dawn. the DNA barcode of the large and small silverfish insects based on the mitochondrial COI gene [ J ]. Jiangsu agricultural science, 2014, 42 (03): 30-32.); tiger et al showed that both 5 '-and 3' -terminal sequences of mtDNA COI gene can effectively distinguish between Rhynchophorus macropteres and Rhus niveus macropteres based on DNA barcode technology (see: Setaria arvensis, Argomphytum robustum, Lonicera caerulea, Lossenbin, Dawny, Wangxin, Heyao, Guo Yang. identification of molecules of Rhus macropteres and Rhus niveus based on DNA barcode [ J ]. plant quarantine, 2020, 34 (01): 30-34.). However, the real-time fluorescent PCR rapid identification technology research is not carried out in the identification of the bark beetle molecules at present.
Real-time fluorescence quantitative PCR is a highly sensitive nucleic acid quantitative technique developed on the basis of PCR technology, and has been widely applied to various fields of biology, medicine, agriculture, food, environmental protection and the like in recent years due to the advantages of accuracy, specificity, sensitivity, rapidness and the like. The method is widely applied in the field of insect quarantine, for example, according to the COI gene sequence of fire ant, such as old rock, a primer and a TaqMan probe which have specificity to the fire ant are designed, and the identification of the fire ant with different insect states can be carried out (see: Chenyan, Chengning, Zhushuiyang, Chenhongjun. fire ant real-time fluorescent molecule detection method [ J ] plant quarantine, 2005 (04): 204. plus 206.); feng et al designed 1 pair of primers and specific TaqMan probe according to the conserved region sequence of leaf miner and established a real-time fluorescence quantitative PCR identification system of leaf miner, which can accurately identify various insect states of leaf miner (see: Xiaoan Feng, Chen Nai-Zhong, Ma Jun, Zhu Shui-fang, Hu Xue-nan. molecular identification of Liriomyza trifolii (Burgess) (Dipt., Agromozidae) base on real-time PCR [ J ] nal Applied Entomogy, 2007, 131 (8)); ginger sail based on DNA bar code sequence, 27 kinds of Chinese main quarantine fruit fly species specific primers and specific TaqMan-MGB probes (see the ginger sail. the research of Chinese quarantine fruit fly molecule identification technology system [ D ]. university of agriculture, 2015 ]); xulang and the like realize the rapid and accurate identification of the new pineapple gray scale and the pineapple gray scale by using a TaqMan real-time fluorescence PCR method (see detail: Xulang, Linwei, Huangduling, Zhang Weifeng, Luxiayu, Wangying, Zhengzheng, Jiayi, Lou Ding Feng, Yudao Jiang.
The inventor takes the yellow fir bark beetle as a research object, selects the same genus bark beetles which are frequently intercepted by the port, such as the black resin bark beetle D.terebrans, the red resin bark beetle D.valens, the red wing bark beetle D.rufipennis, the mountain pine bark beetle D.armandi, the south pine bark beetle D.frontalis and the other bark beetle, namely the bark beetle Gnatrotrichus retusus as a contrast, designs a specific primer pair and a Taq probe according to the mtDNA COI gene sequence, and establishes a molecular identification technology aiming at the yellow fir bark beetle by utilizing the TaqMan real-time fluorescence PCR technology to obtain the invention.
Disclosure of Invention
The invention aims to provide a method for quickly and accurately identifying bark beetles of yellow fir, which facilitates the quarantine of wood pests.
Based on the above purpose, the invention provides a method for detecting bark beetle of yellow fir, which comprises the following steps,
(1) designing a primer and a probe;
the sequences of the primer and the probe are as follows:
the upstream primer HS-F sequence is as follows: 5'-GGAATTGACTAGTTCCTT-3', respectively;
the downstream primer HS-R sequence is as follows: 5'-ATTGCGTAGTCAACAGAGG-3', respectively;
the probe primer HS-P sequence is as follows: 5'-CTACTCTTAAGAAGAATTG-3', respectively;
the 5 'end of the probe primer is connected with a fluorescent group FAM, and the 3' end of the probe primer is connected with a non-fluorescent quenching group.
(2) Extracting genome DNA of bark beetle of yellow fir;
(3) and (2) performing real-time fluorescent PCR detection by using the primers and the probes in the step (1), and distinguishing the bark beetle from other quarantine bark beetle insects according to an amplification curve.
As a preferable technical method of the invention, the step (2) is implemented by the following steps: taking the foot or cephalothorax muscle tissue of the test yellow fir bark beetle specimen to extract the genome DNA.
As a preferable technical method, the reaction system of the real-time fluorescence PCR detection method in the step (3) comprises the following components: premix Ex Taq (Probe qPCR) (2X) 10. mu.L, forward primer HS-F (10. mu.M) 0.4. mu.L, reverse primer HS-R (10. mu.M) 0.4. mu.L, Probe HS-P (10. mu.M) 0.8. mu.L, ROX Reference Dye II (50X) 0.2. mu. L, DNA template 2. mu.L, and sterilized water 6.2. mu.L.
As a preferable technical method, the reaction procedure of the real-time fluorescence PCR detection method in the step (3) is as follows: pre-denaturation at 95 ℃ for 30sec, denaturation at 95 ℃ for 5sec, annealing at 59 ℃ for 30sec, and 40 cycles of denaturation-annealing.
The real-time fluorescent PCR detection method adopted by the invention adopts a Taq-Man MGB probe. The Taq-Man MGB probe is a novel probe developed on the basis of a common Taq-Man probe, and has the following advantages compared with the common probe: the 3' end is added with Minor Groove Binder (MGB miniature Groove Binder) molecules which can be combined with a target template, the Tm value of the probe is increased, so that the length of the probe is shorter and is only 13-18bp, and the possibility of nonspecific hybridization is obviously reduced; the 3' end adopts a quenching group which does not emit light per se and is closer to the fluorescent reporter group, so that the non-specific fluorescent background signal is reduced, the experimental result is more accurate, and the detection sensitivity is improved; the optimization of the detection components is simpler, the time for establishing a detection system is reduced, and the detection working efficiency is improved.
In addition, the invention also provides a primer for detecting the bark beetle of the yellow fir, which has the specific sequence as follows:
the upstream primer HS-F sequence is as follows: 5'-GGAATTGACTAGTTCCTT-3', respectively;
the downstream primer HS-R sequence is as follows: 5'-ATTGCGTAGTCAACAGAGG-3', respectively;
in addition, the invention also provides a probe for detecting the bark beetle of the yellow fir, which has the specific sequence as follows:
5'-CTACTCTTAAGAAGAATTG-3', wherein the 5 'end is connected with a fluorescent group FAM and the 3' end is connected with a non-fluorescence quenching group.
In addition, the invention also provides a kit for detecting the ips typographus, which comprises a primer and a probe, wherein the specific sequences of the primer and the probe are as follows:
the upstream primer HS-F sequence is as follows: 5'-GGAATTGACTAGTTCCTT-3', respectively;
the downstream primer HS-R sequence is as follows: 5'-ATTGCGTAGTCAACAGAGG-3', respectively;
the probe primer HS-P sequence is as follows: 5'-CTACTCTTAAGAAGAATTG-3', respectively;
the 5 'end of the probe is connected with a fluorescent group FAM, and the 3' end of the probe is connected with a quenching group.
The invention also provides an application of the real-time fluorescence PCR detection method for the bark beetle of the yellow fir in quarantine of wood pests, and particularly relates to the quarantine of the wood pests of genus Yew Pseudotsuga, genus Larix and genus Taxus Tsuga.
Compared with the prior art, the detection method of the bark beetle of the yellow fir has the following beneficial effects or advantages:
the invention provides a method for detecting bark beetle of yellow fir, and the combination of specific primers (HS-F and HS-R) and a probe (HS-P) which are screened by tests can achieve the aim of identifying the bark beetle in a Taq-Man real-time fluorescent quantitative PCR method. The invention is suitable for the quarantine identification of bark beetles in different states, makes up the defects of the conventional morphological identification method, and compared with the DNA bar code identification technology applied in the prior art, the primer and the MGB probe have strong specificity, high sensitivity, simple experimental operation and short time; the identification result is visual, and the result can be obtained by observing the amplification curve. The research can meet the original requirement of rapid clearance in and out of the border, improve the detection rate, effectively prevent the invasion and diffusion of the dangerous bark beetle and have important significance for protecting the agriculture and forestry production and ecological safety of China.
Drawings
FIG. 1 is a schematic diagram showing the positions of specific primers (HS-F and HS-R) and probe (HS-P) on the bark beetle mtDNA of the invention.
FIG. 2 shows the result of real-time fluorescent quantitative PCR specificity detection of combinations of primers (HS-F and HS-R) and probe (HS-P) for bark beetle of the yellow fir.
In the figure, A is a Japanese beetle, B is a red bark beetle, C is a red wing bark beetle, D is a Pinus armandi bark beetle, E is a Pinus armandi bark beetle, F is a black bark beetle, G is a blunt bark beetle, and H is ddH2O。
FIG. 3 shows the result of the real-time fluorescence PCR detection of bark beetle in yellow fir.
In the figure, the concentrations of A, B, C, D, E, F corresponding to the template DNAs were 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 0.1 ng/. mu.L, and 0.001 ng/. mu.L, respectively, and the concentration of G corresponding to the template ddH2O。
Detailed Description
The technical aspects of the present invention will be further explained below with reference to the drawings, but the present invention is not limited to the following embodiments.
A real-time fluorescence PCR detection method for bark beetle of yellow fir comprises the following steps:
(1) design of primers and probes
The following is the design and synthesis process of the specific primer and probe used in the detection method of the bark beetle of the yellow fir.
The MeGA6.0 software was used to align the D.pseudocotsugae, D.southern pine bark beetle, D.frontalis, D.valens red-fat bark beetle, D.rufipennis red-wing bark beetle, D.armandi bark beetle, D.terebrans, D.adjacentus, D.apium bark beetle, D.approximatus, D.brix bark beetle, D.brevicornis, D.brevicominis, D.jeffreyi, D.sancaja bark beetle, D.murrayanae, D.podnderrosae, D.puncatus bark beetle, D.podereus bark beetle, D.podagratus, D.punctatus, D.deciduous bark beetle, D.sikonjax, D.meyerba bark beetle, D.parviensis, and D.codylensis size. According to the comparison result, the specific site of the bark beetle of the yellow fir is manually searched, and species specific primers are designed on the upstream and downstream of the specific site. The designed primers were checked for the presence of mismatches, dimers and hairpin structures using Primer Premier 5.0 software and for the presence of homologous sequences using the Blast program provided in NCBI. Then, in the specific primer range (including primer sequences) of the bark beetle, artificially searching mutation sites which can be distinguished from other species; probes were designed at the site of the species-specific site by Primer Express 3.0 software (applied biosystems) in combination with the TaqMan-MGB probe design principle. The designed probe has a fluorescent reporter group at the 5' end labeled as FAM (carboxyfluorescein). The specific primers and MGB probes used in the invention are synthesized by Beijing Optimalaceae Biotechnology Co.
The invention obtains a group of specific primers and probe primers which can be used for identifying the bark beetle after screening, the position of the primers on the bark beetle mtDNA of the bark beetle is shown in figure 1, and the specific sequences are as follows:
the upstream primer HS-F sequence is as follows: 5'-GGAATTGACTAGTTCCTT-3', respectively;
the downstream primer HS-R sequence is as follows: 5'-ATTGCGTAGTCAACAGAGG-3', respectively;
the probe primer HS-P sequence is as follows: 5'-CTACTCTTAAGAAGAATTG-3', respectively;
wherein, the 5 'end of the probe primer is connected with a fluorescent group FAM, and the 3' end of the probe primer is connected with a non-fluorescent quenching group.
(2) Extracting genome DNA of bark beetle of yellow fir
Specifically, the tested insects in the invention are all from size silverfish specimens obtained by port capture and scientific research communication, the specific information is shown in table 1, and all the specimens are soaked in absolute ethyl alcohol and stored in a refrigerator at-20 ℃:
table 1 summary of insect source collection information for test
After the test insects are cleaned and surface moisture is sucked, the test insects are fully ground by using an electric tissue grinder (Tiangen), the genomic DNA of the test insects is extracted by using a Tiangen blood/cell/tissue genomic DNA extraction kit (DP304), and the using method is changed according to the actual situation. The specific process is as follows:
1) the pipette sucks 20. mu.L of buffer GA into a clean 1.5mL centrifuge tube;
2) cutting the feet of the test insects, washing the feet with sterilized water, sucking off excessive water, placing the feet in a centrifugal tube in the step 1), fully grinding the feet by using an electric tissue grinder, cleaning a grinding rod by using 180 mu L of buffer solution GA, and adding the cleaned grinding rod into the centrifugal tube;
3) adding 20 mu L of protease K solution, uniformly mixing, carrying out water bath at 56 ℃ for at least 3h, and centrifuging briefly after the tissue is completely dissolved;
4) adding 200 μ L buffer solution GB, fully reversing and mixing, standing at 70 deg.C for 10min, and centrifuging briefly;
5) adding 20 μ L of anhydrous ethanol, shaking thoroughly, mixing for 15sec, and centrifuging briefly;
6) adding all the solution obtained in the step 4) into an adsorption column CB3 (placing the adsorption column into a collection tube), centrifuging at 12000rpm for 30sec, discarding the waste liquid, and placing the adsorption column back into the collection tube;
7) adding 500 μ L buffer GD into adsorption column CB3, centrifuging at 12000rpm for 30sec, discarding waste liquid, and returning the adsorption column to the collection tube;
8) adding 600 μ L of rinsing solution PW into adsorption column, centrifuging at 12000rpm for 30sec, pouring off waste liquid, and placing adsorption column back into collecting tube;
9) repeating step 8);
10) idling at 12000rpm for 2min, pouring off waste liquid, and placing the adsorption column at room temperature for 10min to completely dry the rinsing liquid remained in the adsorption column;
11) placing the adsorption column into a clean 1.5mL centrifuge tube, suspending and dropwise adding 50 μ L ddH to the middle part of the adsorption membrane2O, standing at room temperature for 3min, centrifuging at 12000rpm for 2min, and collecting the solution into a centrifuge tube;
12) adding the solution obtained in the step 11) into the adsorption column again, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, and collecting the obtained solution;
13) the resulting DNA solutions were labeled, and 1. mu.L of each was used for agarose gel electrophoresis detection and concentration determination with a nucleic acid concentration meter, and the remaining solutions were stored at-20 ℃ for further use.
(3) Real-time fluorescent PCR detection
The invention finally establishes a two-step real-time fluorescence PCR identification technical method of a yellow fir bark beetle TaqMan-MGB probe by continuously optimizing a real-time fluorescence reaction system and amplification conditions, wherein the reaction system comprises the following steps:
premix Ex Taq (Probe qPCR) (2X) 10. mu.L, forward primer HS-F (10. mu.M) 0.4. mu.L, reverse primer HS-R (10. mu.M) 0.4. mu.L, Probe HS-P (10. mu.M) 0.8. mu.L, ROX Reference Dye II (50X) 0.2. mu. L, DNA template 2. mu.L, and sterilized water 6.2. mu.L.
The reaction procedure is as follows:
pre-denaturation at 95 ℃ for 30sec, denaturation at 95 ℃ for 5sec, annealing at 59 ℃ for 30sec, and 40 cycles of denaturation-annealing.
The TaqMan MGB probe HS-P and the primer HS-F/HS-R designed by the invention are combined for real-time fluorescent quantitative PCR detection. As shown in FIG. 2, there is specific amplification only in the amplification curve of bark beetle of the species Abies junior, the CT value is 29.44, i.e., the fluorescence signal increases exponentially from the 29 th cycle, while other species of bark beetles and the other species of bark beetles and ddH2No fluorescence increase signal was observed before the 40 th cycle, indicating that the probe and primer combination is species-specific only for the bark beetle.
The real-time fluorescence quantitative sensitivity detection results are shown in FIG. 3, when the template concentrations are respectively 100ng/μ L, 10ng/μ L, 1ng/μ L and 0.1ng/μ L, the CT values are respectively 26.88, 30.35, 34.00 and 37.85, namely when the template DNA concentration is more than or equal to 1ng/μ L, a yellow fir bark beetle specific fluorescence signal is generated.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications made to the technical solution of the present invention by those skilled in the art without departing from the design spirit of the present invention should fall within the protection scope determined by the present invention.
Claims (10)
1. The real-time fluorescence PCR detection method of the bark beetle of the yellow fir is characterized by comprising the following steps:
(1) designing a primer and a probe;
(2) extracting genome DNA of bark beetle of yellow fir;
(3) and (2) carrying out real-time fluorescent PCR detection by adopting the primers in the step (1), and distinguishing the bark beetle from other quarantine bark beetle insects according to results.
2. The detection method according to claim 1, wherein the primer of step (1) comprises an upstream primer and a downstream primer,
the sequence of the upstream primer HS-F is as follows: 5'-GGAATTGACTAGTTCCTT-3', respectively;
the downstream primer HS-R sequence is as follows: 5'-ATTGCGTAGTCAACAGAGG-3' are provided.
3. The detection method according to claim 1, wherein the primer HS-P sequence of the probe in the step (1) is: 5'-CTACTCTTAAGAAGAATTG-3' are provided.
4. The detection method according to claim 3, wherein the probe primer is connected to a fluorescent group FAM at the 5 'end and a non-fluorescent quenching group at the 3' end.
5. The detection method according to claim 1, wherein the step (2) is implemented by: taking the foot or cephalothorax muscle tissue of the test yellow fir bark beetle specimen to extract the genome DNA.
6. The real-time fluorescent PCR detection method according to claim 1, wherein the reaction system components of the real-time fluorescent PCR detection method of step (3) comprise: premix Ex Taq (Probe qPCR) (2X) 10. mu.L, forward primer HS-F (10. mu.M) 0.4. mu.L, reverse primer HS-R (10. mu.M) 0.4. mu.L, Probe HS-P (10. mu.M) 0.8. mu.L, ROX Reference Dye II (50X) 0.2. mu. L, DNA template 2. mu.L, and sterilized water 6.2. mu.L.
7. The real-time fluorescence PCR detection method according to claim 1, wherein the reaction procedure of the real-time fluorescence PCR detection method of step (3) is as follows: pre-denaturation at 95 ℃ for 30sec, denaturation at 95 ℃ for 5sec, annealing at 59 ℃ for 30sec, and 40 cycles of denaturation-annealing.
8. The real-time fluorescent PCR detection method according to claim 1, wherein the kit comprises primers and probes as follows:
the upstream primer HS-F sequence is as follows: 5'-GGAATTGACTAGTTCCTT-3', respectively;
the downstream primer HS-R sequence is as follows: 5'-ATTGCGTAGTCAACAGAGG-3', respectively;
the probe primer HS-P sequence is as follows: 5'-CTACTCTTAAGAAGAATTG-3', respectively;
the 5 'end of the probe primer is connected with a fluorescent group FAM, and the 3' end of the probe primer is connected with a non-fluorescent quenching group.
9. The use of the real-time fluorescent PCR detection method of ips typographus of any one of claims 1 to 8 in the quarantine of wood pests.
10. The use of claim 9, wherein the test method is used for the quarantine of wood pests of the genera xanthophyll Pseudotsuga, Larix, Tsuga.
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