CN102559922B - Molecular biological method for distinguishing striped rice borer from chilotraea auricilia dudgeon - Google Patents
Molecular biological method for distinguishing striped rice borer from chilotraea auricilia dudgeon Download PDFInfo
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- CN102559922B CN102559922B CN 201210064547 CN201210064547A CN102559922B CN 102559922 B CN102559922 B CN 102559922B CN 201210064547 CN201210064547 CN 201210064547 CN 201210064547 A CN201210064547 A CN 201210064547A CN 102559922 B CN102559922 B CN 102559922B
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- Prior art keywords
- rice borer
- striped rice
- auricilia
- chilotraea
- dudgeon
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Abstract
The invention relates to a molecular biological method for distinguishing striped rice borer from chilotraea auricilia dudgeon and belongs to the technical field of biology. The method comprises the following steps of: taking a genome DNA (deoxyribonucleic acid) as a template, carrying out PCR (polymerase chain reaction) amplification by use of specific primers, namely F: 5'-GGGCAAGAAAGTAGACGCCTGGTT-3'and R: 5'-AAAACCCAAAGAGGCGACACGGTA-3', and carrying out enzyme digestion on an amplified product by use of restriction enzyme MspA1 I, wherein the detected sample is explained to be the striped rice borer if only one segment with the length of 400bp is obtained; and the detected sample is explained to be the chilotraea auricilia dudgeon if two segments with the lengths of 247bp and 153bp areobtained. The method is high in stability, short in period, and high in sensitivity, and is specially used for distinguishing the striped rice borer from the chilotraea auricilia dudgeon with high sensitivity and quick detection.
Description
One, technical field
The present invention relates to the molecular biology differentiating method of rice grub striped rice borer (Chilo suppressalis walker) and goldrimmed moth (Chilo auricilia Dudgeo), belong to biological technical field.
Two, background technology
Striped rice borer and goldrimmed moth are the normal property the sent out insects on the paddy rice, owing to all belong to borer pest, difficult control usually causes dropping in production over a large area of paddy rice.
The hazard approach of striped rice borer and goldrimmed moth is similar, and larva all is earlier in the harm of leaf sheath place, again in leaf sheath channel intrusion rice stem.Also closely similar to the harm state that paddy rice is caused, all can form symptoms such as withered sheath, the withered heart, withered booting, according to the hazard conditions in the rice field, we can't judge accurately that the harm that striped rice borer causes still is the harm that goldrimmed moth causes.The larva of striped rice borer and goldrimmed moth all was 6 ages usually, and there are 5 brown ordinates at the similar and back of larva body colour, utilized visual inspection to be difficult to both are made a distinction.Moreover after striped rice borer or goldrimmed moth were by infected by microbes, polypide completely lost original morphological specificity, causes judging to be which kind of snout moth's larva.
Present evaluation for striped rice borer and goldrimmed moth, normally distinguish by means of morphological feature, this crucial morphological feature that must guarantee sample to be tested is excellent, but as mentioned above, after the morphological specificity forfeiture, then can't accurately distinguish striped rice borer and goldrimmed moth, this has greatly limited the research of striped rice borer and goldrimmed moth association area.For example, if will study the microbial infection situation of somewhere striped rice borer or goldrimmed moth, but because which kind of snout moth's larva can't accurately distinguish by the sample of microbial infection be, so the research in this field has been subjected to very big restriction.In order to solve this difficult problem, we have invented this molecular biology differentiating method.
Three, summary of the invention
1, goal of the invention
The molecular biology method of a kind of accurate differentiation striped rice borer and goldrimmed moth is provided, the differentiating method of a kind of reliable striped rice borer and goldrimmed moth is provided for the related personnel of non-entomological taxonomy education background.
2, technical scheme
Genomic dna with sample to be tested is template, utilizes special primer F:5 '-GGGCAAGAAAGTAGACGCCTGGTT-3 ' and R:5 '-AAAACCCAAAGAGGCGACACGGTA-3 ' to carry out pcr amplification.Amplified production carries out enzyme with restriction endonuclease MspA1 I and cuts digestion, carries out agarose gel electrophoresis then.If have only the fragment of a 400bp, the sample striped rice borer that then detects; Be respectively 247bp and 153bp if obtain two bar segment, then the sample that detects is goldrimmed moth.
3, beneficial effect
Method of the present invention is compared with traditional method, and major advantage has: (1) accuracy height: traditional method can be because different identifiers causes qualification result error to occur to the inaccurate of each characteristic of division assurance.And method of the present invention is based on the molecular detection technology of PCR, and that round pcr has developed is very ripe, has the experiment flow of stdn and foolization, has guaranteed result's high accuracy.(2) highly sensitive: traditional method can't be distinguished the sample of forfeiture morphological feature.And based on method of the present invention, as long as can extract the genomic dna of trace from sample to be tested, just can increase obtains target P CR product, thereby finishes the evaluation to sample to be tested.
Four, embodiment
1, the extraction of sample genomic dna
Utilize Shanghai to give birth to the genomic dna that worker UNIQ-10 pillar animal gene group DNA extraction agent box extracts sample, concrete operations are as follows:
(1) sample to be detected is clayed into power in liquid nitrogen, and shifts and put in the centrifuge tube of 1.5ml;
(2) Proteinase K of adding 300 μ l ACL Solution and 20 μ l.(Proteinase K was put 37 ℃ of incubations 1 hour in advance, is beneficial to the activity of activator enzyme K);
(3) vortex concussion mixing is 1 minute, places 55 ℃ of water-baths to place then 1-3 hour, can suitably take out mixing during this period, helps abundant cracking;
(4) take out sample, rock mixing gently after waiting to be down to room temperature, then 12, centrifugal 5 minutes of 000rpm;
(5) draw 300 μ l supernatants to UNIQ-10Column, and then draw among the AB Solution to UNIQ-10Column of 300 μ l, mixing 2-3 time of turning upside down, room temperature left standstill 3 minutes;
Centrifugal 3 minutes of (6) 3,000rpm take off UNIQ-10Column, outwell waste liquid in the collection tube;
(7) UNIQ-10Column is put back in the collection tube, add 500 μ l Wash Solution, 8,000rpm, centrifugal 30 seconds of room temperature;
(8) repeating step (7) once;
(9) take off UNIQ-10Column, discard the waste liquid in the collection tube, UNIQ-10Column is put back in the collection tube, 10,000rpm, centrifugal 30 seconds of room temperature is to remove residual Wash Solution;
(10) UNIQ-10Column is put into the centrifuge tube of clean 1.5ml, add 50 μ l Elution Buffer in UNIQ-10Column central authorities, room temperature was placed 2-3 minute.Again 10,000rpm, centrifugal 1 minute of room temperature.Liquid in the centrifuge tube is the genomic dna of sample, and is standby in-20 ℃ of preservations.
2, the preparation of PCR system
Use the PCR reaction system of 25 μ l, comprising: 10 * PCR Buffer (Mg of 2.5 μ l
2+Free), the MgCl of the dNTPs of 2 μ l (2.5mmol/L), 1.5 μ l
2(25mmol/L), the Taq DNA Polymerase (TakaRa) of each 1 μ l of upstream and downstream primer (10 μ mol/L), sample genomic dna 1 μ l, 0.125 μ l, add aqua sterilisa again and complement to 25 μ l.
3, pcr amplification program
94 ℃ 3 minutes; 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 50 seconds (32 circulations); 72 ℃ 8 minutes.
4, the MspA1 I enzyme of PCR product is cut digestion
The enzyme of 10 μ l is cut system, comprising: the MspA1 I enzyme of 10 * Buffer 4 of 1 μ l, 100 * BSA of 0.1 μ l, 0.25 μ l, PCR product≤0.5 μ g add aqua sterilisa and complement to 10 μ l.Afterwards 37 ℃ incubation 1-2 hour.
5, enzyme is cut the electrophoresis detection of product
Enzyme is cut product and is carried out electrophoresis at 2% sepharose, current stabilization 80mA, and electrophoresis 40 minutes, ethidium bromide (EB) dyeing back is observed down in ultraviolet etc., judges the size of electrophoretic band according to DNA Marker.
If electrophoresis showed has only the fragment of a long 400bp, represent that then sample to be tested is striped rice borer; If electrophoresis showed has two bar segment, long 247bp and 153bp represent that then institute's test sample originally is goldrimmed moth respectively.
Embodiment
In November, 2011, we collect 3 parts by the sample of microbial infection in the Heyuan field, can't determine that by morphological observation sample is striped rice borer or goldrimmed moth, then utilizes side of the present invention to identify, the result shows that this three increment originally is goldrimmed moth.Detected result is seen the Fig. 2 in the Figure of description.
Five, description of drawings
The characteristic electrophoretogram of Fig. 1 striped rice borer and goldrimmed moth
The detected result of sample is gathered in Fig. 2 Heyuan field
In Fig. 1, what " Csu " indicated is the striped rice borer sample, and what " Cau " indicated is the goldrimmed moth sample.In Fig. 2, A, B, three indicated swimming lanes of C are represented 3 parts of test sample.In Fig. 1 and Fig. 2, " M " indicated swimming lane is DNA Marker, is followed successively by 1000bp, 700bp, 500bp, 400bp, 300bp, 200bp, 100bp from top to bottom.
Figure in " Figure of abstract " Word document is the Fig. 1 in " Figure of description " Word document.
Claims (1)
1. method of utilizing molecular biology method accurately to distinguish striped rice borer and goldrimmed moth, it is characterized in that: utilize special primer F:5 '-GGGCAAGAAAGTAGACGCCTGGTT-3 ' and R:5 '-AAAACCCAAAGAGGCGACACGGTA-3 ' that striped rice borer and goldrimmed moth genomic dna are carried out pcr amplification, the sample amplification product after the MspA1I enzyme is cut still for 400bp for striped rice borer, amplified production then is goldrimmed moth for 247bp and 153bp two bands through the MspA1I enzyme is cut after.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201210064547 CN102559922B (en) | 2012-03-13 | 2012-03-13 | Molecular biological method for distinguishing striped rice borer from chilotraea auricilia dudgeon |
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| CN 201210064547 CN102559922B (en) | 2012-03-13 | 2012-03-13 | Molecular biological method for distinguishing striped rice borer from chilotraea auricilia dudgeon |
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| CN102559922B true CN102559922B (en) | 2013-09-25 |
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| CN103224993A (en) * | 2013-05-22 | 2013-07-31 | 江苏省农业科学院 | Detection method of target resistance mutation of Taiwan rice stem borer for organic phosphorus pesticide |
| CN108588233A (en) * | 2018-05-03 | 2018-09-28 | 南京农业大学 | A kind of early molecule detection of 8 kinds of trichogrammas of Trichogrammatidae and identification method and application |
| CN109652561B (en) * | 2018-12-10 | 2021-09-21 | 华中农业大学 | Method for identifying chilo suppressalis rice population and cane shoot population based on timeout gene |
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| CN102229993B (en) * | 2011-05-27 | 2013-07-31 | 宁波检验检疫科学技术研究院 | PCR-RFLP method for identifying mucronate form of Bursaphelenchus xylophilus and round-tailed form of Bursaphelenchus xylophilus |
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Granted publication date: 20130925 |