CN101838687A - Codling moth identification primer, identification method and application - Google Patents
Codling moth identification primer, identification method and application Download PDFInfo
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- CN101838687A CN101838687A CN201010000966A CN201010000966A CN101838687A CN 101838687 A CN101838687 A CN 101838687A CN 201010000966 A CN201010000966 A CN 201010000966A CN 201010000966 A CN201010000966 A CN 201010000966A CN 101838687 A CN101838687 A CN 101838687A
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Abstract
The invention relates to a codling moth identification primer, which comprises a primer 1F: 5'-ATTACCCCCATCTATTATACTT-3' and a primer 1R: 5'-TGGTAATGATAAAAGAAGTAAAAGAGC-3'. A method for identifying codling moths by use of the primer comprises the following steps: (1) extracting a DNA template of an identification sample; (2) using the codling moth identification primer in right claim 1 to perform PCR amplification on template DNA; and (3) detecting the existence of 289bp band through electrophoresis. The codling moth identification primer provided by the invention has good amplification effect on target fragments of codling moths, can identify samples only according to the existence of the 289bp band when the primer is used for identification, and has the advantages of quickness, convenience, clear results and accuracy.
Description
Technical field
The present invention relates to a kind of carpocapsa pononella primers designed and utilize this specificity primers designed that sample to be identified is carried out the method that Rapid identification is discerned carpocapsa pononella, the invention still further relates to above-mentioned carpocapsa pononella primers designed in the application of distinguishing on carpocapsa pononella, small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect.
Background technology
Carpocapsa pononella Cydia pomonella L., different name Laspeyresia Pomonolla L., be commonly called as codling moth, belong to lepidopteran, Tortricidae, multiple fruit trees such as main harm apple, pears, Chinese pear-leaved crabapple, Malus spectabilis, Lee, peach, hawthorn are the crushing insects of a kind of fruit, such as apple, pear, etc. and tone fruit trees, belong to worldwide quarantine harmful organism.This worm is caused harm with young wormy fruit, causes a large amount of wormed fruit, has a strong impact on the quality of fruit; Larva also has changes the really habit of harm, causes the preceding a large amount of sheddings of fruit harvesting and rotten, and the borer fruit rate of larva about 50%, can reach 70%-100% when serious usually.Carpocapsa pononella originates in the European southeast, 69 countries and regions now all over the world.The fifties, this worm is found in Xinjiang of China; The mid-80 has entered Dunhuang, Gansu Province and area, Jiuquan; Entered morningstar lily in 2006, and the trend of further expanding is eastwards arranged.If carpocapsa pononella spreads to the big provinces of apple such as Shaanxi, Shandong, the outlet of the apple of China will be restricted, and financial loss will be extremely serious.Therefore, further raising is significant for preventing that exotic invasive insect carpocapsa pononella from further expanding in China to identification, the identification capacity of carpocapsa pononella, and the rapid identification method of carpocapsa pononella also becomes the important means that prevents that carpocapsa pononella from further invading.
Small heart-eating peach worm Carposina sasakii, oriental fruit months Grapholitha molestaBusck and Lee's small kernel-eating insect Grapholita funebrana are 3 kinds of common heart-eating worms of China, low instar larvae of these 3 kinds of heart-eating worms and carpocapsa pononella larva are externally closely similar on the form, do not have significant difference.At present, China's sanitary authority identifies it mainly is that formalness feature with mature larva or adult is as foundation to the classification of carpocapsa pononella.But in the actual quarantine in port, low instar larvae often or the ovum intercepted and captured, the common practice is it to be carried out indoor feeding wait to obtain to identify behind the mature larva again, has so not only prolonged to import and export being open to the custom the time of article, and has increased health officer's working strength.Therefore carpocapsa pononella being identified quickly and accurately, is on the port quarantine or all significant in control.
Summary of the invention
The object of the invention is to provide a kind of carpocapsa pononella primers designed, uses this primers designed to identify method and the application of this primers designed on differentiation carpocapsa pononella, small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect of carpocapsa pononella.
The present invention at first provides a kind of carpocapsa pononella primers designed, and the sequence of described primers designed is:
Primer 1F:5 '-ATTACCCCCATCTATTATACTT-3 ',
Primer 1R:5 '-TGGTAATGATAAAAGAAGTAAAAGAGC-3 ',
This primer can amplify the fragment of a 289bp.The present invention utilizes the COI universal primer that carpocapsa pononella, small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect sample that picks up from national different areas carried out pcr amplification, order-checking obtains the sequence that length is 502bp, the COI sequence of 4 kinds of heart-eating worms is compared with CLUSTALX software, find out the difference site of carpocapsa pononella and other 3 kinds of heart-eating worms, utilize Primer5.0 software design primer, and utilize Blast routine check primer specificity, thereby design specificity primers designed at carpocapsa pononella.All can amplify the bright band of 289bp when stating primers F/R in the use from 10 the geographic carpocapsa pononella samples in the whole nation, small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect sample then can not amplify band when stating primer in the use.
The present invention also provides a kind of method of using above-mentioned carpocapsa pononella primers designed to identify carpocapsa pononella, may further comprise the steps:
(a). the extraction of sample DNA template to be identified;
(b). use the described carpocapsa pononella primers designed of claim 1 that template DNA is carried out pcr amplification;
(c). electrophoresis detection has or not the 289bp band.
As preferably, the method for above-mentioned evaluation carpocapsa pononella, in the step (a), the extracting method of sample DNA template to be identified is a salting-out process.
As preferably, the method for above-mentioned evaluation carpocapsa pononella, in the step (a), described salting-out process may further comprise the steps:
(1). the sample to be identified that alcohol is preserved, remove alcohol after, dry;
(2). the air dried sample is transferred in the centrifuge tube that extracting solution is housed and fully grinds;
(3). homogenate is moved to 60～70 ℃ of water-bath 15～30min;
(4). in homogenate, add NaAc, ice bath;
(5). the mixed solution behind the ice bath is centrifugal;
(6). go up blue or green liquid and be transferred in another centrifuge tube, centrifugal behind the adding equal-volume Virahol, abandon supernatant liquor;
(7). in leaving sedimentary centrifuge tube, add 70% washing with alcohol DNA, centrifugal, abandon supernatant;
(8). step (7) repeats 1～2 time;
(9). after drying naturally, in centrifuge tube, add sterilized water dissolving DNA, freezing preservation.
As preferably, the method for above-mentioned evaluation carpocapsa pononella, in the step (4), described NaAC is the cold NaAc of 8mol/I, ice bath time 10～20min; In the step (9), the temperature of described freezing preservation is-20 ℃.
As preferably, the method for above-mentioned evaluation carpocapsa pononella, in the step (2), described extracting solution is mixed with by 0.2M sucrose, 0.1M Tris, 0.1M NaCl, 0.05M EDTA, 0.5%SDS, pH8.5～9.5.
As preferably, the method for above-mentioned evaluation carpocapsa pononella, described centrifugal under 4 ℃ in step (5) and (6), with the centrifugal 15～20min of the speed of 10000rp/min; In the step (7), described centrifugal under 4 ℃, with the centrifugal 4～6min of the speed of 10000rp/min.
As preferably, the method for above-mentioned evaluation carpocapsa pononella, in the step (b), the condition of described pcr amplification is conventional PCR system, and annealing temperature is 55 ℃～57 ℃, and annealing time is 30s.
As preferably, the method for above-mentioned evaluation carpocapsa pononella, in the step (c), described electrophoresis detection is that 1～1.5% (mass percent) agarose gel electrophoresis detects.
As preferably, the method for above-mentioned evaluation carpocapsa pononella, above-mentioned electrophoresis detection utilize UV-light to detect electrophoresis result.
The present invention also further provides above-mentioned carpocapsa pononella primers designed in the application of distinguishing on carpocapsa pononella, small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect.
Carpocapsa pononella primers designed provided by the invention is simple in structure, and the purpose fragment of carpocapsa pononella is had good expanding effect; When utilizing this Auele Specific Primer to identify to distinguish carpocapsa pononella, small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect, fast and convenient, qualification result is accurate, and the PCR reaction is normal condition, success ratio height; Experimental result presents with the form of electrophorogram, only according to the having or not of 289bp band, can identify that the result is clear to sample, does not need that low instar larvae or ovum are carried out indoor feeding and identifies to mature larva again.Carpocapsa pononella primers designed provided by the invention and utilize this primer to identify that carpocapsa pononella is applied in the actual quarantine in port can not only shorten being open to the custom the time of import and export article greatly, and can also reduce health officer's working strength.
Description of drawings
Fig. 1 is that the carpocapsa pononella special primer is to gathering the amplification from 10 parts of total DNA of carpocapsa pononella sample of China different areas.Among Fig. 1,
M is marker;
1-10 is followed successively by collection from Zhangye, Gansu, Jiuquan, Dunhuang, the carpocapsa pononella sample of Hami, Urumchi, Kuerle, Keshen, Kuitun, Jinghe, Yi Li;
11-15 is followed successively by collection from Pingliang, Gansu, Zhangye, Shaanxi Yang Ling, Feng County, the small heart-eating peach worm sample in Shenyang, Liaoning;
16-19 is followed successively by collection from Zhangye, Gansu, Urumchi, Xinjiang, Keshen, the oriental fruit months sample of Shaanxi Yang Ling;
20-22 is followed successively by the Lee small kernel-eating insect sample of collection from Urumchi, Xinjiang, Kuitun, Keshen.
The sequence table explanation
Sequence table 1 is Gansu, 6 regional carpocapsa pononellas of Xinjiang two provinces and the U.S., Byelorussia, Australia, German 4 ground carpocapsa pononella COI sequence alignments.Wherein, GS-JQ represents the Jiuquan, and GS-DH represents Dunhuang, Gansu, and XJ-HM represents Hami, and XJ-KEL represents the Kuerle, Xinjiang, and XJ-KS represents Kashi, and XJ-YL represents Xinjiang Yi Li.
Sequence table 2 is the comparison of carpocapsa pononella and oriental fruit months, Lee's small kernel-eating insect and small heart-eating peach worm COI sequence.It in the frame position of specificity carpocapsa pononella primers designed.
Embodiment
The present invention is based on present Protocols in Molecular Biology means, from the dna profiling of sample to be identified extract, acquisition three aspects of the design of carpocapsa pononella special primer and electrophoretogram start with, by develop new method with reach make carpocapsa pononella authentication method more simply, purpose fast and accurately.
The extracting method of sample DNA template wherein to be identified is a salting-out process, and concrete steps are as follows:
1. random choose single head heart-eating worm sample (straight alcohol preservation), larva is got head, and adult is got foot or chest muscle; Sample takes out in the back immersion sterilized water and removes alcohol, and dries on thieving paper;
2. be transferred to after sample dries in the 1.5ml centrifuge tube that 100 μ L extracting solutions are housed and fully grind;
3. homogenate is moved to 60～70 ℃ of water-bath 15-30min;
4. in homogenate, add the cold 8mol/LNaAc of 10 μ L, ice bath 15min;
5. with 4 ℃ of the mixed solutions behind the ice bath, 10000rp/min, centrifugal 20min;
6. supernatant liquor is transferred in another 1.5ml centrifuge tube, adds the equal-volume Virahol, and the centrifugal 15min of 10000rp/min abandons supernatant;
7. add 70% ethanol 1ml washing DNA in leaving sedimentary centrifuge tube, the centrifugal 5min of 10000rp/min abandons supernatant;
8. step 7 repeats 1～2 time.
9. dry the back naturally and in pipe, add 50 μ L sterilized water dissolving DNAs, preserve standby in-20 ℃ of refrigerators.
The agarose gel electrophoresis that electrophoresis detection is used is ordinary method.
The carpocapsa pononella primers designed be designed to core of the present invention, in order to obtain the special and primers designed accurately of carpocapsa pononella, we have carried out following research:
1. conservative property research in the carpocapsa pononella COI gene kind: by gathering Chinese Gansu, Xinjiang two provinces totally 6 geographic carpocapsa pononella adults and larva sample, with the COI universal primer:
mtD7.2F?5′-GGAGGATTTGGAAATTGATTAGTTCC-3′
mtD9.2R?5′-CCCGGTAAAATTAAAATATAAACTTC-3′
Obtain carpocapsa pononella chondriogen COI fragment sequence through conventional pcr amplification and conventional order-checking.
Simultaneously, also from 4 carpocapsa pononella correlated serieses of NCBI search, sequence number is respectively FJ217763, FJ217761, FJ217754, FJ217752 for we, and the relevant sample of sequence derives from the U.S., Byelorussia, Australia and German respectively.With CLUSTAL X software above-mentioned carpocapsa pononella COI sequence is compared, find that the carpocapsa pononella of different sources is very high in the conservative property in COI zone, thereby guaranteed the accuracy (sequence table 1) of primer.
2. conservative property research in small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect COI gene kind: by small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect adult and the larva sample of gathering Chinese different areas, with the COI universal primer:
mtD72F?5′-GGAGGATTTGGAAATTGATTAGTTCC-3′
mtD9.2R?5′-CCCGGTAAAATTAAAATATAAACTTC-3′
Obtain this 3 kinds of heart-eating worm COI fragment sequences through conventional pcr amplification and conventional order-checking.
Simultaneously, we have also searched for the correlated series of these 3 kinds of heart-eating worms from NCBI, by above-mentioned 3 kinds of heart-eating worm COI sequences are compared respectively, find that the COI zone of 3 kinds of heart-eating worms all has very high conservative property.
3. the COI sequence alignment of carpocapsa pononella and other three kinds of heart-eating worms (small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect): in order to design the special primer at carpocapsa pononella, we have carried out comparing (sequence table 2) to the COI sequence of 4 kinds of heart-eating worms.
By sequence table 1, sequence table 2 as can be seen, the selected zone of design carpocapsa pononella special primer is region of variability between planting, and there is certain base difference in this zone, and very conservative in planting, therefore, this regional sequence can be used as the special primer of carpocapsa pononella.
4. by the specificity of pcr amplification experimental verification carpocapsa pononella special primer and to the segmental expanding effect of carpocapsa pononella purpose, concrete steps are as follows:
(1) PCR reaction system: reaction system is reference with 50 μ L, and primer transfers to 10pmol/ μ L with deionized water, and each reagent dosage is respectively:
10 * TaqDNA polymerase buffer (Mg
2+) 5 μ L
dNTP(10mM) 4μL
TaqDNA polysaccharase (5U/ μ L) 0.4 μ L
ddH
2O 34.6μI
(2) PCR reaction conditions: be reflected on the PCR instrument and carry out, 95 ℃ of pre-sex change 2min, carry out circulating reaction 40 times by following parameter then:
95 ℃ of 15s of sex change
55 ℃ of 30s anneal
Extend 72 ℃ of 45s
10min is extended in 72 ℃ of compensation after the loop ends, and reaction product is kept at 4 ℃.
Experimental result shows (Fig. 1), only carpocapsa pononella has bright band at the 289bp place, its excess-three kind heart-eating worm does not all have band in this position, illustrate that the carpocapsa pononella special primer is special to carpocapsa pononella, thereby the purpose fragment that reaches carpocapsa pononella from the specificity that has experimentally proved this primer has good expanding effect.
In sum, the present invention utilizes the sequence difference of carpocapsa pononella and other three kinds of heart-eating worms, designs the special primer at carpocapsa pononella, and is core with this primer, develops a kind of this primer that utilizes and carries out the method for pcr amplification with quick discriminating carpocapsa pononella.The advantage of this method is: carpocapsa pononella primers designed high specificity, the purpose fragment of carpocapsa pononella is had good expanding effect, and guaranteed the accuracy of qualification result; The PCR reaction is normal condition, success ratio height; Experimental result presents with the electrophorogram form, only according to the having or not of 289bp band, can identify that the result is clear to sample.
Embodiment
Utilize specific carpocapsa pononella primers designed IF/R that carpocapsa pononella, small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect sample of gathering from Chinese different areas carried out Rapid identification.
Primer 1F:5 '-ATTACCCCCATCTATTATACTT-3 '
Primer 1R:5 '-TGGTAATGATAAAAGAAGTAAAAGAGC-3 '
Step 1: the total DNA extraction of heart-eating worm to be identified
1. collection of specimens information: gather 10 parts of carpocapsa pononella samples, 5 parts of small heart-eating peach worm samples, 4 parts of oriental fruit months samples and 3 parts of Lee's small kernel-eating insect samples from China different areas.
2.DNA the extraction of template
Random choose single head heart-eating worm sample (straight alcohol preservation), larva is got head, and adult is got foot or chest muscle; Sample takes out in the back immersion sterilized water and removes alcohol, and dries on thieving paper; Be transferred to after sample dries in the 100 μ L extracting solutions and fully grind; Homogenate is moved to 60～70 ℃ of water-bath 15～30min; In homogenate, add the cold 8mol/L NaAc of 10 μ L, ice bath 15min; Centrifugal 20min (4 ℃ of 10000rp/min); Draw supernatant liquor, add the equal-volume Virahol in supernatant liquor, centrifugal 15min (10000rp/min) abandons supernatant; Add 70% ethanol 1ml washing DNA, centrifugal 5min (10000rp/min) abandons supernatant; Add 70% ethanol 1ml washing DNA again, centrifugal 5min (10000rp/min) abandons supernatant; Naturally dry the back and add 50 μ L sterilized water dissolving DNAs, this dna solution can be used for pcr amplification.
Step 2:PCR amplification
PCR reaction the primer is specificity carpocapsa pononella primers designed IF/R provided by the invention
Primer 1F:5 '-ATTACCCCCATCTATTATACTT-3 '
Primer 1R:5 '-TGGTAATGATAAAAGAAGTAAAAGAGC-3 '
50 μ L standards systems are adopted in the PCR reaction, contain 10 * Taq buffer (Mg
2+) 5 μ L, 10mmoldNTP 4 μ L mixed solutions, each 1 μ L of primer, TaqDNA polysaccharase (5U/ μ L) 0.4 μ L, template DNA solution 4 μ L, ddH
2O 34.6 μ L.Reaction conditions is: 95 ℃ of pre-sex change 2min, and 95 ℃ of 15s, 55 ℃ of 30s, 72 ℃ of 45s, 10min is extended in 40 back 72 ℃ of compensation of circulation.
Step 3: agarose gel electrophoresis and ultraviolet detection
The used sepharose concentration of agarose gel electrophoresis is 1%, contains a small amount of EB, total applied sample amount 4 μ L (3 μ LPCR products, 1 μ Lloading buffer), and electrophoretic voltage is 120v, the time is 30min.
Detect electrophoresis result and take pictures with the UV-light detector, result such as accompanying drawing 1 show that only carpocapsa pononella amplifies the bright band of 289bp, and small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect sample then can not amplify band.
The above embodiment is the preferred embodiment that proves absolutely that the present invention lifts, and protection scope of the present invention is not limited thereto.Being equal to that those skilled in the art are done on basis of the present invention substitutes or conversion, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
Sequence table 1
Sequence table 2
Claims (10)
1. carpocapsa pononella primers designed, it is characterized in that: the sequence of described primers designed is:
Primer 1F:5 '-ATTACCCCCATCTATTATACTT-3 ';
Primer 1R:5 '-TGGTAATGATAAAAGAAGTAAAAGAGC-3 '.
2. use the described carpocapsa pononella primers designed of claim 1 to identify the method for carpocapsa pononella, it is characterized in that: may further comprise the steps:
(a). the extraction of sample DNA template to be identified;
(b). use the described carpocapsa pononella primers designed of claim 1 that template DNA is carried out pcr amplification;
(c). electrophoresis detection has or not the 289bp band.
3. the method for evaluation carpocapsa pononella according to claim 2, it is characterized in that: in the step (a), the extracting method of sample DNA template to be identified is a salting-out process.
4. the method for evaluation carpocapsa pononella according to claim 3, it is characterized in that: described salting-out process may further comprise the steps:
(1). the sample to be identified that alcohol is preserved, remove alcohol after, dry;
(2). the air dried sample is transferred in the centrifuge tube that extracting solution is housed and fully grinds;
(3). homogenate is moved to 60～70 ℃ of water-bath 15～30min;
(4). in homogenate, add NaAc, ice bath;
(5). the mixed solution behind the ice bath is centrifugal;
(6). supernatant liquor is transferred in another centrifuge tube, and is centrifugal behind the adding equal-volume Virahol, abandons supernatant liquor;
(7). in leaving sedimentary centrifuge tube, add 70% washing with alcohol DNA, centrifugal, abandon supernatant;
(8). step (7) repeats 1～2 time;
(9). after drying naturally, in centrifuge tube, add sterilized water dissolving DNA, freezing preservation.
5. the method for evaluation carpocapsa pononella according to claim 4, it is characterized in that: in the step (4), described NaAC is the cold NaAc of 8mol/L, ice bath time 10～20min; In the step (9), the temperature of described freezing preservation is-20 ℃.
6. the method for evaluation carpocapsa pononella according to claim 4 is characterized in that: described centrifugal under 4 ℃ in step (5) and (6), with the centrifugal 15～20min of the speed of 10000rp/min; In the step (7), described centrifugal under 4 ℃, with the centrifugal 4～6min of the speed of 10000rp/min.
7. the method for evaluation carpocapsa pononella according to claim 2, it is characterized in that: in the step (b), the condition of described pcr amplification is conventional PCR system, and annealing temperature is 55 ℃～57 ℃, and annealing time is 30s.
8. according to the method for each described evaluation carpocapsa pononella of claim 2～7, it is characterized in that: in the step (c), described electrophoresis detection is that 1～1.5% (mass percent) agarose gel electrophoresis detects.
9. the method for evaluation carpocapsa pononella according to claim 8 is characterized in that: described electrophoresis detection is to utilize UV-light to detect.
10. the described carpocapsa pononella primers designed of claim 1 is in the application of distinguishing on carpocapsa pononella, small heart-eating peach worm, oriental fruit months and Lee's small kernel-eating insect.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103882116A (en) * | 2014-01-22 | 2014-06-25 | 西北农林科技大学 | Molecular identification primer for four fruit tree core-eating insects and using method of molecular identification primer |
| CN109609609A (en) * | 2019-01-25 | 2019-04-12 | 河北出入境检验检疫局检验检疫技术中心 | A kind of real-time fluorescence PCR detection method of Lee's Grapholita spp |
| CN109628614A (en) * | 2019-01-28 | 2019-04-16 | 河北出入境检验检疫局检验检疫技术中心 | A kind of the PCR detection primer and detection method of Lee's Grapholita spp |
| CN110257528A (en) * | 2018-12-21 | 2019-09-20 | 中国农业科学院植物保护研究所 | Carpocapsa pononella specificity SS-COII primer and its application |
| CN114891788A (en) * | 2022-06-21 | 2022-08-12 | 青岛农业大学 | LAMP method-based codling moth identification primer group, identification method and identification kit |
-
2010
- 2010-01-21 CN CN201010000966A patent/CN101838687B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103882116A (en) * | 2014-01-22 | 2014-06-25 | 西北农林科技大学 | Molecular identification primer for four fruit tree core-eating insects and using method of molecular identification primer |
| CN103882116B (en) * | 2014-01-22 | 2015-12-02 | 西北农林科技大学 | Four planting fruit-trees heart-eating worm Molecular Identification primer and using method |
| CN110257528A (en) * | 2018-12-21 | 2019-09-20 | 中国农业科学院植物保护研究所 | Carpocapsa pononella specificity SS-COII primer and its application |
| CN109609609A (en) * | 2019-01-25 | 2019-04-12 | 河北出入境检验检疫局检验检疫技术中心 | A kind of real-time fluorescence PCR detection method of Lee's Grapholita spp |
| CN109609609B (en) * | 2019-01-25 | 2022-04-19 | 河北出入境检验检疫局检验检疫技术中心 | Real-time fluorescence PCR detection method for plum fruit borer |
| CN109628614A (en) * | 2019-01-28 | 2019-04-16 | 河北出入境检验检疫局检验检疫技术中心 | A kind of the PCR detection primer and detection method of Lee's Grapholita spp |
| CN114891788A (en) * | 2022-06-21 | 2022-08-12 | 青岛农业大学 | LAMP method-based codling moth identification primer group, identification method and identification kit |
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|---|---|
| CN101838687B (en) | 2012-09-26 |
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