CN101285104A - Kit for detecting bean pod mottle virus and detection process thereof - Google Patents
Kit for detecting bean pod mottle virus and detection process thereof Download PDFInfo
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- CN101285104A CN101285104A CNA2008100711544A CN200810071154A CN101285104A CN 101285104 A CN101285104 A CN 101285104A CN A2008100711544 A CNA2008100711544 A CN A2008100711544A CN 200810071154 A CN200810071154 A CN 200810071154A CN 101285104 A CN101285104 A CN 101285104A
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Abstract
The invention relates to a detection reagent kit for bean pod mottle viruses and a detection method thereof, which are specially used for the detection of the bean pod mottle viruses. The detection reagent kit comprises a primer, virus extraction reagent, RT Buffer, an RNase inhibiting factor, inverse transcriptase, dNTPs, PCR Buffer, Mgcl2, Taq DNA polymerase, positive control, negative control, PBST buffer solution and RNase-free ddH2O. The method designs a specific primer according to conservative areas of bean pod mottle virus genes, utilizes a TC-RT-PCR technology to detect the bean pod mottle viruses, does not need fussy RNA extraction steps, directly makes reverse transcription and PCR to virus extract solution, and has simple and convenient operation, steady result, strong specificity, high sensitivity and relatively lower detection cost, so the method has higher utility value.
Description
Technical field
The present invention relates to a kind of detection kit and detection method thereof of bean pod mottle virus, it belongs to biological technical field, is exclusively used in the rapid detection to bean pod mottle virus on the soybean such as Check and Examination of Port quarantine and agriculture plant quarantine department.
Background technology
Bean pod mottle virus (BPMV) belongs to Comoviridae (Comoviridae), Comovirus (Comovirus) member, and viral genome is the linear sense single stranded rna of dyad, and RNA-1 is about 6kb, and RNA-2 is about 3.6kb.BPMV was reported on the Kidney bean Tendergreen kind of U.S. Charleston in 1948, now mainly be distributed in north America region, widely distributed in the soybean planting areas such as the Arkansas State, the North Carolina state, Virginia, the Kentucky State, the state of Mississippi, Louisiana, Iowa, Illinois and Indiana State of the U.S..Except that the U.S., Canada, Brazil, Peru also have the report that BPMV takes place on soybean in recent years.Bean pod mottle virus infects the bean pod mottle disease that soybean causes, not only influences the normal growth of soybean, causes production loss, and the soybean seeds quality is obviously descended.According to statistics, it is 3%-52% that BPMV infects the production loss scope that soybean causes, and time of infection more early, lose serious more, if with soybean mosaic virus (Soybean Mosaic Virus, SMV) compound infecting, then the soybean yields loss can bring serious threat to soybean production up to 85%.In addition, after BPMV infects soybean, also can cause soybean to be more vulnerable to the harm of Phomopsis (Phomopsis) fungi, cause the soybean seeds quality further to reduce.BPMV has formally been listed in the newly-increased Plant Quarantine venereal disease poison register that enters the territory of China as dangerous harmful organism.Up to now, China does not see the report that BPMV takes place as yet, but along with China's Foreign Trade and soyabean processing industry develop rapidly, annual from the external a large amount of soybean of (the particularly U.S.) import, BPMV is increasing with the risk that imported soybean imports into.For avoiding BPMV to import China into, already bring great risk for China's agriculture production, particularly soybean planting, press for the port quarantine of reinforcement to BPMV, therefore improving this virus detection techniques has extremely important realistic meaning.
Current, the method that is used for the plant virus detection comprises that mainly Biological Detection, Electronic Speculum detect, serology detects and molecular Biological Detection.Can detect BPMV by the inoculation plant indicator, produce mottled symptom behind this virus inoculation soybean, the Kidney bean Tendergreen, and produce local lesion after inoculation Kidney bean Pinto and the Bountiful kind, but this method is long detection time, and the result is subject to the influence of human factor and envrionment conditions.Electron microscopic observation is owing to instrument that needs costliness and special operator, and the material pre-treatment is comparatively complicated, therefore has on the rapid detection of BPMV than big limitation.Serology detects has simple, easy and simple to handle, the with low cost and sensitive advantage of required equipment, has become the most frequently used detection method of BPMV at present, but the use prerequisite of this method be need cost an arm and a leg, the antiserum(antisera) of high specificity.Compare with the serology detection method, RT-PCR molecular detecting method based on nucleic acid level has advantages such as sensitivity is higher, specificity is stronger, the result is more reliable, have many pieces both at home and abroad about utilizing the RT-PCR method to detect the research report of BPMV, comprise (Gu etc. such as common RT-PCR, immunocapture nido RT-PCR and half-nest type RT-PCR, Phytopathology, 2002,92 (4): 446～452; Yu Cui etc., Plant Quarantine, 2006,20 (4): 201～204; Hear big just etc., Plant Pathology, 2006,36 (4): 296～300).But, do not see the report of using TC-RT-PCR (test tube is caught RT-PCR) technology for detection BPMV so far as yet, more do not see to be specifically designed to the TC-RT-PCR detection kit that detects BPMV.
Summary of the invention
The detection kit and the detection method thereof that the purpose of this invention is to provide a kind of bean pod mottle virus, it can carry out quick, accurate and effective quarantine to bean pod mottle virus on inward soybean seeds in port and the field soybean and identify.
Technical solution of the present invention is made of following two portions:
(1) a kind of detection kit of bean pod mottle virus is characterized in that it consists of:
(1) 1
#Liquid, contain viral extraction agent: the PBS damping fluid, concentration 1 *, pH7.4;
(2) 2
#Liquid contains bean pod mottle virus upstream primer: 5 '-GGCAATTTTAGAGCAAGTGTTG-3 ', concentration 10 μ mol/L;
(3) 3
#Liquid contains bean pod mottle virus downstream primer: 5 '-GCATTTGGCATATTCATGAGAAT-3 ', concentration 10 μ mol/L;
(4) 4
#Liquid contains RT Buffer, concentration 5 *;
(5) 5
#Liquid contains the RNA enzyme inhibition factor, concentration 40U/ μ L;
(6) 6
#Liquid contains ThermoScript II, concentration 200U/ μ L;
(7) 7
#Liquid contains dNTPs, concentration 10mmol/L;
(8) 8
#Liquid contains PCR Buffer, concentration 10 *;
(9) 9
#Liquid contains Mgcl
2, concentration 25mmol/L;
(10) 10
#Liquid contains the Taq archaeal dna polymerase, concentration 5U/ μ L;
(11) 11
#Liquid contains the bean pod mottle virus positive control;
(12) 12
#Liquid contains the bean pod mottle virus negative control;
(13) 13
#Liquid contains the PBST damping fluid, concentration 1 *;
(14) 14
#Liquid contains RNase-free ddH
2O.
Above-mentioned 1
#Liquid-14
#Liquid is respectively 1 pipe, wherein 1
#The content of liquid is 10mL, 2
#The content of liquid is 100 μ L, 3
#The content of liquid is 100 μ L, 4
#The content of liquid is 150 μ L, 5
#The content of liquid is 25 μ L, 6
#The content of liquid is 25 μ L, 7
#The content of liquid is 100 μ L, 8
#The content of liquid is 100 μ L, 9
#The content of liquid is 100 μ L, 10
#The content of liquid is 10 μ L, 11
#The content of liquid is 2mL, 12
#The content of liquid is 2mL, 13
#The content of liquid is 10mL, 14
#The content of liquid is 10mL.
(2) detection method of the detection kit of the described bean pod mottle virus of scheme () is characterized in that comprising the steps:
(1) preparation of viral extracting solution:
If 1. testing sample is a soybean leaves, then take by weighing a certain amount of leaf tissue, be that the ratio of 1: 10 (grams per milliliter) adds viral extraction agent 1 in the weightmeasurement ratio of sample and extraction buffer
#Liquid, after fully grinding, 4 ℃ of centrifugal 10min of following 10000g get supernatant liquor as viral extracting solution;
If 2. or testing sample be soybean seeds, behind the 2h that then earlier soybean seeds is soaked in water, strip its kind skin as sample, be that the ratio of 1: 10 (grams per milliliter) adds viral extraction agent 1 in the weightmeasurement ratio of sample and extraction buffer again
#Liquid, after fully grinding, 4 ℃ of centrifugal 10min of following 10000g get supernatant liquor as viral extracting solution;
(2) reverse transcription reaction: the viral extracting solution 50 μ L bag of getting step (1) preparation is managed by PCR, and with 50 μ L 11
#Liquid and 50 μ L 12
#Liquid is managed by PCR as positive control and negative control bag respectively, hatches 3h, 13 under 37 ℃ of every pipes
#Liquid is washed pipe 3 times and 14
#Liquid then carries out reverse transcription reaction after washing and managing 2 times; The reverse transcription reaction program is: add 1 μ L 3 by every pipe
#Liquid and 9 μ L 14
#Liquid places cooled on ice 3min behind 70 ℃ of water-bath 10min immediately, adds 5 μ L 4 by every pipe again
#Liquid, 1 μ L 5
#Liquid, 1 μ L 6
#Liquid, 2 μ L 7
#Liquid, through 42 ℃ of 1h, 70 ℃ of synthetic cDNA of 10min;
(3) PCR reaction: with viral extracting solution, 11
#Liquid and 12
#Liquid is template by each 5 μ L cDNA that reverse transcription reaction obtains respectively, adds 2.5 μ L 8 by every pipe
#Liquid, 2 μ L 9
#Liquid, 0.5 μ L 7
#Liquid, 2 μ L 2
#Liquid, 2 μ L 3
#Liquid, 10.75 μ L14
#Liquid and 0.25 μ L 10
#Liquid carries out the PCR reaction then; The PCR response procedures is to enter following circulation behind 94 ℃ of sex change 3min: 94 ℃ of sex change 30s, 52 ℃ of annealing 45s, 72 ℃ extend 1min, totally 35 circulations, last is taken turns after the loop ends 72 ℃ and extends 10min;
(4) after pcr amplification product electrophoresis detection: PCR reaction finishes, get product 10 μ L, detect, on gel imaging system, observe behind the ethidium bromide staining and the record experimental result with 1.5% agarose gel electrophoresis.
The electrophoresis detection result who contains the bean pod mottle virus sample is for bright DNA band occurring at the 542bp place.
The present invention is according to bean pod mottle virus gene conservative zone design Auele Specific Primer, utilize TC-RT-PCR (test tube is caught RT-PCR) technology that bean pod mottle virus is detected, compared with prior art, the detection kit of bean pod mottle virus provided by the present invention and detection method thereof have following advantage:
1) the present invention uses the TC-RT-PCR technology, has saved RNA extraction step loaded down with trivial details among the conventional RT-PCR, can directly carry out RT-PCR to viral extracting solution, not only simplifies procedures, reduces operational requirement, and avoided with an organic solvent, reduces environmental pollution; Compare with IC-RT-PCR (immunocapture RT-PCR), the present invention does not need expensive antiserum(antisera), but directly removes viral adsorption with the PCR pipe, has saved the detection cost greatly.
2) detection method provided by the invention detects bean pod mottle virus, amount of samples is few, high specificity, highly sensitive, accuracy good, easy and simple to handle, the result stable, can not only detect the simple grain soybean seeds, is suitable for the rapid detection of a large amount of samples simultaneously.
3) the detection kit making method of the present invention's design is simple, use is economical, has important actual application value.
Description of drawings
Fig. 1 is the detected result of soybean leaves in spite of illness.M:DNA molecular weight standard (100bp) wherein; 1: test sample (soybean leaves in spite of illness); The 2:BPMV positive control; The 3:BPMV negative control;
Fig. 2 is the detected result of soybean seeds in spite of illness.M:DNA molecular weight standard (100bp) wherein; 1: test sample (soybean seeds in spite of illness); The 2:BPMV positive control; The 3:BPMV negative control.
Fig. 3 is the detected result of soybean seedling in spite of illness.M:DNA molecular weight standard (100bp) wherein; 1: test sample I (soybean seedling in spite of illness); 2: test sample II (soybean seedling in spite of illness); 3: test sample III (soybean seedling in spite of illness); The 4:BPMV positive control; The 5:BPMV negative control.
Embodiment
Below the invention will be further described by specific embodiment.
Embodiment 1: the detection kit of bean pod mottle virus
A kind of detection kit of bean pod mottle virus is characterized in that it consists of:
(1) 1
#Liquid, contain viral extraction agent: the PBS damping fluid, concentration 1 *, pH7.4;
(2) 2
#Liquid contains bean pod mottle virus upstream primer: 5 '-GGCAATTTTAGAGCAAGTGTTG-3 ', concentration 10 μ mol/L;
(3) 3
#Liquid contains bean pod mottle virus downstream primer: 5 '-GCATTTGGCATATTCATGAGAAT-3 ', concentration 10 μ mol/L;
(4) 4
#Liquid contains RT Buffer, concentration 5 *;
(5) 5
#Liquid contains the RNA enzyme inhibition factor, concentration 40U/ μ L;
(6) 6
#Liquid contains ThermoScript II, concentration 200U/ μ L;
(7) 7
#Liquid contains dNTPs, concentration 10mmol/L;
(8) 8
#Liquid contains PCR Buffer, concentration 10 *;
(9) 9
#Liquid contains Mgcl
2, concentration 25mmol/L;
(10) 10
#Liquid contains the Taq archaeal dna polymerase, concentration 5U/ μ L;
(11) 11
#Liquid contains the bean pod mottle virus positive control;
(12) 12
#Liquid contains the bean pod mottle virus negative control;
(13) 13
#Liquid contains the PBST damping fluid, concentration 1 *;
(14) 14
#Liquid contains RNase-free ddH
2O.
Described 1
#Liquid-14
#Liquid is respectively 1 pipe, wherein 1
#The content of liquid is 10mL, 2
#The content of liquid is 100 μ L, 3
#The content of liquid is 100 μ L, 4
#The content of liquid is 150 μ L, 5
#The content of liquid is 25 μ L, 6
#The content of liquid is 25 μ L, 7
#The content of liquid is 100 μ L, 8
#The content of liquid is 100 μ L, 9
#The content of liquid is 100 μ L, 10
#The content of liquid is 10 μ L, 11
#The content of liquid is 2mL, 12
#The content of liquid is 2mL, 13
#The content of liquid is 10mL, 14
#The content of liquid is 10mL.
The detection method of 2 pairs of bean pod mottle virus of embodiment
The detection kit of above-mentioned bean pod mottle virus is used to detect the detection method of bean pod mottle virus (BPMV), comprising:
1) taking by weighing the sick leaf of 0.05g soybean, is the viral extraction agent 1 of 1: 10 (grams per milliliter) adding by the weightmeasurement ratio of sample and extraction buffer
#Liquid, after fully grinding, 4 ℃ of centrifugal 10min of following 10000g get supernatant liquor as viral extracting solution;
2) get the sick leaf supernatant liquors of 50 μ L soybean (viral extracting solution) bag and managed by PCR, and with 50 μ L11
#Liquid, 50 μ L 12
#Liquid is managed by PCR as positive control and negative control bag respectively.Hatch 3h, 13 under 37 ℃ of every pipes
#Liquid is washed pipe 3 times and 14
#Liquid carries out reverse transcription reaction after washing and managing 2 times; The reverse transcription reaction program is: add 1 μ L 3 by every pipe
#Liquid and 9 μ L 14
#Liquid places cooled on ice 3min behind 70 ℃ of water-bath 10min immediately, adds 5 μ L 4 by every pipe again
#Liquid, 1 μ L 5
#Liquid, 1 μ L 6
#Liquid, 2 μ L7
#Liquid.Through 42 ℃ of 1h, 70 ℃ of synthetic cDNA of 10min;
3) get with viral extracting solution, 11
#Liquid and 12
#Liquid is template by each 5 μ L cDNA that reverse transcription reaction obtains respectively, adds 2.5 μ L 8 by every pipe
#Liquid, 2 μ L 9
#Liquid, 0.5 μ L 7
#Liquid, 2 μ L 2
#Liquid, 2 μ L 3
#Liquid, 10.75 μ L 14
#Liquid and 0.25 μ L 10
#Liquid carries out the PCR reaction then.The PCR response procedures is to enter following circulation behind 94 ℃ of sex change 3min: 94 ℃ of sex change 30s, 52 ℃ of annealing 45s, 72 ℃ extend 1min, totally 35 circulations, last is taken turns after the loop ends 72 ℃ and extends 10min;
4) after the PCR reaction finishes, get product 10 μ L, detect, on gel imaging system, observe behind the ethidium bromide staining and the record experimental result with 1.5% agarose gel electrophoresis.
Contain the DNA band (Fig. 1) of electrophoresis detection result for occurring at the 542bp place becoming clear of bean pod mottle virus (BPMV) sample, otherwise do not have.
The inward detection of soybean seeds in spite of illness of the U.S. is intercepted and captured at embodiment 3 ports
Be that with embodiment 2 differences detected object is the seed of soybean.Earlier soybean seeds is soaked in water behind the 2h, strips its kind skin as sample, the weightmeasurement ratio by sample and extraction buffer is that 1: 10 (grams per milliliter) adds viral extraction agent 1 again
#Liquid, after fully grinding, 4 ℃ of centrifugal 10min of following 10000g get supernatant; All the other steps contain the DNA band (Fig. 2) of electrophoresis detection result for occurring at the 542bp place becoming clear of bean pod mottle virus (BPMV) sample, otherwise do not have with embodiment 2.
The detection of embodiment 4 inward U.S. soybean seedling
In inward soybean isolation implant base, picked at random has the soybean seedling of mottled symptom, and the blade of per 5 strain seedling is as a duplicate samples, totally 3 parts.All the other steps contain the DNA band (Fig. 3) of electrophoresis detection result for occurring at the 542bp place becoming clear of bean pod mottle virus (BPMV) sample, otherwise do not have with embodiment 2.
Sequence table .SEQ.txt 2008-5-30
SEQUENCE?LISTING
＜110〉Inspection ﹠ Quarantine Technology Center of Fujian Entry-Exit Inspection ﹠ Quar
＜120〉detection kit of bean pod mottle virus and detection method thereof
<130>None
<160>2
<170>PatentIn?version?3.3
<210>1
<211>22
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉primer
<400>1
ggcaatttta?gagcaagtgt?tg 22
<210>2
<211>23
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉primer
<400>2
gcatttggca?tattcatgag?aat 23
Claims (4)
1, a kind of detection kit of bean pod mottle virus is characterized in that it consists of:
(1) 1
#Liquid, contain viral extraction agent: the PBS damping fluid, concentration 1 *, pH7.4;
(2) 2
#Liquid contains bean pod mottle virus upstream primer: 5 '-GGCAATTTTAGAGCAAGTGTTG-3 ', concentration 10 μ mol/L;
(3) 3
#Liquid contains bean pod mottle virus downstream primer: 5 '-GCATTTGGCATATTCATGAGAAT-3 ', concentration 10 μ mol/L;
(4) 4
#Liquid contains RT Buffer, concentration 5 *;
(5) 5
#Liquid contains the RNA enzyme inhibition factor, concentration 40U/ μ L;
(6) 6
#Liquid contains ThermoScript II, concentration 200U/ μ L;
(7) 7
#Liquid contains dNTPs, concentration 10mmol/L;
(8) 8
#Liquid contains PCR Buffer, concentration 10 *;
(9) 9
#Liquid contains Mgcl
2, concentration 25mmol/L;
(10) 10
#Liquid contains the Taq archaeal dna polymerase, concentration 5U/ μ L;
(11) 11
#Liquid contains the bean pod mottle virus positive control;
(12) 12
#Liquid contains the bean pod mottle virus negative control;
(13) 13
#Liquid contains the PBST damping fluid, concentration 1 *;
(14) 14
#Liquid contains RNase-free ddH
2O.
2. the detection kit of bean pod mottle virus according to claim 1 is characterized in that: described 1
#Liquid-14
#Liquid is respectively 1 pipe, wherein 1
#The content of liquid is 10mL, 2
#The content of liquid is 100 μ L, 3
#The content of liquid is 100 μ L, 4
#The content of liquid is 150 μ L, 5
#The content of liquid is 25 μ L, 6
#The content of liquid is 25 μ L, 7
#The content of liquid is 100 μ L, 8
#The content of liquid is 100 μ L, 9
#The content of liquid is 100 μ L, 10
#The content of liquid is 10 μ L, 11
#The content of liquid is 2mL, 12
#The content of liquid is 2mL, 13
#The content of liquid is 10mL, 14
#The content of liquid is 10mL.
3, the detection method of the detection kit of the described bean pod mottle virus of claim 1 is characterized in that comprising the steps:
(1) preparation of viral extracting solution:
If 1. testing sample is a soybean leaves, then take by weighing a certain amount of leaf tissue, be that the ratio of 1: 10 (grams per milliliter) adds viral extraction agent 1 in the weightmeasurement ratio of sample and extraction buffer
#Liquid, after fully grinding, 4 ℃ of centrifugal 10min of following 10000g get supernatant liquor as viral extracting solution;
If 2. or testing sample be soybean seeds, behind the 2h that then earlier soybean seeds is soaked in water, strip its kind skin as sample, be that the ratio of 1: 10 (grams per milliliter) adds viral extraction agent 1 in the weightmeasurement ratio of sample and extraction buffer again
#Liquid, after fully grinding, 4 ℃ of centrifugal 10min of following 10000g get supernatant liquor as viral extracting solution;
(2) reverse transcription reaction: the viral extracting solution 50 μ L bag of getting step (1) preparation is managed by PCR, and with 50 μ L 11
#Liquid and 50 μ L 12
#Liquid is managed by PCR as positive control and negative control bag respectively, hatches 3h, 13 under 37 ℃ of every pipes
#Liquid is washed pipe 3 times and 14
#Liquid then carries out reverse transcription reaction after washing and managing 2 times; The reverse transcription reaction program is: add 1 μ L 3 by every pipe
#Liquid and 9 μ L 14
#Liquid places cooled on ice 3min behind 70 ℃ of water-bath 10min immediately, adds 5 μ L 4 by every pipe again
#Liquid, 1 μ L 5
#Liquid, 1 μ L 6
#Liquid, 2 μ L 7
#Liquid, through 42 ℃ of 1h, 70 ℃ of synthetic cDNA of 10min;
(3) PCR reaction: with viral extracting solution, 11
#Liquid and 12
#Liquid is template by each 5 μ L cDNA that reverse transcription reaction obtains respectively, adds 2.5 μ L 8 by every pipe
#Liquid, 2 μ L 9
#Liquid, 0.5 μ L 7
#Liquid, 2 μ L 2
#Liquid, 2 μ L 3
#Liquid, 10.75 μ L14
#Liquid and 0.25 μ L 10
#Liquid carries out the PCR reaction then; The PCR response procedures is to enter following circulation behind 94 ℃ of sex change 3min: 94 ℃ of sex change 30s, 52 ℃ of annealing 45s, 72 ℃ extend 1min, totally 35 circulations, last is taken turns after the loop ends 72 ℃ and extends 10min;
(4) after pcr amplification product electrophoresis detection: PCR reaction finishes, get product 10 μ L, detect, on gel imaging system, observe behind the ethidium bromide staining and the record experimental result with 1.5% agarose gel electrophoresis.
4, according to the detection method of the detection kit of the described bean pod mottle virus of claim 3, it is characterized in that: the DNA band of electrophoresis detection result that contains the bean pod mottle virus sample for occurring at 542bp place becoming clear.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100711544A CN101285104B (en) | 2008-05-30 | 2008-05-30 | Kit for detecting bean pod mottle virus and detection process thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100711544A CN101285104B (en) | 2008-05-30 | 2008-05-30 | Kit for detecting bean pod mottle virus and detection process thereof |
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| Publication Number | Publication Date |
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| CN101285104A true CN101285104A (en) | 2008-10-15 |
| CN101285104B CN101285104B (en) | 2010-06-09 |
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|---|---|---|---|
| CN2008100711544A Expired - Fee Related CN101285104B (en) | 2008-05-30 | 2008-05-30 | Kit for detecting bean pod mottle virus and detection process thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429562B (en) * | 2008-11-10 | 2011-09-07 | 宁波检验检疫科学技术研究院 | Fluorescence quantitative RT-PCR detecting agent for bean pod mottle virus, preparation method and application thereof |
| CN106755592A (en) * | 2017-02-16 | 2017-05-31 | 中国检验检疫科学研究院 | Method based on RPA technology for detection bean pod mottle virus, RPA primers and kit |
-
2008
- 2008-05-30 CN CN2008100711544A patent/CN101285104B/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429562B (en) * | 2008-11-10 | 2011-09-07 | 宁波检验检疫科学技术研究院 | Fluorescence quantitative RT-PCR detecting agent for bean pod mottle virus, preparation method and application thereof |
| CN106755592A (en) * | 2017-02-16 | 2017-05-31 | 中国检验检疫科学研究院 | Method based on RPA technology for detection bean pod mottle virus, RPA primers and kit |
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|---|---|
| CN101285104B (en) | 2010-06-09 |
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