CN106191047B - Nucleic acid test strip method for detecting cucumber bacterial angular leaf spot and application thereof - Google Patents
Nucleic acid test strip method for detecting cucumber bacterial angular leaf spot and application thereof Download PDFInfo
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- 230000003321 amplification Effects 0.000 claims description 25
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Abstract
The invention discloses a nucleic acid test strip method for detecting cucumber bacterial angular leaf spot and application thereof. The invention firstly provides a specific primer group, which consists of a primer PSPL-BF, a primer PSPL-MBF, a primer PSPL-DF, a primer PSPL-CPR and a primer PSPL-BR, wherein each primer is shown as a sequence 1 to a sequence 5 in a sequence table in sequence. The specific primer group is used for identifying the cucumber bacterial angular leaf spot germs or detecting whether a plant sample to be detected contains the cucumber bacterial angular leaf spot germs. The invention has the advantages of good specificity, high sensitivity, simple operation, high detection efficiency and the like, and can meet the basic requirements of port and field detection.
Description
Technical field
The invention belongs to technical field of plant quarantine, are related to Measurement for Biotechnique, and in particular to one kind is used for cucumber bacterium
Property angular leaf spot fungus nucleic acid test strip detection method and application this method identification cucumber bacterial angular leaf spot bacterium or containing cucumber it is thin
The method of the vegetable material of bacterium property angular leaf spot fungus.
Background technique
Cucumber bacterial angular leaf spot is one of important disease of cucumber, there is generation throughout our country, with the big face of cucumber
Product plantation, cucumber bacterial angular leaf spot aggravate cucumber underproduction situation year by year.The pathogen of cucumber bacterial angular leaf spot is
Pseudomonas syringae pv.Lachrymans, the main host plants of the pathogenic bacteria are cucumber, can additionally infect cucurbit,
Muskmelon, cucurbita pepo and sponge gourd.There is the spot of yellow chlorisis in the cucumber initial stage of catching an illness, and blade back is water soaking mode scab, catch an illness the later period by
In the blocking of vein, irregular polygon is presented in scab, and as scab cracks, blade is withered to fall off.
The detection method of cucumber bacterial angular leaf spot bacterium be based primarily upon morphological feature, Pathogenicity, cultural colony and
Serological reaction identification and real-time fluorescence PCR etc., time-consuming, sensitivity is low or needs expensive operation instrument, is unfavorable for
The popularization and use of grass-roots unit.
Summary of the invention
The object of the present invention is to provide a kind of nucleic acid test strip method for for detecting cucumber bacterial angular leaf spot bacterium and
It is applied.
Present invention firstly provides a species-specific primers groups, by primer PSPL-BF, primer PSPL-MBF, primer PSPL-
DF, primer PSPL-CPR and primer PSPL-BR composition；
The primer PSPL-BF is following (a1) or (a2)；
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) sequence 1 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1
The DNA molecular of identical function；
The primer PSPL-MBF is following (a3) or (a4)；
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) sequence 2 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of identical function；
The primer PSPL-DF is following (a5) or (a6)；
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) sequence 3 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3
The DNA molecular of identical function；
The primer PSPL-CPR is following (a7) or (a8)；
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) sequence 4 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4
The DNA molecular of identical function；
The primer PSPL-BR is following (a9) or (a10)；
(a9) single strand dna shown in the sequence 5 of sequence table；
(a10) sequence 5 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5
The DNA molecular of identical function.
The special primer group is the primer sets designed based on cross primer constant-temperature amplification principle.
The end 5' of primer PSPL-MBF carries out biotin labeling.
The end 5' of primer PSPL-DF carries out 6-FAM label.
The purposes of the special primer group is following (b1) or (b2) or (b3) or (b4):
(b1) cucumber bacterial angular leaf spot bacterium is identified；
(b2) kit for identifying cucumber bacterial angular leaf spot bacterium is prepared；
(b3) it detects in plant sample to be measured and whether contains cucumber bacterial angular leaf spot bacterium；
(b4) preparation for detect in plant sample to be measured whether the kit containing cucumber bacterial angular leaf spot bacterium.
The present invention also protects the application of the special primer group, for as follows (b1) or (b2) or (b3) or (b4):
(b1) cucumber bacterial angular leaf spot bacterium is identified；
(b2) kit for identifying cucumber bacterial angular leaf spot bacterium is prepared；
(b3) it detects in plant sample to be measured and whether contains cucumber bacterial angular leaf spot bacterium；
(b4) preparation for detect in plant sample to be measured whether the kit containing cucumber bacterial angular leaf spot bacterium.
The present invention also protects the kit containing the special primer group；The purposes of the kit be following (c1) or
(c2):
(c1) cucumber bacterial angular leaf spot bacterium is identified；
(c2) it detects in plant sample to be measured and whether contains cucumber bacterial angular leaf spot bacterium.
The kit may also include the reagent for cross primer constant-temperature amplification, such as Bst archaeal dna polymerase.
The kit may also include disposable nucleic acid detection apparatus.
The present invention also protects the preparation method of the kit, includes the steps that individually packing each primer.
The present invention also protects a kind of method for identifying cucumber bacterial angular leaf spot bacterium, includes the following steps:
(1) genomic DNA of tested bacteria is extracted；
(2) genomic DNA obtained using step (1) carries out cross primer constant temperature using the special primer group as template
Amplification, then makes the following judgment:
It is to be measured if the special primer group is used to may be implemented using the genomic DNA as the specific amplification of template
Bacterium is or candidate is cucumber bacterial angular leaf spot bacterium；If the special primer group is used to can not achieve with the genome
DNA is the specific amplification of template, and tested bacteria is or candidate is non-cucumber bacterial angular leaf spot bacterium.
The present invention also protects a kind of method for identifying cucumber bacterial angular leaf spot bacterium, includes the following steps:
Using tested bacteria as template, cross primer constant-temperature amplification is carried out using the special primer group, is then carried out as follows
Judge: if the special primer group is used to may be implemented using the tested bacteria as the specific amplification of template, tested bacteria
For or candidate be cucumber bacterial angular leaf spot bacterium；If the special primer group is used to can not achieve using the tested bacteria as mould
The specific amplification of plate, tested bacteria is or candidate is non-cucumber bacterial angular leaf spot bacterium.
The present invention also protects a kind of method for identifying cucumber bacterial angular leaf spot bacterium, includes the following steps: to detect to be measured thin
The target sequence for whether containing the special primer group in the genomic DNA of bacterium, then makes the following judgment: if tested bacteria
Contain the target sequence of the special primer group in genomic DNA, tested bacteria is or candidate is cucumber bacterial angular leaf spot bacterium；Such as
The target sequence of the special primer group is not contained in the genomic DNA of fruit tested bacteria, tested bacteria is or candidate is non-cucumber
Bacterial angular leaf spot bacterium.
The present invention also protect it is a kind of identify vegetable material to be measured whether the method containing cucumber bacterial angular leaf spot bacterium, including
Following steps:
(1) total DNA of vegetable material to be measured is extracted；
(2) total DNA obtained using step (1) carries out cross primer constant-temperature amplification using the special primer group as template,
Then it makes the following judgment:
If the special primer group is used to may be implemented using the total DNA as the specific amplification of template, to measuring plants
Material contains or doubtful containing cucumber bacterial angular leaf spot bacterium；If using the special primer group can not achieve with described total
DNA is the specific amplification of template, and vegetable material to be measured does not contain or doubtful without containing cucumber bacterial angular leaf spot bacterium.
The present invention also protect it is a kind of identify vegetable material to be measured whether the method containing cucumber bacterial angular leaf spot bacterium, including
Following steps:
The target sequence for whether containing the special primer group in the total DNA of vegetable material to be measured is detected, is then carried out as follows
Judgement: if containing the target sequence of the special primer group in the total DNA of vegetable material to be measured, vegetable material to be measured contains or doubts
Seemingly contain cucumber bacterial angular leaf spot bacterium；If not containing the target sequence of the special primer group in the total DNA of vegetable material to be measured
Column, vegetable material to be measured do not contain or doubtful without containing cucumber bacterial angular leaf spot bacterium.
The present invention also protects the application of the target sequence of the special primer group or the target sequence of the special primer group；
The application is following (c1) or (c2):
(c1) cucumber bacterial angular leaf spot bacterium is identified；
(c2) it detects in plant sample to be measured and whether contains cucumber bacterial angular leaf spot bacterium.
In any description above method, in the reaction system of cross primer constant-temperature amplification, the concentration of primer PSPL-CPR is
1.5 μm of ol/L, primer PSPL-MBF concentration be 0.9 μm of ol/L, the concentration of primer PSPL-DF is 0.9 μm of ol/L, primer
The concentration of PSPL-BF is 0.3 μm of ol/L, the concentration of primer PSPL-BR is 0.3 μm of ol/L.
In any description above method, in the reaction system of cross primer constant-temperature amplification, the concentration of primer PSPL-CPR is
The concentration of primer PSPL-MBF of 1.5 μm of ends ol/L, 5' with biotin labeling is that 0.9 μm of end ol/L, 5' has 6-FAM
The concentration of the primer PSPL-DF of label is 0.9 μm of ol/L, the concentration of primer PSPL-BF is 0.3 μm of ol/L, primer PSPL-BR
Concentration is 0.3 μm of ol/L.
In any description above method, the reaction system of cross primer constant-temperature amplification is following (20 μ l): primer PSPL-
The 0.9 μm of end ol/L, 5' primer PSPL-MBF of the CPR1.5 μm of end ol/L, 5' with biotin labeling is marked with 6-FAM
0.9 μm of ol/L of primer PSPL-DF, 0.3 μm of ol/L of primer PSPL-BF, primer PSPL-BR0.3 μm of ol/L, dNTP
0.4mmol, 10 × Thermopol buffer, 2 μ l, Bst archaeal dna polymerase 8 units, MgSO42mmol, template 2 μ l, it is remaining
Amount is sterile water.
In any description above method, the reaction condition of cross primer constant-temperature amplification concretely: 62 DEG C, 60min.
The tested bacteria is Pseudomonas fluorescens (Pseudomonas fluorescens), tomato bacterial leaf spot
Bacterium (Pseudomonas syringae pv.tomato), P.stwartii subsp.stewartii (Pantoea stewartii
Subsp.stewartii), Kidney bean bacterial wilting germ (Curtobacterium flaccumfaciens
Pv.flaccumfaci), Acidovorax avenae oat subspecies (Acidovorax avenae subsp.avenae), orchid brown spot
Dizzy epidemic disease bacterium (the Pseudomonas of bacterium (Acidovorax avenae subsp.cattleyae), Kidney bean
savastanoipv.phaseolicola)、Acidovorax konjaci、Pseudomonas syringae
pv.syringae、Pseudomonas syringae pv.tabaci、Acidovorax delafieldii、
Acidovoraxvalerianellae or cucumber bacterial angular leaf spot bacterium (Pseudomonas syringae
pv.Lachrymans)。
The bacterial strain of the Pseudomonas fluorescens (Pseudomonas fluorescens) concretely NCPPB No.967.
The pseudomonas syringae pv.tomato (Pseudomonas syringae pv.tomato) concretely NCPPB No.269:
Bacterial strain.The P.stwartii subsp.stewartii (Pantoea stewartii subsp.stewartii) concretely NCPPB
No.449: bacterial strain.Kidney bean bacterial wilting germ (the Curtobacterium flaccumfaciens
Pv.flaccumfaci) concretely NCPPB No.178: bacterial strain.The Acidovorax avenae oat subspecies (Acidovorax
Avenae subsp.avenae) concretely NCPPB No.359: bacterial strain.Orchid brown patch germ (the Acidovorax
Avenae subsp.cattleyae) concretely NCPPB 4196 bacterial strain.The Kidney bean is swooned epidemic disease bacterium (Pseudomonas
Savastanoipv.phaseolicola) the concretely bacterial strain of ICMP 2740.The Acidovorax konjaci is specific
It can be the bacterial strain of ATCC 33996.The Pseudomonas syringae pv.syringae concretely Belgian microorganism
The bacterial strain that Culture Collection Center number is LMG 5083.The Pseudomonas syringae pv.tabaci concretely compares
The bacterial strain that Culture Collection number is LMG2130 when sharp.The Acidovorax delafieldii is concretely
The bacterial strain that Belgian Culture Collection number is LMG 1792.The Acidovoraxvalerianellae is specific
It can be the bacterial strain that New Zealand's International Plant Organism Depositary number is ICMP13406.The cucumber bacterial angular leaf spot bacterium
(Pseudomonas syringaepv.Lachrymans) concretely bacterial strain NCPPB540, bacterial strain NCPPB542, bacterial strain
NCPPB277, bacterial strain NCPPB467 or bacterial strain NCPPB1428.
Any description above vegetable material to be measured concretely cucumber material, more specifically can be cucumber leaves.
After completing cross primer constant-temperature amplification, disposable nucleic acid detection apparatus can be used and detected.Test strip and matter
Control band develops the color, and result is the positive.Quality Control band develops the color and test strip does not develop the color, and result is feminine gender.Quality Control band is not shown
Color judges that detection process is wrong, need to detect again.
The present invention provides the cucumber bacterial angular leaf spot sclerotium acid test paper established based on cross primer isothermal amplification technology
Detection method.This method carries out isothermal amplification reactions using sample total DNA or bacterium solution as template, after reaction using primary
Property the nucleic acid test strip that includes of nucleic acid tests device carry out result judgement.The present invention has specific good, high sensitivity, operation letter
Just, the advantages that detection efficiency is high is able to satisfy the basic demand of port and Fields detection.
Detailed description of the invention
Fig. 1 is the result of versatility test and specific test；1 represents the blank that bacteria suspension is replaced with isometric sterile water
Control.
Fig. 2 is the result of sensitivity test.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.Disposable nucleic acid detection apparatus (Disposable Nucleic Acid Detection Device): the excellent think of in Hangzhou reaches
Bioisystech Co., Ltd, model D001-3.10 × Thermopol buffer is included for BstDNA polymerase.
Embodiment 1, the design and synthesis of primer
By a large amount of sequence analyses, sequence design, artificial screening optimization, compliance test result, one group is finally obtained for reflecting
Determine the special primer group of cucumber bacterial angular leaf spot bacterium.Special primer group is by primer PSPL-BF, primer PSPL-MB, primer
PSPL-DF, primer PSPL-CPR and primer PSPL-BR composition.
PSPL-BF (sequence 1): 5'-GCATCCTGCACCACCAATT-3'；
PSPL-MBF (sequence 2): 5'-ACCACCATCCATGCCTACA-3'；
PSPL-DF (sequence 3): 5'-CAACGACCAGAACCTGATT-3'；
PSPL-CPR (sequence 4): 5'-ACCACCATCCATGCCTACATAGGTACGGATCGGTGTGATA-3'；
PSPL-BR (sequence 5): 5'-ATTGATTGCGTCGCCGAAC-3'.
The end 5' of PSPL-MBF carries out biotin labeling, and the end 5' of PSPL-DF carries out 6-FAM label.
PSPL-BF is positive displacement primer.PSPL-MBF is detection probe 1.PSPL-DF is detection probe 2.PSPL-CPR
For cross primer.PSPL-BR is backward-substitition primer.
The foundation of embodiment 2, method
One, identify tested bacteria whether be cucumber bacterial angular leaf spot bacterium method
1, the bacteria suspension of tested bacteria is prepared.
2, the bacteria suspension prepared using step 1 carries out the expansion of cross primer constant temperature using the primer sets that embodiment 1 designs as template
Increase.
Reaction system (20 μ l): the 1.5 μm of ends ol/L, 5' primer PSPL-CPR have the primer PSPL- of biotin labeling
The 0.9 μm of end ol/L, 5' MBF has 0.3 μm of 0.9 μm of ol/L of primer PSPL-DF, primer PSPL-BF ol/ of 6-FAM label
L, 0.3 μm of ol/L of primer PSPL-BR, dNTP 0.4mmol, 10 × Thermopol buffer, 2 μ l, Bst archaeal dna polymerase 8
A unit, MgSO42mmol, 2 μ l of template, surplus is sterile water.
Reaction condition: 62 DEG C, 60min.
3, the product for taking step 2 is detected (by specification operation) using disposable nucleic acid detection apparatus.Test strip
It develops the color with Quality Control band, result is the positive；Quality Control band develops the color and test strip does not develop the color, and result is feminine gender；Quality Control band
It does not develop the color, judges that detection process is wrong, need to detect again.
In practical application, also can extract tested bacteria genomic DNA, using Genomic DNA solution replace bacteria suspension as
Template carries out above-mentioned steps.
Two, identify vegetable material to be measured whether the method containing cucumber bacterial angular leaf spot bacterium
1, the total DNA of vegetable material to be measured is extracted.
2, the total DNA extracted using step 1 carries out the expansion of cross primer constant temperature using the primer sets that embodiment 1 designs as template
Increase.Reaction system and reaction condition with step 12.
3, the product for taking step 2 is detected (by specification operation) using disposable nucleic acid detection apparatus.Test strip
It develops the color with Quality Control band, result is the positive；Quality Control band develops the color and test strip does not develop the color, and result is feminine gender；Quality Control band
It does not develop the color, judges that detection process is wrong, need to detect again.
Embodiment 3, versatility test
Tested bacteria: bacterial strain NCPPB540, bacterial strain NCPPB542, bacterial strain NCPPB277, bacterial strain NCPPB467 or bacterial strain
NCPPB1428 is existing Pseudomonas syringae pv.Lachrymans bacterial strain.Refer to above each bacterial strain
Document: Wang Zhe, Chen Qing, the application PCR method such as field madder quickly detect cucumber bacterial angular leaf spot bacterium [J] plant quarantine, and 2011,
25(6):29-32。
Using the bacteria suspension of tested bacteria as template, detected according to the step of embodiment 2 one method.
The 3 of the result is shown in Figure 1 of bacterial strain NCPPB540, the 4 of the result is shown in Figure 1 of bacterial strain NCPPB542, bacterial strain NCPPB277's
The 5 of the result is shown in Figure 1, the 6 of the result is shown in Figure 1 of bacterial strain NCPPB467, the 7 of the result is shown in Figure 1 of bacterial strain NCPPB1428.The result shows that
The testing result of each tested bacteria is the positive.
Embodiment 4, specific test
Tested bacteria is as follows:
Pseudomonas fluorescens (Pseudomonas fluorescens): NCPPB No.967；
Pseudomonas syringae pv.tomato (Pseudomonas syringae pv.tomato): NCPPB No.269:；
P.stwartii subsp.stewartii (Pantoea stewartii subsp.stewartii): NCPPB No.449:；
Kidney bean bacterial wilting germ (Curtobacterium flaccumfaciens pv.flaccumfaci):
NCPPB No.178:；
Acidovorax avenae oat subspecies (Acidovorax avenae subsp.avenae): NCPPB No.359:；
Orchid brown patch germ (Acidovorax avenae subsp.cattleyae): NCPPB 4196；
Kidney bean is swooned epidemic disease bacterium (Pseudomonas savastanoipv.phaseolicola): ICMP2740；
Acidovorax konjaci:ATCC 33996；
Pseudomonas syringae pv.syringae: Belgian Culture Collection, number LMG
5083；
Pseudomonas syringaepv.tabaci: Belgian Culture Collection, number LMG 2130；
Acidovorax delafieldii: Belgian Culture Collection, number LMG 1792；
Acidovoraxvalerianellae: New Zealand's International Plant Organism Depositary, number ICMP13406.
NCPPB: United Kingdom National plant pathogenetic bacteria collection (National Collection of Plant
Pathogenic Bacteria).ATCC: American Type Culture collection (American type culture
collection)。
Using the bacteria suspension of tested bacteria as template, detected according to the step of embodiment 2 one method.
The 2 of the result is shown in Figure 1 of pseudomonas syringae pv.tomato.
The result shows that the testing result of each tested bacteria is feminine gender.
Embodiment 5, sensitivity test
The bacteria suspension of cucumber bacterial angular leaf spot bacteria strain NCPPB1428 is subjected to ten times of gradient dilutions with sterile water, point
Not obtaining bacteria concentration is 0.54 × 105Bacteria suspension 1, the bacteria concentration of cfu/ml is 0.54 × 104Bacteria suspension 2, the bacteria concentration of cfu/ml
It is 0.54 × 103The bacteria suspension 3 and bacteria concentration of cfu/ml is 0.54 × 102The bacteria suspension 4 of cfu/ml.
Using each bacteria suspension as template (by 2 μ l sterile waters as blank control), according to the step of embodiment 2 one method
It is detected.
As a result see Fig. 2.In Fig. 2,1 it is blank control, 2 be concentration is 0.54 × 105The bacteria suspension of cfu/ml, 3 are concentration
It is 0.54 × 104The bacteria suspension of cfu/ml, 4 be concentration are 0.54 × 103The bacteria suspension of cfu/ml, 5 be concentration be 0.54 ×
102The bacteria suspension of cfu/ml.The result shows that bacteria concentration is 0.54 × 103The bacterium solution testing result of cfu/ml or more is the positive, instead
The additional amount for answering bacteria suspension in system is 2 μ l, is 1.08 bacteriums through conversion sensitivity minimization.
Embodiment 6, detection plant sample to be measured
Several cucumber leaves with cucumber bacterial angular leaf spot disease symptom are taken, as vegetable material to be measured.It is each to
Measuring plants material is derived from different cucumber plants.
Vegetable material to be measured is taken, is detected according to the step of embodiment 2 two method.Result is the positive.
Vegetable material to be measured is taken, bacterial strain is isolated and purified and carries out morphological feature identification, pathogenic identification, cultural colony mirror
The identification of fixed and serological reaction, result is the positive.
The above result shows that each vegetable material tool to be measured contains cucumber bacterial angular leaf spot bacterium, side provided by the invention
The accuracy of method is 100%.
Claims (7)
1. special primer group, by primer PSPL-BF, primer PSPL-MBF, primer PSPL-DF, primer PSPL-CPR and primer
PSPL-BR composition；
The primer PSPL-BF is single strand dna shown in the sequence 1 of sequence table；
The primer PSPL-MBF is single strand dna shown in the sequence 2 of sequence table；
The primer PSPL-DF is single strand dna shown in the sequence 3 of sequence table；
The primer PSPL-CPR is single strand dna shown in the sequence 4 of sequence table；
The primer PSPL-BR is single strand dna shown in the sequence 5 of sequence table.
2. the application of special primer group described in claim 1, for as follows (b1) or (b2) or (b3) or (b4):
(b1) cucumber bacterial angular leaf spot bacterium is identified；
(b2) kit for identifying cucumber bacterial angular leaf spot bacterium is prepared；
(b3) it detects in plant sample to be measured and whether contains cucumber bacterial angular leaf spot bacterium；
(b4) preparation for detect in plant sample to be measured whether the kit containing cucumber bacterial angular leaf spot bacterium.
3. the kit containing special primer group described in claim 1；The purposes of the kit is following (c1) or (c2):
(c1) cucumber bacterial angular leaf spot bacterium is identified；
(c2) it detects in plant sample to be measured and whether contains cucumber bacterial angular leaf spot bacterium.
4. the preparation method of kit described in claim 3 includes the steps that individually packing each primer.
5. a kind of method for identifying cucumber bacterial angular leaf spot bacterium, includes the following steps:
(1) genomic DNA of tested bacteria is extracted；
(2) genomic DNA obtained using step (1) carries out cross primer using special primer group described in claim 1 as template
Then constant-temperature amplification makes the following judgment:
If the special primer group is used to may be implemented using the genomic DNA as the specific amplification of template, tested bacteria
For or candidate be cucumber bacterial angular leaf spot bacterium；It is with the genomic DNA if the special primer group is used to can not achieve
The specific amplification of template, tested bacteria is or candidate is non-cucumber bacterial angular leaf spot bacterium.
6. a kind of method for identifying cucumber bacterial angular leaf spot bacterium, includes the following steps:
Using tested bacteria as template, cross primer constant-temperature amplification is carried out using special primer group described in claim 1, is then carried out
Judge as follows: to be measured if the special primer group is used to may be implemented using the tested bacteria as the specific amplification of template
Bacterium is or candidate is cucumber bacterial angular leaf spot bacterium；If the special primer group is used to can not achieve with the tested bacteria
For the specific amplification of template, tested bacteria is or candidate is non-cucumber bacterial angular leaf spot bacterium.
7. it is a kind of identify vegetable material to be measured whether the method containing cucumber bacterial angular leaf spot bacterium, include the following steps:
(1) total DNA of vegetable material to be measured is extracted；
(2) total DNA obtained using step (1) carries out cross primer constant temperature using special primer group described in claim 1 as template
Amplification, then makes the following judgment:
If using the special primer group to may be implemented using the total DNA as the specific amplification of template, vegetable material to be measured
Contain or doubtful containing cucumber bacterial angular leaf spot bacterium；It is with the total DNA if the special primer group is used to can not achieve
The specific amplification of template, vegetable material to be measured do not contain or doubtful without containing cucumber bacterial angular leaf spot bacterium.
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|---|---|---|---|---|
| CN101638685A (en) * | 2008-07-29 | 2010-02-03 | 杭州优思达生物技术有限公司 | Method for amplifying target nucleic acid sequence by using cross primer and kit for amplifying target nucleic acid sequence and application thereof |
| CN102181522A (en) * | 2011-03-07 | 2011-09-14 | 中国检验检疫科学研究院 | Primer for detecting cucumber bacterial angular leaf-spot germ |
| CN102758005A (en) * | 2012-04-13 | 2012-10-31 | 中国检验检疫科学研究院 | Primers and method for cross primer isothermal amplification detection of acidovorax citrulli |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638685A (en) * | 2008-07-29 | 2010-02-03 | 杭州优思达生物技术有限公司 | Method for amplifying target nucleic acid sequence by using cross primer and kit for amplifying target nucleic acid sequence and application thereof |
| CN102181522A (en) * | 2011-03-07 | 2011-09-14 | 中国检验检疫科学研究院 | Primer for detecting cucumber bacterial angular leaf-spot germ |
| CN102758005A (en) * | 2012-04-13 | 2012-10-31 | 中国检验检疫科学研究院 | Primers and method for cross primer isothermal amplification detection of acidovorax citrulli |
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