CN107338312A - A kind of method and kit that rice leaf spot bacteria is detected using digital pcr - Google Patents
A kind of method and kit that rice leaf spot bacteria is detected using digital pcr Download PDFInfo
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- CN107338312A CN107338312A CN201710675084.2A CN201710675084A CN107338312A CN 107338312 A CN107338312 A CN 107338312A CN 201710675084 A CN201710675084 A CN 201710675084A CN 107338312 A CN107338312 A CN 107338312A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention provides a kind of method and kit that rice leaf spot bacteria is detected using digital pcr.Kit provided by the present invention contains a pair of specific primers pair and a probe of the both ends with fluorescence labeling, and the nucleotide sequence of the specific primer pair is as shown in SEQ ID NO.1 2, and the nucleotide sequence of the probe is as shown in SEQ ID NO.3.Rhs family gene design of the primer of the present invention based on rice leaf spot bacteria, utilize above-mentioned specific primer pair and probe, rice leaf spot bacteria is detected using PCR method, specific good and high sensitivity, testing result accuracy is high, the detection of the germ suitable for rice paddy seed sample, there is important application value.
Description
Technical field
The present invention relates to plant protection and technical field of molecular biological detection, specifically, is related to bacterial blight of rice
Bacterium number word PCR detection method.
Background technology
Rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae), is a kind of Xanthomonas campestris section, Huang Dan
The bacterium of born of the same parents' Bacillus, careless rice, green hair sandbur, Bermuda grass, barnyard grass, Leersia Sw, Rong, not plump shell grass, moleplant seed, big can be infected
The grass family host plants such as broomcorn millet, ditch millet, Ba Lacao, wild rice stem, America wild rice, Korea lawn grass, additionally include difformed galingale herb, rhizoma cyperi
The sedges such as son.The germ found first in 1884 in Japanese Fukuoka area, it be found earliest on rice it is thin
Fungal disease.Since the 1950s, morbidity scope constantly expands.At present, bacterial blight of rice turns into Asia and peace
The important disease of foreign rice region, bacterial blight of rice are found, with lesion seed in Nanjing Suburb in China first in nineteen fifty
Output and allocation and transportation, it occurs and the getting worse that causes harm, soon extension sprawling comprehensively；Now in addition to Xinjiang, all parts of the country have
Different degrees of generation, but it is more universal and serious with south China, East China, the generation of Central China rice region.After rice is aggrieved, typically cause leaf
Piece dries up, and shrivelled kernel increases, mass of 1000 kernel decline, and rice matter is crisp, and loss caused by giving Rice Production every year typically reachable 10%~
30%, even up to more than 50%.
At present, the detection method for rice leaf spot bacteria mainly have including nursery observation on Growth method, direct method of isolation,
Traditional detection method, Virus monitory method and the PCR detection methods of Pathogenicity etc..Traditional detection method step is complicated, time-consuming, not
Adapt to the needs of quick detection.It is most widely used with ELISA in Virus monitory method, a large amount of samples can be detected in a short time
Product, but the antibody of different strains is not readily available, the cross reaction in course of reaction, easily cause false positive or false negative phenomenon
Problem.PCR detection techniques can fast and effectively detect rice leaf spot bacteria from rice paddy seed sample, no matter to the country
Outlet of the plant quarantine still to ensureing China's rice paddy seed and rice is significant, and oneself has a variety of different PCR at present
Detection technique is applied to the detection and identification of leaf spot bacteria, but bacterial bearing rate not high sample in part can not be made reliably
Diagnosis, causes false negative result.
Digital pcr (Digital-PCR) is a kind of new PCR detection method, and its operation principle is DNA or cDNA
Sample segment is many independent, parallel PCR reactions, and these reactions of part contain target molecules (positive), and other are not wrapped
Containing (feminine gender).Individual molecule can be amplified 1,000,000 times or more, it is possible to increase the performance of existing TaqMan measure, so as to real
Now higher sensitivity, the degree of accuracy, avoid the appearance of false negative result.
The content of the invention
It is an object of the invention to provide a kind of method and kit that rice leaf spot bacteria is detected using digital pcr.
The specific primer pair of detection rice leaf spot bacteria provided by the present invention and specific probe combination, it is described to draw
Thing sense primer Xoo119F and anti-sense primer Xoo119R to being made up of.The sense primer, anti-sense primer and specific probe
Sequence is as follows：
Sense primer Xoo119F：5′-AACGGATCGATGGACTTTACG-3′(SEQ ID NO.1)
Anti-sense primer Xoo119R：5′-AATGTGCACTTCCGCTATGA-3′(SEQ ID NO.2)
Specific probe Xoo119P：5’-FAM-TTCGTGTGAGGTTACCCTGC-TAMRA-3’(SEQ ID NO.3)
The present invention further provides the digital pcr detection method of rice leaf spot bacteria, comprise the following steps：
(1) genomic DNA of testing sample is extracted；
(2) using the genomic DNA of extraction as template, combined using described primer pair and specific probe, carry out numeral
Pcr amplification reaction；
(3) pcr amplification product is detected.
Those skilled in the art can use the primer pair of the present invention and probe to be detected from suitable PCR.It is preferred that
Ground, present invention selection digital pcr are expanded.
In above-mentioned steps (2), the reaction system of " the carry out digital pcr " is specially system 2.
System 1 is：10 μ l 2 × SuperMix, 1 μ l sense primers Xoo119F (10uM), 1ul anti-sense primers Xoo119R
(10uM), 0.5 μ l specific probes Xoo119P (10uM), 2 μ l genomic DNAs, ddH2O complements to 20 μ l.The system is premix
Liquid system.
System 2 is：System 1 reacts the reaction micro system prepared with oil on drop generators with 70ul digital pcrs.
Above, the 2 × SuperMix and digital pcr reaction oil can be Bole BIO-RAD companies of the U.S.
Product.
According to currently preferred, the program of digital pcr amplified reaction is in the step (2)：95 DEG C of pre-degenerations
10min；95 DEG C of denaturation 10s, 58 DEG C of annealing and extension 60s, totally 40 circulations；98 DEG C of solidification droplet 10min.
Detection pcr amplification product is specific as follows in above-mentioned steps (3)：
There is the amplification for being clearly distinguishable from negative reaction hole (genomic DNA is 2 μ l ddH2O replacements in negative reaction hole)
Signal, the reacting hole is designated as positive amplification hole, shows to carry rice leaf spot bacteria in testing sample.
Rhs family genes design for rice leaf spot bacteria detects the specific primer pair and probe of the germ,
And the method using Digital Detecting rice leaf spot bacteria is established based on the specific primer pair and probe.It is demonstrated experimentally that adopt
With method Digital Detecting rice leaf spot bacteria provided by the present invention, specificity is good (can be from rice leaf spot bacteria
(Xanthomonas oryzae pv.oryzae), xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae
Pv.oryzicola), citrus processing (Xanthomonas axonopodis pv.citri), pepper bacterial leaf spot
Bacterium (Xanthomonas axonopodis pv.vesicatoria), Burkholderia glumae (Burkholderia
Glumae), oat bites sour bacterium oat pvs oryzae and oryzicola (Acidovorax avenae subsp.avenae), pantoea agglomerans
Precise Identification goes out X.oryzae pv.oryzae in (Pantoea agglomerans)), and high sensitivity is (in 20ul reactants
In system, for test limit up to 28 copies, i.e. sensitivity is 1.4 copies/ul), suitable for the low rice of rice leaf spot bacteria content
The detection of seed sample, there is important application value.
Brief description of the drawings
Fig. 1 is the specificity experiments result of embodiment 3.A04 passage X.oryzae pv.oryzae produce positive droplet, its
Its bacterial strain and blank control do not produce positive droplet, i.e., only the testing result of X.oryzae pv.oryae bacterial strains is the positive.
Fig. 2 is the sensitivity experiment result of embodiment 4.Positive droplet is produced in A01, B01, C01, D01 and E01, and
Positive droplet number gradually successively decreases by gradient, and the stoichiometric number 28 in numerical analysis F01 20ul systems copies, F01, G11 and
H11 does not produce positive droplet.
Fig. 3 is that rice paddy seed experimental result is detected in embodiment 5.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, it is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Strain X .oryzae pv.oryzae (NCPPB1153), X.oryzae are used in following embodiments
pv.oryzicola(NCPPB1151)、X.axonopodis pv.citri(NCPPB3607)、X.axonopodis
pv.vesicatoria(NCPPB2594)、B.glumae(NCPPB2391)、A.avenae subsp.avenae
(NCPPB522), P.agglomerans (NCPPB179) is preserved in National Collection of Plant
(abbreviation NCPPB, network address are Pathogenic Bacteria：http://ncppb.fera.defra.gov.uk/), Gong Zhongke
To be obtained from the collection warehousing.Healthy rice paddy seed is purchased from Anhui Huarun Biotechnology Co., Ltd., and the rice paddy seed that carries disease germs is by Anhui
Entry-Exit Inspection and Quarantine Bureau provides.Digital pcr correlated response reagent consumptive material and Bio-Rad numeral reaction systems (Bio-Rad
QX200) be Bio-Rad companies of the U.S. product, digital pcr correlated response reagent consumptive material include 2 × SuperMix, numeral
PCR droplets generation card and digital pcr reaction oil；Bio-Rad digital pcrs reaction system (Bio-Rad QX200) includes
Droplet Generator drop generators and Droplet Reader droplets read instrument.PCR instrument is purchased from American AB I companies (type
Number：96-Well Thermal Cycler9902).Plant genome DNA extracts kit (catalog number (Cat.No.)：DP305-
02), purchased from TIANGEN Biotech (Beijing) Co., Ltd..Bacterial genomes DNA extraction kit (catalog number (Cat.No.)：DP302-02),
Purchased from TIANGEN Biotech (Beijing) Co., Ltd..
Embodiment 1 is used for the primer pair of rice leaf spot bacteria digital pcr detection and the design of specific probe combination
Devised according to the rhs family genes of rice leaf spot bacteria and be used for rice leaf spot bacteria digital pcr as follows
4 primer pairs and the specific probe combination of detection：
Sense primer Xoo119F：5′-AACGGATCGATGGACTTTACG-3′
Anti-sense primer Xoo119R：5′-AATGTGCACTTCCGCTATGA-3′
Specific probe Xoo119P：5’-FAM-TTCGTGTGAGGTTACCCTGC-TAMRA-3’
Probe Xoo119P 5 ' ends have FAM fluorescence labelings, and 3 ' ends have TAMRA fluorescence labelings.
Sense primer Xoo114F：5′-GATCCGCTCGGTCGCAATGTG-3′
Anti-sense primer Xoo114R：5′-GGCTCGCCCCAGTCCGTCGTA-3′
Specific probe Xoo114P：5’-FAM-TGCAGGGTAACCTCACACGAATGG-TAMRA-3’
Probe Xoo114P 5 ' ends have FAM fluorescence labelings, and 3 ' ends have TAMRA fluorescence labelings.
Sense primer Xoo105F：5′-TTTACGGCTCGCCCCAG-3′
Anti-sense primer Xoo105R：5′-CAATGTGCACTTCCGCTATGAC-3′
Specific probe Xoo105P：5’-FAM-TTCGTGTGAGGTTACCCTGC-TAMRA-3’
Probe Xoo105P 5 ' ends have FAM fluorescence labelings, and 3 ' ends have TAMRA fluorescence labelings.
Sense primer Xoo 106F：5′-TGGACTTCACTATCCGACCT-3′
Anti-sense primer Xoo 106R：5′-GTGTACTTCCGCTACGACTTAC-3′
Specific probe Xoo 106P：5’-FAM-TGCGCGAAAGATTGCCTTGC-TAMRA-3’
Probe Xoo106P 5 ' ends have FAM fluorescence labelings, and 3 ' ends have TAMRA fluorescence labelings.
Through experiment screening, only primer pair Xoo119F/Xoo119R and probe Xoo119P combinations have preferably specificity.
The foundation of the rice leaf spot bacteria digital pcr detection method of embodiment 2
1st, reaction system reaction system 20ul is prepared, by 2 × SuperMix 10ul, primer Xoo119F1ul, primer
Xoo119R 1ul, probe Xoo119P 0.5ul, the genomic DNA 2ul and ddH of testing sample2O 5.5ul, in blank control
Genomic DNA ddH2O is replaced.
2nd, after completing step 1, reaction system is transferred in the system well of digital pcr droplet generation card, and anti-
Using the digital pcr reaction oil that 70ul is added in oily well, digital pcr droplet generation card is transferred to Droplet
In Generator drop generators, start instrument, obtain micro system.
3rd, after completing step 2, the micro system is transferred in 96 orifice plates, sealer；
4th, after completing step 3,96 orifice plate is placed in PCR instrument, carries out pcr amplification reaction, response procedures：95 DEG C pre-
It is denatured 10min；95 DEG C of denaturation 10s, 58 DEG C of annealing and extension 60s, totally 40 circulations；98 DEG C of solidification droplet 10min.
5th, after completing step 4,96 orifice plate is taken out, is placed in Droplet Reader droplets reading instrument and carries out droplet
Fluorescence is read, and then passes through the single droplet fluorescence signal of FAM single channel phosphor collections.
6th, after completing step 5, instrument determines that fluorescence threshold limits and determines that negative droplet and the positive are micro- by analyzing scatter diagram
Drop.Then make the following judgment：If testing sample produces positive droplet signal, testing sample contains bacterial blight of rice
Bacterium；If testing sample does not produce positive droplet signal, testing sample does not contain rice leaf spot bacteria.
The method that embodiment 3 is established according to embodiment 2 carries out specific detection
The genomic DNA of the 7 kinds of bacterial strains obtained with the extraction of bacterial genomes DNA extraction kit from NCPPB collection warehousings.
According to the method for embodiment 2, specific detection is carried out.Experimental result see Fig. 1 (A04 is X.oryzae pv.oryzae,
B04 is that X.oryzae pv.oryzicola, C04 are X.axonopodis pv.citri, D04 X.axonopodis
Pv.vesicatoria, E04 B.glumae, F04 are A.avenae subsp.avenae, G04 P.agglomerans,
H04 is blank control).As a result show, only X.oryzae pv.oryzae produce positive droplet, and other bacterial strains and blank control are equal
It (is now 1366 as the threshold value judged, the droplet instrument less than threshold value is judged as feminine gender, higher than threshold value not produce positive droplet
Droplet instrument be judged as the positive).Therefore, only the testing result of X.oryzae pv.oryzae bacterial strains is the positive, is tied with expected
Fruit is completely the same.
The above results show that the specificity of the reagent set for the detection rice leaf spot bacteria that embodiment 1 is provided is high.
The method that embodiment 4 is established according to embodiment 2 carries out sensitivity technique
1st, the genomic DNA of X.oryzae pv.oryzae bacterial strains is extracted with bacterial genomes DNA extraction kit, initially
Genomic DNA is named as dilution I, and (dilution factor is designated as 100)。
2nd, after completing step 1,1 parts by volume dilution I is taken, adds the ddH of 9 parts by volume2O, it is well mixed, obtains dilution
II (dilution factor notes 10-1), dilution III (dilution factor notes 10 are made by that analogy-2), dilution IV (dilution factor note 10-3), it is dilute
Release liquid V (dilution factor notes 10-4), dilution VI (dilution factor note 10-5), dilution VII (dilution factor note 10-6)。
3rd, after completing step 2, according to the method for the step 2 of embodiment 2, sensitivity technique is carried out.The DNA profiling of testing sample
Dilution II, dilution II, dilution III, dilution IV, dilution V, dilution VI, the dilution VII prepared for step 2.
4th, experimental result is shown in that (A01 is that dilution II, B01 are that dilution II, C01 are that dilution III, D01 are dilution to Fig. 2
Liquid IV, E01 are that dilution V, F01 are that dilution VI, G01 are that dilution VII, H01 are blank control).As a result show, dilution
II, dilution III, dilution IV and dilution V produce positive droplet, and dilution VI, dilution VII and blank control are not
It (is now 296 as the threshold value judged, the droplet instrument less than threshold value is judged as feminine gender, higher than threshold value to produce positive droplet
Droplet instrument is judged as the positive).According to numerical analysis, the reagent for the detection leaf spot bacteria that embodiment 1 is provided is above-mentioned
Detection in 20ul reaction systems is limited to 28 copies.
The method that embodiment 5 is established according to embodiment 2 carries out rice paddy seed detection
The method established using embodiment 2 detects carry disease germs rice paddy seed sample and healthy rice paddy seed sample respectively.
1st, the extraction of rice paddy seed sample DNA：20g testing samples are weighed, are placed in the 50ml centrifuge tubes of sterilizing, are added sterile
For water to testing sample is flooded, 4 DEG C overnight, takes soak 12000rpm to centrifuge 5min, collects precipitation and is carried using plant genome DNA
Kit is taken to carry out sample DNA extraction according to operating instruction.
2nd, according to the method for embodiment 2, rice paddy seed detection is carried out.Experimental result is shown in that (A11 is the rice seed increment that carries disease germs to Fig. 3
Product, B11 are healthy rice paddy seed sample, and C11 is blank control).As a result show, only A11 carry disease germs rice paddy seed sample produce sun
Property droplet, healthy rice paddy seed sample and blank control do not produce positive droplet (be now 284 as the threshold value judged, it is low
It is judged as feminine gender in the droplet instrument of threshold value, the droplet instrument higher than threshold value is judged as the positive).Therefore, the rice paddy seed that carries disease germs is examined
Result is surveyed as the positive, healthy rice paddy seed sample detection result is feminine gender, completely the same with expected results.
The above results show that the specificity of the primer and probe for the detection rice leaf spot bacteria that embodiment 1 is provided is high,
Suitable for the detection of rice paddy seed sample.
SEQUENCE LISTING
<110>Chinese in Inspection ＆ Quarantine Technology Center of Anhui Entry-Exit Inspection ＆ Quarantine Bureau of China Inst. of Quarantine Inspection Sciences
Zhangjiagang Entry-Exit Inspection and Quarantine Bureau of people republic
<120>A kind of method and kit that rice leaf spot bacteria is detected using digital pcr
<130> KH171114399.6
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
aacggatcga tggactttac g 21
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
aatgtgcact tccgctatga 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
ttcgtgtgag gttaccctgc 20
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
gatccgctcg gtcgcaatgt g 21
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<400> 5
ggctcgcccc agtccgtcgt a 21
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence
<400> 6
tgcagggtaa cctcacacga atgg 24
<210> 7
<211> 17
<212> DNA
<213>Artificial sequence
<400> 7
tttacggctc gccccag 17
<210> 8
<211> 22
<212> DNA
<213>Artificial sequence
<400> 8
caatgtgcac ttccgctatg ac 22
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<400> 9
ttcgtgtgag gttaccctgc 20
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
tggacttcac tatccgacct 20
<210> 11
<211> 22
<212> DNA
<213>Artificial sequence
<400> 11
gtgtacttcc gctacgactt ac 22
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
tgcgcgaaag attgccttgc 20
Claims (8)
1. detect the specific primer pair and probe combinations of rice leaf spot bacteria, the nucleotide sequence of the specific primer pair
As shown in SEQ ID NO.1-2, the nucleotide sequence of the probe is as shown in SEQ ID NO.3.
2. specific primer pair as claimed in claim 1 and probe combinations, it is characterised in that 5 ' flag F AM of the probe,
3 ' mark TAMRA.
3. the kit containing specific primer pair described in claim 1 and probe combinations.
4. the kit described in any specific primers pair of claim 1-2 and probe combinations or claim 3 is detecting
Application in rice leaf spot bacteria.
A kind of 5. method that rice leaf spot bacteria is detected using digital pcr, it is characterised in that comprise the following steps：
(1) genomic DNA of testing sample is extracted；
(2) using the genomic DNA of extraction as template, using the specific primer pair and probe combinations described in claim 1, carry out
Digital pcr amplified reaction；
(3) pcr amplification product is detected.
6. according to the method for claim 5, it is characterised in that digital pcr expands its 20 μ l reaction system in step (2)
For：10 μ l 2 × SuperMix, 10 μM of upstream and downstream primer each 1 μ l, 10 μM of μ l of specific probe 0.5,2 μ l genomic DNAs,
ddH2O complements to 20 μ l.
7. according to the method for claim 5, it is characterised in that the program of digital pcr amplified reaction is carried out in step (2)
For：95 DEG C of pre-degeneration 10min；95 DEG C of denaturation 10s, 58 DEG C of annealing and extension 60s, totally 40 circulations；98 DEG C of solidification droplets
10min。
8. according to any described methods of claim 5-7, it is characterised in that in step (3), the amplification obtained according to detection is believed
Number judge testing sample whether carry rice leaf spot bacteria, there is the amplified signal for being clearly distinguishable from negative reaction hole, show
Rice leaf spot bacteria is carried in testing sample.
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| CN108760706A (en) * | 2018-06-08 | 2018-11-06 | 农业部环境保护科研监测所 | A kind of method of quick screening low cadmium-accumulation rice varieties |
| CN109486970A (en) * | 2018-11-16 | 2019-03-19 | 海南大学 | A kind of ring mediated isothermal amplification detection primer group, detection method and the detection kit of rice Xanthomonas |
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| CN111621549A (en) * | 2020-06-05 | 2020-09-04 | 南京农业大学 | LAMP (loop-mediated isothermal amplification) rapid detection method for bacterial blight in rice seeds and application |
| CN113293235A (en) * | 2021-06-24 | 2021-08-24 | 仲恺农业工程学院 | Primer for frog virus detection and application thereof |
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| CN116200514A (en) * | 2023-04-25 | 2023-06-02 | 三亚中国检科院生物安全中心 | Primer probe combination product for detecting rice pathogenic bacteria and application thereof |
| CN116656850A (en) * | 2023-07-27 | 2023-08-29 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof |
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| CN116656850A (en) * | 2023-07-27 | 2023-08-29 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof |
| CN116656850B (en) * | 2023-07-27 | 2023-10-31 | 中国热带农业科学院三亚研究院 | Sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof |
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