CN116179751B - Detection target, LAMP primer, kit and detection method of soybean rust bacteria - Google Patents
Detection target, LAMP primer, kit and detection method of soybean rust bacteria Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- 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
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an LAMP primer composition for detecting soybean rust bacteria, a kit and a detection method thereof. Extracting DNA of a microorganism to be detected, taking a DNA solution as a reaction template, and adding a detection solution in a kit to carry out LAMP reaction, wherein the LAMP reaction procedure is as follows: and (3) carrying out reaction amplification for 70min at 62 ℃, then adding SYBRGreenI into the amplified product, observing color change, and if the color is changed from orange to yellow-green, or fluorescence exists under an ultraviolet lamp, indicating that soybean rust bacteria exist in the object to be detected, and if the color is not changed but is still orange, or no fluorescence exists under the ultraviolet lamp, indicating that the soybean rust bacteria do not exist in the object to be detected. The invention has higher accuracy, sensitivity and effectiveness, is convenient to operate and good in practicability, provides a new technical platform for detecting soybean rust bacteria, and can be used for high-sensitivity rapid detection of soybean rust bacteria.
Description
Technical Field
The invention belongs to the field of biotechnology detection, and relates to a detection target, an LAMP primer, a kit and a detection method of soybean rust bacteria.
Background
Soybean rust germ (Phakopsora pachyrhizi) belongs to basidiomycetes (Phylum Basidiomycota), rust class (Class Urediniomycetes), order of rust (OrderUredinales), family of phakopsoraceae (Family Phakopsoraceae), genus Phakopsora (Genus Phakopsora), and can cause soybean leaf yellowing, abscission and early plant blight; or a reduction in empty pods and pod numbers, resulting in serious economic losses in soybean production. Until now, no commercial disease-resistant variety is put into use, and timely use of bactericides is the most effective measure for preventing and controlling the disease. The early warning and monitoring of the diseases are enhanced, a rapid detection method is established, a basis is provided for risks of the diseases and medication decisions, and the loss caused by soybean rust bacteria is reduced.
Currently, there are few detection techniques for soybean rust. The traditional detection method is mainly based on the morphology of soybean rust bacteria for identification. As soybean rust bacteria are living nutrition specific pathogenic bacteria, the soybean rust bacteria cannot grow on an artificial culture medium, and great difficulty is brought to detection. Along with the development of molecular biology, particularly the popularization of PCR technology, more and more molecular biology technology is developed and applied to the detection of pathogenic bacteria, however, the detection of soybean rust bacteria is slow, in addition, the technology has great improvement on specificity and sensitivity, the detection time is longer, meanwhile, the technology depends on a precise temperature circulating device, the detection process is complex, and the requirement of rapid detection cannot be met.
The Loop-mediated isothermal amplification (LAMP) technology is a novel nucleic acid amplification technology, and is a novel nucleic acid amplification technology capable of replacing PCR due to the advantages of simple and rapid operation, high specificity, low cost and the like. The method designs 4 specific primers aiming at 6 regions of a target gene, causes self-circulation strand displacement reaction under the action of Bst large-fragment polymerase, and generates white magnesium pyrophosphate precipitation which is a byproduct when a large amount of target DNA is synthesized within 80min at 60-65 ℃. Because the LAMP amplification process relies on identifying 6 independent areas of a target sequence, the reaction specificity is strong, the nucleic acid amplification process is carried out under the constant temperature condition, the common water bath or equipment with stable heat source can meet the reaction requirement, and the detection cost is greatly reduced. The LAMP reaction has the characteristics of simplicity, rapidness, high efficiency, economy and the like, so that the LAMP has extremely wide application prospect. Since the establishment of LAMP detection technology, the technology has been widely applied to detection and research on pathogenic bacteria such as bacteria, viruses, fungi, parasites and the like, but few reports on detection of plant pathogenic rust bacteria are provided, and no report on detection of soybean rust bacteria is provided at home and abroad.
Disclosure of Invention
The invention designs the LAMP primer composition specific to the soybean rust bacteria by comparing and analyzing the difference of the soybean rust bacteria and other 60 rust bacteria strains on the genome sequence and taking the gene sequence of Phapa_Oyhb as a target, and establishes an LAMP rapid detection method of the soybean rust bacteria on the basis.
The aim of the invention can be achieved by the following technical scheme:
in a first aspect, the invention provides a specific detection target Phapa_Oyhb of soybean rust bacteria Phapporapachyrhizi, wherein the DNA sequence of the detection target is shown as SEQ ID NO: 1.
ATGCTGAGTACTAGGGTTATCATGAAGCTGATCCTGCTCGGTGTTTTTTCAAACCTTCTGATTGTTGCCTGTGATGAGGGCTTTAACTGTAAGGATCCGTCTAAGGGTCATCCGCACCGTCTGGTGAATGCTACCCAATGTGTAAAGTCAGTAAAAATAAAAATCATCTGATACATCAAACAATCCAATCCTCTTCTCACTATCTGTTTTGAAAGGATAATTAATCAGAAGATACCTAAATAACCTCAAAAGATCTGATCTTCTTCCCCCTCTTTTGTACCATCAGTGCCATCAAATCGTTTCCAGTCATAAATGGTGATTTCTCTGTTTCGTTGAACCCAGCCAAAAGTTACGTACGACACTGTGGGACTTGTAAAGTTAAGTATAAAACATAACGCAAAGATCTATCAGATCGAACATTCGATGATTCAAGCACCAGAGGGAGTGACAAGATGAGAGAATCTTTTTGTGAAGGACTCAGTGTTTGATAAGAATCAAAGAGGATGATAGAAATAGGGATGGAGGAGAGACTGATATAGTTGTTGATAAGACTAACGATCAGAGCTTCTCTCAGACTGATGGGTCATCTTCTTCTCTCTATCAATAAATTAGCTTGAACTGAGTGGATCTGAAAAGGCCAAACAAGCAGGACTGCCTCTGTAAGTTTTCTCTGTTTCCCTCTTTAACAGAAAAACACAATCAATAACAGAGTGAGAGGGATGACGTAAGGTACTGACACCTTTCGGAGCTGGGTCTTCTTCTCTTAATAGTGCAAAAGATGAAACGTTGATCAAGATAAAGGATGCTGTTAACCACTGTAATAACGATGTGTGTAATTTAAAATATAAATAATCTTGTAACTGGCAGTTCATATATGAGCGTTGGCGGCTGACCTTCTATACCCTATAAAAAAAAAGTCGTATGGAGATTTTTCAATGTACAACGAGAGGAGTAAAGTCACTGTTCGAATCGACTGGGAATACTCTACGAAAGGTGCCTACTGCCCC(SEQ ID NO：1)
In a second aspect, the present invention provides an LAMP primer composition for detecting soybean rust Phakopsora pachyrhizi, comprising: the forward inner primer FIP shown as SEQ ID NO.2, the reverse inner primer BIP shown as SEQ ID NO.3, the forward outer primer F3 shown as SEQ ID NO.4, the reverse outer primer B3 shown as SEQ ID NO.5 and the reverse loop primer LB shown as SEQ ID NO. 6.
In a third aspect, the invention also provides application of the primer composition in detection of Pharkopsoapphyshizi sojae.
In a fourth aspect, the invention also provides application of the primer composition in preparation of a kit for detecting Phakopsorapachyrhizi of soybean rust bacteria.
In a fifth aspect, the present invention further provides an LAMP kit for detecting soybean rust bacteria Phakopsoracaachrhizi, wherein the concentrations of the reagents in the kit are as follows: 0.8. Mu.M forward inner primer FIP, 0.8. Mu.M reverse inner primer BIP, 0.1. Mu.M forward outer primer F3, 0.1. Mu.M reverse outer primer B3, 0.1. Mu.M reverse loop primer LB, 0.8M betaine, 1.4mM dNTPs, 20mM Tris-HCl, 10mM KCl, 10mM (NH 4) 2 SO 4 、6mM MgSO 4 1mL of a detection solution was prepared from 0.1% Triton X-100 and Bst DNA polymerase U/. Mu.l using ultrapure water; wherein the forward inner primer FIP is shown as SEQ ID NO.2, the reverse inner primer BIP is shown as SEQ ID NO.3, the forward outer primer F3 is shown as SEQ ID NO.4, the reverse outer primer B3 is shown as SEQ ID NO.5, and the reverse loop primer LB is shown as SEQ ID NO. 6.
In a sixth aspect, the invention also provides an application of the LAMP kit for detecting soybean rust bacteria, wherein the LAMP kit is used for detecting soybean rust bacteria.
In a seventh aspect, the present invention also provides a LAMP detection method for soybean rust bacteria, which uses the LAMP primer composition of claim 2 or the LAMP kit of claim 5 to perform LAMP reaction, and after the reaction, adding SYBR Green I into the amplified product, observing the color change of the reaction solution or whether there is fluorescence under an ultraviolet LAMP to determine whether the microorganism to be detected is soybean rust bacteria.
Further, the method comprises the steps of extracting DNA of a microorganism to be detected, taking 4 mu L of DNA solution as a reaction template, adding 21 mu L of detection solution in an LAMP kit for LAMP, and carrying out the LAMP reaction procedure: and (3) carrying out reaction amplification for 70min at 62 ℃, then adding 0.25 mu L SYBR Green I into the amplified product, observing the color change of the reaction solution, and if the color is changed from orange to yellow-Green or fluorescent under an ultraviolet lamp, indicating that soybean rust bacteria exist in the object to be detected, and if the color is not changed but is still orange or fluorescent under the ultraviolet lamp, indicating that the soybean rust bacteria do not exist in the object to be detected.
Compared with the prior art, the invention has the following technical effects,
(1) The invention discovers that a novel target gene Phapa_Oyhb is used for LAMP detection of soybean rust bacteria, and based on the gene, particularly a specific target region on the gene, the invention designs an LAMP primer composition for detecting soybean rust bacteria.
(2) The operation is convenient: the LAMP method for detecting soybean rust bacteria provided by the invention solves the problems of long period, time and labor waste, complexity and poor specificity required by the biological detection method for soybean rust bacteria in the prior art, and the problems that a thermal cycle instrument is required by a PCR detection technology and soybean rust bacteria cannot be detected rapidly. The detection method can accurately, quickly and efficiently detect the soybean rust bacteria in 70min under the isothermal condition of 62 ℃, has simple requirements on experimental places, does not need other complex instruments, can better meet the field detection of the soybean rust bacteria, and is suitable for popularization and use in basic layers and import and export inspection and quarantine departments.
(3) And (3) isothermal amplification: unlike PCR method, which needs thermal cycle, the method gets rid of the dependence on thermal cycle instrument, and the LAMP reaction can occur only by stable heat source, which greatly expands the application range of LAMP, and the LAMP can react under constant heat source because betaine is added into LAMP reaction liquid, so that double-chain DNA is in dynamic balance of melting, and amplification is realized under the action of BstDNA polymerase.
(4) The accuracy is high: according to the genome sequence of soybean rust bacteria, the genome sequences of 3 soybean rust bacteria are compared with the genome sequences of other 58 rust bacteria strains, a specific LAMP primer is designed by taking a specific and intraspecies conserved gene Phapa_Oyhb among soybean rust bacteria as a target, and the gene is not used as the target in common PCR for designing the identification primer of the soybean rust bacteria. The LAMP reaction specifically recognizes 6 independent regions on the target sequence by 5 primers (FIP, BIP, F, B3, LB), and its specificity is relatively high. In addition, the reverse loop primer LB can improve the reaction rate, and the reverse loop primer LB and other four primers can be used together, so that the soybean rust bacteria detection can be rapidly performed under the condition of ensuring the reaction accuracy.
(5) The sensitivity is high: the LAMP detection method for soybean rust bacteria has very high sensitivity, and can reach 10pg DNA, which indicates that the detection method is sufficient for accurately and rapidly detecting soybean rust bacteria under the condition of low DNA concentration.
For a better understanding and implementation, the present invention is described in detail below with reference to the drawings.
Drawings
FIG. 1 shows the design of specific LAMP primers based on the gene sequence of Phapa-Oyhb of soybean rust as a target.
FIG. 2 is a sensitivity verification of the LAMP detection method of soybean rust.
FIG. 3 shows the universality of LAMP detection method for detection of different soybean rust strains.
FIG. 4 shows that LAMP detection method can only detect soybean rust, but other pathogenic bacteria and non-soybean rust which are common on soybean cannot be detected.
Detailed Description
For the purpose of making the objects and technical solutions of the present invention more clear, the present invention is further described with specific examples, not only these examples.
Example 1: detection of soybean rust by LAMP method
LAMP primer composition for detecting soybean rust bacteria: the forward inner primer FIP is shown as SEQ ID NO.2, the reverse inner primer BIP is shown as SEQ ID NO.3, the forward outer primer F3 is shown as SEQ ID NO.4, the reverse outer primer B3 is shown as SEQ ID NO.5, and the reverse loop primer LB is shown as SEQ ID NO.6, as shown in FIG. 1.
The LAMP kit for detecting soybean rust bacteria has the following reagent concentrations: 0.8. Mu.M forward inner primer FIP, 0.8. Mu.M reverse inner primer BIP, 0.1. Mu.M forward outer primer F3, 0.1. Mu.M reverse outer primer B3, 0.1. Mu.M reverse loop primer LB, 0.8M betaine, 1.4mM dNTPs, 20mM Tris-HCl, 10mM KCl, 10mM (NH) 4 ) 2 SO 4 、6mM MgSO 4 1mL of a detection solution was prepared from 0.1% Triton X-100 and Bst DNA polymerase U/. Mu.l using ultrapure water.
LAMP detection method: extracting DNA of a microorganism to be detected, taking 4 mu L of DNA solution as a reaction template, adding 21 mu L of detection solution in an LAMP kit for LAMP, wherein the LAMP reaction procedure is as follows: and (3) carrying out reaction amplification for 70min at 62 ℃, then adding 0.25 mu L SYBR Green I into the amplified product, observing color change, and if the color is changed from orange to yellow-Green or fluorescent under an ultraviolet lamp, indicating that soybean rust bacteria exist in the object to be detected, and if the color is not changed but is orange or fluorescent under the ultraviolet lamp, indicating that soybean rust bacteria do not exist in the object to be detected.
Example 2: sensitivity test of LAMP reaction of soybean rust bacteria
In order to determine the sensitivity of the LAMP detection method, the extracted DNA of soybean rust bacteria is diluted by a ratio of 10 times after the concentration is measured by a spectrophotometer, the concentration range of the DNA is set to be 10ng-100fg, 4 mu L of diluted DNA diluent with each concentration is taken as a template, and 21 mu L of kit solution is added for LAMP reaction, wherein the reaction procedure is as follows: the reaction was amplified at 62℃for 70min.
The template concentration range of soybean rust bacteria is set to be 10ng-100fg based on the color change of the reaction solution and the sensitivity of the LAMP method for detecting whether fluorescence exists under an ultraviolet LAMP. The results show that: when the template concentration of the soybean rust bacteria is 10ng, 1ng, 100pg and 10pg respectively, the color of the solution in the corresponding reaction tube is changed into yellow green (figure 2) and positive reaction is carried out; when the template concentration of the soybean rust bacteria is 1pg and 100fg respectively, the corresponding solution in the reaction tube is orange, has negative reaction, and is further observed under an ultraviolet lamp to verify the result. The results show that the sensitivity of detection by using the LAMP detection primer of the invention reaches 10pg of primer detection.
Example 3: LAMP detection method for detecting different soybean rust bacteria strains
LAMP detection method as in example 1: DNA of 7 soybean rust strains was extracted, and 4. Mu.LDNA solution was used as a reaction template (ddH was used 2 O as control), the detection solution in the 21 μl amp kit was added for LAMP, the LAMP reaction procedure was: the reaction was amplified at 62℃for 70min, then 0.25. Mu.L SYBR Green I was added to the amplified product to observe the color change, indicating that soybean rust could be detected if the color changed from orange to yellowish Green or there was fluorescence under an ultraviolet lamp, and indicating that soybean rust could not be detected if the color was not changed but was still orange or there was no fluorescence under an ultraviolet lamp. As shown in FIG. 3, each of the 7 strains of soybean rust was able toDetected, whereas the blank control could not. The LAMP detection method is shown to have universality for detecting different soybean rust strains.
Example 4: specificity test of LAMP reaction of soybean rust bacteria
In order to verify the specificity of the LAMP method, soybean rust bacteria and other non-target strains are used as test materials, and LAMP detection results show that: only the color of the solution in the reaction tube taking soybean rust bacteria as a template is changed from orange to yellow-green, and positive reaction is generated (shown in figure 4); the color of the solution in the reaction tube of the other non-target strain and the negative control was not changed, and the reaction was negative (Table 1).
TABLE 1
The numerical values set forth in these examples do not limit the scope of the present invention unless specifically stated otherwise. In all examples shown and described herein, unless otherwise specified, any particular value is to be construed as exemplary only and not as limiting, and thus, other examples of exemplary embodiments may have different values.
Claims (8)
1. A specific detection target phapa_oyhb of soybean rust bacteria phakopsoappachrhizi, which is characterized in that the DNA sequence of the detection target is shown as SEQ ID NO: 1.
2. A LAMP primer composition for detecting phakopsoroylachyrhizi, said primer composition comprising: the forward inner primer FIP shown as SEQ ID NO.2, the reverse inner primer BIP shown as SEQ ID NO.3, the forward outer primer F3 shown as SEQ ID NO.4, the reverse outer primer B3 shown as SEQ ID NO.5 and the reverse loop primer LB shown as SEQ ID NO. 6.
3. Use of the primer composition according to claim 2 for detecting phakopsoroapathy sojae.
4. Use of the primer composition of claim 2 in the preparation of a kit for detecting soybean rust germ Phakopsora pachyrhizi.
5. The LAMP kit for detecting soybean rust bacteria Phakopsporachyrhizi is characterized in that the concentration of each reagent in the kit is as follows: 0.8. Mu.M forward inner primer FIP, 0.8. Mu.M reverse inner primer BIP, 0.1. Mu.M forward outer primer F3, 0.1. Mu.M reverse outer primer B3, 0.1. Mu.M reverse loop primer LB, 0.8M betaine, 1.4mM dNTPs, 20mM Tris-HCl, 10mM KCl, 10mM (NH 4) 2 SO 4 、6mMMgSO 4 1mL of a detection solution was prepared from 0.1% TritonX-100 and BstDNApolymerase 8U/. Mu.l using ultrapure water; wherein the forward inner primer FIP is shown as SEQ ID NO.2, the reverse inner primer BIP is shown as SEQ ID NO.3, the forward outer primer F3 is shown as SEQ ID NO.4, the reverse outer primer B3 is shown as SEQ ID NO.5, and the reverse loop primer LB is shown as SEQ ID NO. 6.
6. The use of the LAMP kit for detecting soybean rust bacteria Phakopsporachyrhizi according to claim 5.
7. A LAMP detection method of soybean rust bacteria Phakopsporachyrhizi is characterized in that the LAMP primer composition of claim 2 or the LAMP kit of claim 5 is utilized for carrying out LAMP reaction, SYBRGreenI is added into amplification products after the reaction is finished, and whether the color of a reaction solution changes or whether fluorescence exists under an ultraviolet LAMP is observed so as to judge whether microorganisms to be detected are soybean rust bacteria.
8. The LAMP detection method according to claim 7, comprising extracting DNA of a microorganism to be detected, taking a 4 μLDNA solution as a reaction template, adding a detection solution in a 21 μLLAMP kit for LAMP, and carrying out the LAMP reaction by the following procedure: and (3) carrying out reaction amplification for 70min at 62 ℃, then adding 0.25 mu LSYBRGrenI into the amplified product, observing the color change of the reaction solution, and if the color is changed from orange to yellow-green or fluorescent under an ultraviolet lamp, indicating that soybean rust bacteria exist in the object to be detected, and if the color is not changed but is still orange or fluorescent under the ultraviolet lamp, indicating that the soybean rust bacteria do not exist in the object to be detected.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101798593A (en) * | 2009-11-19 | 2010-08-11 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Detection primer of phakospora sojae sawada, kit and real-time PCR (polymerase chain reaction) detection method |
| CN101875968A (en) * | 2009-11-19 | 2010-11-03 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Phakopsora pachyrhizi Sydow detection primer, kit and real-time fluorescence PCR (Polymerase Chain Reaction) detection method |
| CN113881804A (en) * | 2021-11-11 | 2022-01-04 | 青海省农林科学院 | Primer pair for wheat stripe rust detection, probe and application |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101798593A (en) * | 2009-11-19 | 2010-08-11 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Detection primer of phakospora sojae sawada, kit and real-time PCR (polymerase chain reaction) detection method |
| CN101875968A (en) * | 2009-11-19 | 2010-11-03 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Phakopsora pachyrhizi Sydow detection primer, kit and real-time fluorescence PCR (Polymerase Chain Reaction) detection method |
| CN113881804A (en) * | 2021-11-11 | 2022-01-04 | 青海省农林科学院 | Primer pair for wheat stripe rust detection, probe and application |
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