CN113151522A - LFD-RPA technology-based rice bacterial leaf streak germ detection kit, primer probe composition and application thereof - Google Patents
LFD-RPA technology-based rice bacterial leaf streak germ detection kit, primer probe composition and application thereof Download PDFInfo
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Abstract
The invention discloses a rice bacterial leaf streak germ detection kit based on an LFD-RPA technology, a primer probe composition and application thereof. The invention specifically discloses a kit for specifically detecting rice bacterial leaf spot pathogen (Xanthomonas oryzae pv. oryzicola) comprising a primer XOC367-3F (SEQ ID No.1), a primer XOC367-3R (SEQ ID No.2) and a Probe (SEQ ID No. 3). The detection method based on the LFD-RPA technology established by the invention is simple, quick, sensitive and specific. The detection sensitivity to the DNA of the pathogenic bacteria can reach 2.59 multiplied by 10‑4ng/mu L, good specificity, no cross reaction with other kindred species and other pathogenic bacteria, and can be applied to rice seed samplesAnd (5) during detection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a rice bacterial leaf streak germ detection kit based on an LFD-RPA technology, a primer probe composition and application thereof.
Background
Xanthomonas oryzae (Xanthomonas oryzae) is an important group of pathogenic bacteria in Xanthomonas, and bacterial blight and bacterial streak disease caused by 2 pathogenic varieties of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae (Xanthomonas oryzae pv. oryzae, Xoc) are two very serious diseases in rice grain production, and seriously threaten the safe production of rice in China. The bacterial blight of rice is mainly prevalent in many rice growing regions in China and some Asian countries, and also occurs in the India subcontinent and in Korea Japan. The bacterial leaf streak disease of rice occurs in a wider range than that of bacterial leaf blight of rice, is firstly mainly found in Philippine (1918), is subsequently found in tropical and subtropical regions of Asia, northern Australia and eastern Africa, and is currently found in rice growing areas all over the world, besides rice, broad leaf rice (Oryza latifolia) and water bamboo (Zizania latifolia) are also harmed, and a plurality of wild rice can be infected to cause the disease. In the last two decades, bacterial leaf streak of rice is rapidly prevalent in southern areas of China, especially in subtropical areas such as Guangxi and Guangdong, and has become a main disease in rice production in southern areas of China. Therefore, compared with bacterial blight of rice, bacterial leaf streak causes more serious harm to rice production in China, and is a quarantine pest of entry plants in China.
At present, the detection method for the pathogenic bacteria mainly comprises a seedling growth observation method, traditional detection methods such as pathogenic bacteria isolation culture and pathogenicity determination, serological detection, PCR detection and the like. However, most of these methods are time-consuming, high in cost, require specialized instruments and operating technicians, and cannot meet the requirement of point-of-care testing (POCT).
Recombinase Polymerase Amplification (RPA) is a highly efficient isothermal nucleic acid amplification technique. The technical principle is as follows: the recombinase, in combination with the primer, forms a protein-DNA complex that is able to search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequences, strand exchange reaction formation occurs and DNA synthesis is initiated, and the target region on the template is exponentially amplified. The entire process is carried out very quickly and detectable levels of amplification product are typically obtained within ten minutes. The technology is different from PCR, does not need an annealing reaction process, can realize nucleic acid index amplification under the condition of normal temperature, can be combined with lateral flow chromatography test strips (LFD) by the RPA technology through labeling with fluorescent groups, realizes the visual detection of amplified products, does not need complex instruments, is suitable for on-site rapid detection, and has extremely wide application prospect. The RPA technology develops rapidly since Piepenburg and the like are reported for the first time in 2006, and is well applied to the fields of animal epidemic disease diagnosis, microorganism and drug resistance detection and the like. For example, the RPA technology is successfully applied to the detection of schistosoma japonicum, porcine kobuvirus, sulfanilamide drug resistance genes and the like, and the detection result is directly visible within 2-5min by combining with LFD. Although the LFD-RPA technology has the defects of easy false positive caused by aerosol pollution and the like, the LFD-RPA technology has the advantages of short detection time, high sensitivity, high specificity, no need of instruments, capability of reacting at normal temperature and the like, and has wide application prospect in the field of basic field detection.
Disclosure of Invention
The technical problem to be solved by the invention is how to conveniently, rapidly and sensitively detect the rice bacterial streak germ (Xanthomonas oryzae pv. oryzicola).
In order to solve the technical problems, the invention firstly provides a kit for detecting rice bacterial leaf streak germs, and the kit comprises a composition for detecting rice bacterial leaf streak germs and LFD test paper; the composition comprises a primer XOC367-3F, a primer XOC367-3R and a Probe Probe; the nucleotide sequence of the primer XOC367-3F is shown as SEQ ID No.1, the nucleotide sequence of the primer XOC367-3R is shown as SEQ ID No.2, and the nucleotide sequence of the Probe is shown as SEQ ID No. 3.
The primer XOC367-3F is a forward primer; the primer XOC367-3R is a reverse primer.
In the kit, the 5' end of the primer XOC367-3R is labeled by biotin.
In the kit, the Probe has any one or a combination of the following modifications:
A1) tetrahydrofuran is labeled at any base position of 30 th to 35 th bases from the 5' end of the Probe;
A2) labeling a fluorescent group at the 5' end of the Probe;
A3) the 3' end of the Probe Probe is labeled with an amine group, a phosphate group or C3-spacer.
Further, the site of the labeled tetrahydrofuran is about 15 bases from the 3' end.
The fluorescent group is selected from at least one of FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED460, LC RED705, Quasar705 or Texas Red.
Further, in the kit, the Probe Probe is labeled with FAM at the 5 ' end, modified with tetrahydrofuran between the bases 33 and 34 from the 5 ' end, and labeled with C3-spacer at the 3 ' end.
The invention also provides a composition for detecting the bacterial leaf streak germs of the rice.
The composition is an LFD-RPA visual detection composition for detecting rice bacterial leaf streak pathogens.
The invention also provides a method for detecting the bacterial leaf streak germs of the rice, which comprises the following steps:
B1) extracting genome DNA of a sample to be detected;
B2) performing LFD-RPA detection by using the composition by using the genomic DNA as a template; and determining whether the sample to be detected contains rice bacterial leaf streak pathogens or not according to the LFD test strip.
In the above method, the method for determining whether the sample to be tested contains rice bacterial leaf streak pathogens according to the LFD test strip comprises the following steps: if the LFD test strip has two purple-red strips, one is positioned in the quality control area, and the other is positioned in the detection area, the result is positive, and the sample to be detected contains rice bacterial leaf streak germs; if the LFD test strip only has a purple-red strip and is positioned in the quality control area, the result is negative, which indicates that the sample to be tested does not contain rice bacterial streak pathogens.
The mauve strip positioned in the quality control area is a quality control line (C line); the purple red strip in the detection area is a detection line (T line).
In the above method, the reaction system for LFD-RPA detection in step B2) is: to the RPA freeze-dried enzyme were added 29.4. mu.L of buffer, 11.5. mu.L of ultrapure water, 2. mu.L of 10. mu.M primer XOC367-3F, 2. mu.L of 10. mu.M primer XOC367-3R 2. mu.L, 0.6. mu.L of 10. mu.M Probe, 2. mu.L of DNA template, and 2.5. mu.L of magnesium acetate.
Further, the method comprises the steps of carrying out amplification reaction in a 39 ℃ water bath box under the LFD-RPA detection system, placing an amplification product in a lateral flow chromatography detection test strip sealing device after 15 minutes, and observing after placing for 5 minutes at room temperature.
The invention also provides application of the kit in detection or auxiliary detection of rice bacterial leaf streak pathogens.
The invention also provides application of the composition in detection or auxiliary detection of rice bacterial leaf streak pathogens.
The invention also provides application of the composition in preparing products for detecting or assisting in detecting rice bacterial leaf streak pathogens.
The rice bacterial streak germ (Xanthomonas oryzae pv. oryzicola, Xoc) is a quarantine harmful organism of the imported plants in China and can be remotely transmitted along with seeds. The rapid and simple detection has important significance for preventing the disease from spreading and spreading. The key to establish the sensitive and efficient LFD-RPA optimal system is to design primers and probes with high amplification efficiency and strong specificity. Due to the particularity of its technical principle, PCR primers are often not suitable for LFD-RPA. Therefore, according to the gene sequence of the rice bacterial leaf streak germ, the specific amplification primer XOC367-3F, the primer XOC367-3R and the Probe Probe are designed, and the recombinase polymerase isothermal amplification (LFD-RPA) detection method of the rice bacterial leaf streak germ based on the flow-measuring chromatography test strip is established. The method is simple, rapid, sensitive and specific, and can specifically detect 3 rice grains from different plant pathogenic bacteriaBacterial streak strain. The detection sensitivity to the DNA of the pathogenic bacteria can reach 2.59 multiplied by 10-4ng/mu L, good specificity and no cross reaction with other closely related species and other pathogenic bacteria. The rice with bacteria is an important primary infection source of the bacterial leaf streak of rice and is the most main mode for long-distance transmission of pathogenic bacteria. Therefore, the detection of rice seeds is an important way for improving the control level of rice diseases, preventing the diseases from spreading and ensuring the continuous and healthy development of the rice industry in China. The nucleic acid visual detection technology for rice bacterial leaf streak pathogens established based on the LFD-RPA technology can be applied to rice seed sample detection, has short nucleic acid amplification time, high efficiency and low requirement on equipment, and is suitable for rapid detection in simple laboratories of basic level.
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FIG. 1 shows the results of sensitivity analysis. 1: 259 ng/. mu.L; 2: 25.9 ng/. mu.L; 3: 2.59 ng/. mu.L; 4: 2.59X 10-1ng/μL；5：2.59×10-2ng/μL；6：2.59×10-3ng/μL；7：2.59×10-4ng/μL；8：2.59×10-5ng/mu L; 9: blank Control (CK).
FIG. 2 shows the results of the specificity analysis.
FIG. 3 shows the LFD-RPA detection results of rice seed samples carrying bacterial leaf streak pathogens.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, reference is made to test strains, reagents and apparatus:
1. test strains
All test strains are publicly available from the following bacterial species pools:
ATCC (American Type Culture Collection) is American Type Culture Collection, and its website is https:// www.atcc.org/.
NCPPB (National Collection of Plant Pathogenic Bacteria, UK) is the National Collection of Plant Pathogenic Bacteria in the UK, with the website https:// www.fera.co.uk/NCPPB.
BCCM (Belgian Co-organized Collections of Micro-Organisms, BCCM/LMG Ba ctaria Collection) is Belgium microbiological Collection with the website https:// BCCM.
TABLE 1 test strains information
2. The test instrument: real-time fluorescent PCR instruments were purchased from Roche diagnostics products (Shanghai) Inc.
3. Test reagents: a bacterial genomic DNA extraction Kit (TIANAmp Bacteria DNA Kit) was purchased from Tiangen Biochemical technology (Beijing) Ltd; the colloidal gold test paper strip type RPA kit is purchased from Shandong Weifang Anpu future biotechnology company; the primers and the probes are synthesized by Shanghai biological engineering Co., Ltd; LFD flow-through chromatography colloidal gold test strips were purchased from Agdia, usa.
Example 1LFD-RPA detection method
1. Culturing and preserving strain
The 9 test strains in Table 1 were cultured using nutrient agar NA solid medium and nutrient broth NB liquid medium. Firstly, using an inoculating loop to dip bacteria liquid preserved in 30% glycerol to streak on an NA solid culture medium, placing a plate on the NA solid culture medium for culturing for 24-48h at 28 ℃, then picking pure single bacterial colony to an NB liquid culture medium, culturing at 28 ℃, 200rpm under shaking overnight, and using the cultured bacteria liquid for extracting genome DNA. The strain is frozen and preserved at-20 ℃ by using 30% of glycerol at ordinary times. The long-term preservation needs to be carried out in an ultra-low temperature refrigerator at minus 80 ℃.
2. Genomic DNA extraction
2-1, extraction of genomic DNA of test strains
Extracting the genome DNA of the strain to be detected by using the bacterial genome DNA extraction kit according to the operation instruction, and concretely comprising the following steps:
(1) taking 1.5mL of the bacterial culture solution, centrifuging at 12000rpm for 2min, sucking out the supernatant by a pipette, and keeping the precipitate.
(2) To the pellet was added 200. mu.L of buffer GA, and the pellet was fully suspended by shaking.
(3) Add 4. mu.L RNaseA (100mg/mL), shake for 15s, and leave at room temperature for 5 min.
(4) Add 20. mu.L proteinase K and mix well with shaking.
(5) Add 220. mu.L buffer GB, shake for 15s, and stand at 70 ℃ for 10 min.
(6) Add 220. mu.L of absolute ethanol and mix well with shaking.
(7) The adsorption column CB3 was put into a collection tube, and the whole of the solution mixed in the previous step was transferred to the adsorption column, centrifuged at 12000rpm for 30 seconds, and the waste liquid in the collection tube was discarded (this step sometimes requires passing through the column several times because the maximum capacity of the adsorption column is generally 700. mu.L).
(8) Adding 500 μ L of buffer GD into which absolute ethyl alcohol is added as required into the adsorption column CB3 after the previous step of centrifugation, centrifuging at 12000rpm for 30s, and discarding the waste liquid.
(9) Adding 600 μ L of rinsing solution PW which is added with absolute ethyl alcohol according to requirements into the adsorption column CB3 after the previous step of centrifugation, centrifuging at 12000rpm for 30s, and discarding the waste liquid.
(10) Repeating the previous step.
(11) And (4) allowing the adsorption column subjected to the two-time rinsing to be separated again at 12000rpm for 2min, pouring waste liquid in the collection tube, opening a tube cover, and airing for several minutes.
(12) The completely air-dried adsorption column CB3 was put into a clean centrifuge tube, 50. mu.L of TE was added thereto, and the mixture was left for 5 minutes and then centrifuged at 12000rpm for 2 minutes.
(13) The DNA obtained by the previous step of centrifugation was again pipetted into adsorption column CB3, and after standing for 2min, it was centrifuged again at 12000rpm for 2 min.
(14) The finally obtained test strain DNA is stored at-20 ℃ for later use.
2-2, extracting genome DNA of seed sample
A. Seed sample treatment: weighing about 50g of seed sample, pouring into a sterilized triangular flask, adding a proper amount of sterile water to completely immerse the seeds, and soaking overnight at 4 ℃. 10mL of the extract was aspirated and centrifuged at 12000rpm for 5min, and the supernatant was discarded, and the pellet was used for DNA extraction.
B. DNA extraction: extracting total DNA of a seed sample by using a novel plant genome DNA extraction kit (centrifugal column type), and specifically performing the following steps:
(1) adding 400 μ L buffer solution LP1 and 6 μ L RNase A into the precipitate of the seed sample leaching solution obtained in the previous step, vortex-shaking for 1min, and standing at room temperature for 10 min.
(2) 130 μ L of buffer LP2 was added and mixed well for 1min with vigorous shaking to resuspend all the pellet.
(3) Followed by centrifugation at 12000rpm for 5min and aspiration of the supernatant into a new 2mL centrifuge tube.
(4) To the supernatant was added 1.5 times the volume of buffer LP3 to which a corresponding amount of absolute ethanol had been added as required, and immediately mixed thoroughly for 15 sec.
(5) The adsorption column CB3 was placed in a collection tube, and the whole amount of the solution mixed in the previous step was transferred to the adsorption column, centrifuged at 12000rpm for 30sec, and the waste liquid in the collection tube was discarded (this step was sometimes carried out in multiple passes because the maximum capacity of the adsorption column was generally 700. mu.L).
(6) Adding 600 μ L of rinsing solution PW which is added with absolute ethyl alcohol according to requirements into the adsorption column CB3 after the previous step of centrifugation, centrifuging at 12000rpm for 30s, and pouring out waste liquid in the collecting pipe.
(7) And repeating the previous step.
(8) And (4) allowing the adsorption column subjected to the two-time rinsing to be separated again at 12000rpm for 2min, pouring waste liquid in the collection tube, opening a tube cover, and airing for several minutes.
(9) The completely air-dried adsorption column CB3 was put into a clean centrifuge tube, 50. mu.L of elution buffer TE was added thereto, and after standing for 5min, it was centrifuged at 12000rpm for 2 min.
(10) The DNA obtained by the previous step of centrifugation was again pipetted into adsorption column CB3, and after standing for 2min, it was centrifuged again at 12000rpm for 2 min.
(11) The finally obtained seed sample DNA is stored at-20 ℃ for later use.
2-3, determination of DNA concentration
The quality of the DNA was checked by electrophoresis and its concentration was determined using a NanoDrop spectrophotometer.
3. Design and screening of primers and probes
According to a presumed membrane protein gene GHV42-03765 with the length of about 700bp in the whole genome of rice bacterial leaf spot (Xanthomonas oryzae pv. oryzae) as a target sequence, a primer XOC367-3F, a primer XOC367-3R and a Probe Probe of LFD-RPA are designed. The LFD-RPA primer has the same sequence as the common RPA primer except that the 5' end of the LFD-RPA reverse primer carries a biotin (biotin) label. The nucleotide sequence of the primer XOC367-3F is shown as SEQ ID No.1, the nucleotide sequence of the primer XOC367-3R is shown as SEQ ID No.2, and the nucleotide sequence of the Probe is shown as SEQ ID No. 3. The 5' end of the primer XOC367-3R is labeled with biotin. The Probe is labeled with FAM at the 33 rd base from the 5 ' end, modified with tetrahydrofuran between the 33 th base and the 34 th base from the 5 ' end, and labeled with C3-spacer at the 3 ' end.
Specific primer and probe sequences are shown in Table 2.
TABLE 2LFD-RPA amplification primers and Probe sequence information
4. LFD-RPA detection system and method
Adding 29.4 mu L of buffer solution, 11.5 mu L of ultrapure water, 10 mu M of primer XOC367-3F2 mu L, 10 mu M of primer XOC367-3R2 mu L, 10 mu M of Probe Probe 0.6 mu L, 2 mu LDNA template and 2.5 mu L of magnesium acetate into RPA freeze-drying enzyme, fully mixing uniformly, putting the mixture into a 39 ℃ water bath box for amplification reaction, putting the amplification product into a lateral flow chromatography detection test paper strip sealing device after 15 minutes, and observing the amplification product after 5 minutes of room temperature. And determining whether the sample to be detected contains rice bacterial leaf streak pathogens or not according to the LFD test strip. If the LFD test strip has two purple-red strips, one is positioned in the quality control area, and the other is positioned in the detection area, the result is positive, and the sample to be detected contains rice bacterial leaf streak germs; if the LFD test strip only has a purple-red strip and is positioned in the quality control area, the result is negative, which indicates that the sample to be tested does not contain rice bacterial streak pathogens.
Example 2LFD-RPA detection Performance analysis
The concentration of the mixture was 259 ng/. mu.L, 25.9 ng/. mu.L, 2.59X 10-1ng/μL、2.59×10-2ng/μL、2.59×10-3ng/μL、2.59×10-4ng/μL、2.59×10-5Determining the sensitivity of the LFD-RPA technology by ng/mu L of DNA of the rice bacterial leaf streak strain LMG 654; the specificity of the LFD-RPA system is verified by rice bacterial blight bacteria, rice bacterial blight bacteria and other related species and common pathogenic bacteria on rice;
1. sensitivity analysis
The concentration of the mixture was 259 ng/. mu.L, 25.9 ng/. mu.L, 2.59X 10-1ng/μL、2.59×10-2ng/μL、2.59×10-3ng/μL、2.59×10-4ng/μL、2.59×10-5The DNA of ng/. mu.L of bacterial rice bacterial leaf streak strain LMG654 was subjected to sensitivity analysis by LFD-RPA technique, and the template DNA was replaced with an equal amount of water as a blank (CK). As a result, as shown in FIG. 1, the LFD-RPA detection sensitivity was 2.59X 10-4ng/. mu.L. The LFD-RPA can realize the observation result between naked eyes without any instrument, has simple and convenient operation and rapid reaction, and well embodies the advantages of the LFD-RPA in field or field application.
2. Specificity analysis
Respectively selecting 3 positive strains (the numbers of which are respectively LMG654, LMG658 and NCPPB1150), 4 same-genus pathogenic bacteria (the numbers of which are respectively NCPPB793, LMG817, NCPPB4351 and LMG559) and 2 other-genus pathogenic bacteria (the numbers of which are respectively LMG3189 and NCPPB2309), verifying the LFD-RPA system specificity by taking the genome DNA as a template, and replacing the template DNA with equal amount of water to be used as a blank Control (CK). The results are shown in fig. 2, the results of the LFD flow-measuring chromatography test strip show that only the bacterial leaf spot of rice (the numbers of which are respectively LMG654, LMG658 and NCPPB1150) are positive results, the DNA detection results of the other pathogenic bacteria are negative, only one quality control line appears on the test strip, namely, only one mauve strip appears on the LFD test strip and is positioned in a quality control area, and the results are negative, which indicates that the sample to be detected does not contain the bacterial leaf spot of rice.
EXAMPLE 3 LFD-RPA detection of seed samples
1. Preparation of simulated germ-carrying seed sample
(1) Preparation of bacterial suspension for inoculation: activating positive strain LMG654 of rice bacterial streak germ on NA plate, culturing at 28 deg.C for 24-48h, and preparing pure cultured strain into 10 with sterile water6Bacterial suspension of about cfu/mL.
(2) Preparing simulated bacteria-carrying seeds: firstly, detecting the purchased healthy rice seeds by using a conventional detection method to ensure that the healthy rice seeds do not carry the rice bacterial streak germs, then soaking the healthy rice seeds in the bacterial suspension prepared by the method in the step (1) for 1 hour at room temperature, pouring off the bacterial suspension, and air-drying for 2 days at room temperature to prepare a banding strain seed sample.
2. LFD-RPA detection of seed samples
Genomic DNA of the mock bring bacteria seed sample prepared in step 1 of example 3 was extracted according to the method of step 2-2 of example 1, and LFD-RPA detection was performed using the primer and probe composition designed according to the present invention using the same as the template according to the system and method described in step 4 of example 1.
2 parts of rice seed samples artificially inoculated with rice bacterial leaf streak germs and 2 parts of healthy rice seeds were tested by using the established LFD-RPA detection system for rice bacterial leaf streak germs, and the detection results are shown in FIG. 3. As can be seen from FIG. 3, the results of the seed tests inoculated with bacterial leaf spot of rice are all positive, and the treatment group and the blank Control (CK) using healthy rice seeds as negative control are both negative (only C line appears, i.e. only one quality control line appears on the test paper). The system can be successfully applied to the detection and analysis of bacterial streak pathogens in rice seed samples.
In the research, a nucleic acid visual detection technology aiming at the rice bacterial leaf streak pathogens is established based on an LFD-RPA technology, and a primer probe set is designed and the detection efficiency is evaluated. Proved by verification, the system has high detection sensitivity which can reach 2.59 multiplied by 10-4ng/mu L, good specificity and no cross reaction with other closely related species and other pathogenic bacteria.
The rice with bacteria is an important primary infection source of the bacterial leaf streak of rice and is the most main mode for long-distance transmission of pathogenic bacteria. Therefore, the detection of rice seeds is an important way for improving the control level of rice diseases, preventing the diseases from spreading and ensuring the continuous and healthy development of the rice industry in China. The results prove that the LFD-RPA detection system established in the research can be applied to rice seed samples.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
SEQUENCE LISTING
<110> scientific research institute of Chinese inspection and quarantine
<120> LFD-RPA technology-based rice bacterial leaf streak germ detection kit, primer probe composition and application thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
caacgaggcg gtgcttccat gtcaaccgca g 31
<210> 2
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
acctttttgc catcgcctca ttccttgcat ac 32
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<211> 50
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
agcaccagac caagcacctg aggtgtcaag ggtcttcccg agtggatcgg 50
Claims (10)
1. The kit for detecting the rice bacterial leaf streak germs is characterized by comprising a composition for detecting the rice bacterial leaf streak germs and LFD test paper; the composition comprises a primer XOC367-3F, a primer XOC367-3R and a Probe Probe; the nucleotide sequence of the primer XOC367-3F is shown as SEQ ID No.1, the nucleotide sequence of the primer XOC367-3R is shown as SEQ ID No.2, and the nucleotide sequence of the Probe is shown as SEQ ID No. 3.
2. The kit according to claim 1, wherein the 5' end of the primer XOC367-3R is labeled with biotin.
3. The kit according to claim 1 or 2, wherein the Probe has any one or a combination of the following modifications:
A1) tetrahydrofuran is labeled at any base position of 30 th to 35 th bases from the 5' end of the Probe;
A2) labeling a fluorescent group at the 5' end of the Probe;
A3) the 3' end of the Probe Probe is labeled with an amine group, a phosphate group or C3-spacer.
4. The kit according to any one of claims 1 to 3, wherein the Probe Probe is labeled with FAM at the 5 ' end, modified with tetrahydrofuran between the 33 th and 34 th bases from the 5 ' end, and labeled with C3-spacer at the 3 ' end.
5. The composition of any one of claims 1-4.
6. The method for detecting the bacterial leaf streak germs of the rice is characterized by comprising the following steps:
B1) extracting genome DNA of a sample to be detected;
B2) performing LFD-RPA detection using the composition of any one of claims 1-4 using the genomic DNA as a template; and determining whether the sample to be detected contains rice bacterial leaf streak pathogens or not according to the LFD test strip.
7. The method of claim 6, wherein the determination of whether the test sample contains rice bacterial leaf streak disease is performed by using LFD strip: if the LFD test strip has two purple-red strips, one is positioned in the quality control area, and the other is positioned in the detection area, the result is positive, and the sample to be detected contains rice bacterial leaf streak germs; if the LFD test strip only has a purple-red strip and is positioned in the quality control area, the result is negative, which indicates that the sample to be tested does not contain rice bacterial streak pathogens.
8. Use of a kit according to any one of claims 1 to 4 for the detection or the auxiliary detection of bacterial leaf streak disease in rice.
9. Use of a composition according to claim 5 in the detection or in the auxiliary detection of bacterial leaf streak disease in rice.
10. Use of a composition according to claim 5 for the preparation of a product for the detection or the auxiliary detection of bacterial leaf streak disease in rice.
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