CN112662794A - Fluorescence recombinase mediated isothermal amplification detection kit for wilting bacteria in China in corn - Google Patents
Fluorescence recombinase mediated isothermal amplification detection kit for wilting bacteria in China in corn Download PDFInfo
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Abstract
The invention belongs to the technical field of detection, and discloses a fluorescence recombinase mediated isothermal amplification detection kit for wilting virus in China, which comprises: the forward primer is SEQ ID NO: 1; reverse primer SEQIDNO: 2; and probe seq id no: 3. the kit construction method comprises the following steps: activating the strain by using an NA culture medium, collecting thalli by using an inoculating ring scraper, extracting bacterial DNA, verifying the extraction effect by using a nucleic acid tester, and storing at-20 ℃ for later use; designing and synthesizing a primer probe, screening a primer, establishing a system and verifying the system; verifying the specificity of the fluorescent RAA detection method; and (5) verifying the detection effects of the simulated sample and the real sample to obtain the fluorescent recombinase-mediated isothermal amplification detection kit for the wilting disease in the state of corn. The invention has the advantages of rapid reaction, short reaction time of only 20min, strong specificity and high sensitivity, and passes the detection verification of actual samples and simulated samples.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a fluorescence recombinase mediated isothermal amplification detection kit for wilting bacteria in China.
Background
At present, the clavibacterium michiganensis subspecies causes the wilting disease in the state of corn, can cause the loss of the corn by up to 44 percent, is an important bacterial pathogen on the corn and is also a quarantine pathogen which is focused to China. Cmn is firstly found in the middle of Nebraska in the United states and then gradually expands to parts of regions of multiple states and Canada in the United states, and China has not been reported yet. The Cmn is mainly transmitted in a long distance through a seed transmission band, and is predicted and displayed by using a GRAP model, so that the Cmn is suitable for field planting in most corn planting areas in China. Although the corn yield in China is stabilized to be more than 2 hundred million tons for many years, a large amount of imported corn is still needed every year along with the rapid increase of domestic industrial consumption demand and feed consumption demand. With the development of the import trade of corn, the risk of introducing Cmn into China increases sharply, and once the Cmn is invaded, the corn production in China is threatened seriously, and the food production safety in China is influenced. Therefore, establishing and mastering an accurate and rapid detection method is important for effectively monitoring the Cmn diffusion condition.
Currently, detection aiming at Cmn mainly focuses on traditional separation culture, enzyme-linked immunoassay and conventional molecular biology detection. The separation culture and serological detection have long time consumption, complex operation and low sensitivity, and are difficult to meet the requirement of on-site rapid detection. The traditional molecular biology detection methods developed based on polymerase chain reaction, including common PCR, nested PCR, real-time fluorescence PCR and the like, all need to undergo temperature change processes such as denaturation, annealing, extension and the like, and the temperature control is strict, so that the traditional molecular biology detection methods need to be supported by a specific instrument and consume a long time, and are not beneficial to rapid detection in a field laboratory. Loop-mediated isothermal amplification (LAMP) is an in vitro isothermal amplification technology widely applied at present, and Bst DNA polymerase is utilized to generate a complementary sequence through a strand replacement reaction of 4-6 primers so as to realize DNA amplification under the condition of constant temperature (60-65 ℃). The establishment of the LAMP detection method for the wilting disease in China in corn realizes the rapid detection of Cmn under the constant temperature condition, the method has the advantages of strong specificity, high sensitivity and the like, and can be used for detecting the corn samples on site through the verification of actual samples. However, products obtained by the method are a series of bands with different sizes, cloning and sequencing cannot be carried out, and the detection result cannot be further verified. The recombinase-assisted isothermal amplification (RAA) technology effectively overcomes the defect, and can realize exponential amplification of a target fragment under a constant temperature condition, and a product is a single sequence band. RAA uses recombinase extracted from bacteria or fungi, recombinases and primer DNA are combined into polymer under constant temperature condition, template DNA is unzipped with the help of single-stranded DNA binding protein (SSB), new complementary strand is formed under the action of DNA polymerase, and the whole reaction process can be completed only by 20 min. The RAA technology is used for detecting pathogenic microorganisms such as bacteria, viruses, mycoplasma and the like, has the advantages of rapidness, sensitivity, specificity and the like, and is a hot spot for detection research at present. Tambong James T and the like screen partial sequences of cellulase genes by methods such as genome analysis and the like, and can be used as target sequences for molecular biological identification of the wilting bacteria in the interior of corn.
Through the above analysis, the problems and defects of the prior art are as follows: the separation culture and serological detection have long time consumption, complex operation and low sensitivity; the traditional molecular biology detection method requires strict temperature control, needs a specific instrument for support and consumes a long time; the LAMP detection method has the advantages that products are a series of bands with different sizes, and further verification such as clone sequencing and the like cannot be performed on the results.
The difficulty in solving the above problems and defects is: the RAA technology can realize that the detection reaction can be quickly finished within 20min under the constant temperature condition of 39 ℃, and has simple and convenient operation and quick reaction; the difficulty is target sequence screening and primer design.
The significance of solving the problems and the defects is as follows: establishing a detection method for rapidly detecting the wilting germs in the state of the corn under the condition of constant temperature in vitro.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a fluorescence recombinase mediated isothermal amplification detection kit for wilting disease in China.
The invention is realized in such a way that the fluorescence recombinase mediated isothermal amplification detection kit for the wilting disease of the interior of the corn comprises the following components:
forward primer SEQ ID NO: 1; reverse primer SEQ ID NO: 2; and probe SEQ ID NO: 3.
another object of the present invention is to provide a method for constructing a fluorescence recombinase mediated isothermal amplification detection kit for wilting virus in maize, the method comprising:
activating a strain by using an NA culture medium, collecting thalli by using an inoculating loop scraper, extracting bacterial DNA, verifying the extraction effect by using a nucleic acid tester, and storing at-20 ℃ for later use;
designing and synthesizing a primer probe, and screening and establishing a system; using sterilized ddH2O, diluting the determined DNA with the lowest detection concentration, performing fluorescence RAA detection of the optimal primer combination, and determining the sensitivity of the fluorescence RAA detection method;
step three, verifying the specificity of the fluorescence RAA detection method; and (5) verifying the detection effects of the simulated sample and the real sample to obtain the fluorescent recombinase-mediated isothermal amplification detection kit for the wilting disease in the state of corn.
Further, in step one, the strain activation comprises: activation was carried out at 28 ℃ for 48 h.
Further, in the second step, the designing and synthesizing of the primer probe comprises:
(1) analyzing the difference of cellulase gene sequences among C.microganiensis subspecies closely, and selecting a CelB sequence as a template to design a primer and a probe;
(2) designing primers Cmn1, Cmn2 to amplify target sequences with reference to Genbank sequence HE614873, aligning the Cmn1/Cmn2 sequence with the full database sequence using BLAST, determining if the primer combination recognizes the Cmn strain 100% and does not match other sequence data;
(3) if the primer combination identifies the Cmn strain 100% and is not matched with other sequence data, the Cmn DNA is used as a template, a target sequence is amplified by using a primer Cmn1/Cmn2, and a PCR product is subjected to gel cutting recovery through 1.5% agarose gel electrophoresis and is subjected to clone sequencing;
(4) with reference to the RAA principle, forward primers 1F, 2F, 3F, 4F, 5F, 6F, 7F, reverse primers 1R, 2R, 3R, 4R, 5R, 6R, 7R and probe P were designed.
Further, in the step (3), the amplification comprises:
the reaction system is a 50-mu-L system and comprises: 2 × Taq PCR StarMix with Loading Dye 25. mu.L, 10. mu.M (cmn1/cmn2) 2. mu.L, DNA template 2. mu.L, ddH2O 19. mu.L;
the amplification condition is 94 ℃ for 3 min; 30s at 94 ℃, 30s at 62 ℃, 45s at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min.
Further, in step two, the performing primer screening comprises:
cmn DNA extraction to sterilize ddH2Performing 10-fold equal gradient dilution on O, randomly selecting a primer combination, performing fluorescent RAA detection by respectively taking Cmn DNAs with different concentrations as templates, and determining the lowest detection concentration of the fluorescent RAA to the Cmn; taking the DNA with the lowest detection concentration as a template, firstly combining the upstream primers 1F, 2F, 3F and 4F with the downstream primers 1R, 2R, 3R and 4R, carrying out fluorescence RAA detection, and screening out the forward primer, the reverse primer and the primer combination with the highest amplification efficiency; secondly, combining the screened forward primers, 5F, 6F and 7F with the screened reverse primers, 5R, 6R and 7R respectively, and exploring the primer combination with the highest amplification efficiency; finally, the screened primer combinations are comparatively analyzed, and the primer combinations with good stability and high amplification efficiency are screened out.
Further, the gradient concentrations are 26.1, 2.6 and 2.6 multiplied by 10 respectively-1、2.6×10-2、2.6×10-3、2.6×10-4、2.6×10-5、2.6×10-6ng/μL。
Further, in step two, the system establishment comprises:
reaction system: buffer ABuffer40.9 μ L, B Buffer 2.5 μ L, upstream primer and downstream primer each 2.0 μ L (10mM), fluorescent probe 0.6 μ L (10mM), DNA template 2.0 μ L;
amplification conditions: 30s at 39 ℃ for 40 cycles.
Further, in step three, the verifying the specificity of the fluorescent RAA detection method includes:
extracting DNA of Cmm, Cmi, Pssy, Mt, Pss, Pan, Pag, Xo, Ba, Dz and Ecz, determining the concentration, taking 11 reference strain DNA as a template and Cmn DNA with different concentrations as a positive control, sterilizing ddH2And O is negative control, and the fluorescence RAA detection is carried out to verify the specificity of the fluorescence RAA detection method.
Further, in the third step, the verification of the detection effect of the simulated sample and the real sample comprises:
1) using sterilized ddH2O, uniformly mixing the Cmn thalli collected by 1.2.1 to prepare a bacterial suspension; the concentration of the bacterial suspension is determined to be 5.7 multiplied by 10 by using a plate coating method5cfu/mL；
2) Screening a feeding sample with a negative test result by a quarantine identification method for wilting disease in China in corn, grinding the feeding sample into powder and dividing the powder into 4 parts, wherein each part is 10 g;
3) adding the prepared bacterial suspension into a ground sample, and uniformly mixing to prepare a positive simulation sample;
4) extracting the DNA of 4 positive simulation samples and 24 real corn samples by adopting a novel plant genome DNA extraction kit, and carrying out fluorescence RAA detection; meanwhile, GB/T36840-: and (4) rechecking and verifying the quarantine identification method of the wilting disease in China in the corn.
Another objective of the present invention is to provide an application of the fluorescence recombinase-mediated isothermal amplification detection kit for detecting wilting disease in maize.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention utilizes the fluorescent RAA technology, takes the CelB sequence of the State wilting bacteria in the corn as a template, designs and screens primers and probes, and constructs the fluorescent RAA rapid detection method of the State wilting bacteria in the corn.
The invention designs primers and probes aiming at CelB sequence segments of the wilting bacteria in China, and establishes a fluorescent RAA detection method for the wilting bacteria in China through experiments such as primer screening, specificity verification, sensitivity determination and the like. The invention has the advantages of rapid reaction, long reaction time of only 20min, strong specificity and high sensitivity up to 130 fg/mu L, and passes the detection verification of actual samples and simulated samples. The fluorescent RAA detection method is rapid, specific, sensitive and simple, meets the requirements of accurate and rapid field detection, and can provide a new idea for effectively monitoring the wilting germs in the corn. The invention analyzes and compares Cmn and the homologous subspecies cellulase gene sequences thereof, screens out a Cmn conserved sequence as a target sequence, designs and screens primers and probes by utilizing a fluorescent RAA technology, and constructs a fluorescent RAA rapid detection method for the wilting disease in China.
Compared with the LAMP detection method, the fluorescent RAA detection method for the wilting disease in China built by the invention has the advantages of constant temperature, sensitivity and specificity, is simpler and faster, only one pair of primers is needed for RAA, the specific fragment can be amplified for 20min, and the product is a single strip. By combining with a fluorescent probe, the RAA technology can realize dynamic monitoring of the amplification process, and quantitative detection is carried out. The invention tries to use template DNA 10 times gradient diluent to carry out fluorescence RAA quantitative detection research, and takes the concentration of template LOG as abscissa and relative fluorescence intensity delta Rn>A standard curve was established for the ordinate at 20000 cycles, showing: when the sample concentration is selected to be 26.1, 2.6X 10-1ng/. mu.l of data was fitted to a standard curve, which was found to be-3.5 x +21.792(R2 is 0.9932), but with decreasing concentration of the selected samples, the confidence of the fitted standard curve was R2<0.95, for analytical reasons or in relation to insufficient uniformity of dilution of the template gradient, it was necessary to prepare standard plasmids for fluorescent RAA quantitative detection studies.
The invention utilizes RAA amplification technology to detect the wilting disease in the corn, and designs forward primers 1F, 2F, 3F, 4F, 5F, 6F and 7F, reverse primers 1R, 2R, 3R, 4R, 5R, 6R and 7R and a probe P according to the RAA principle. The fluorescence RAA detection experiment is carried out by utilizing different primer combinations, and the following results are found: although the primer combinations tried were all amplifiable, the difference in amplification efficiency was significant (fig. 2-4). For analytical reasons, or in connection with the RAA principle of primer binding to recombinase proteins as dimers, different primer sequences bind to recombinase proteins with different efficiencies. Therefore, it is necessary to further study the binding mechanism between the primer and the recombinase protein in the RAA to guide the primer design and improve the efficiency.
The fluorescent RAA detection method established by screening the primer combination and combining the probe P can detect the wilting disease in the corn within 20 min. The primer specificity detection result shows that the significant amplification phenomenon of the wilting disease in China in two different concentrations of corn is positive, the other 11 control strains including 2 similar subspecies have no significant amplification phenomenon and are negative, and the specificity of the fluorescence RAA detection method is strong. The detection sensitivity of the fluorescent RAA detection method to the DNA of the wilting disease in China is 130 fg/mu L, which is slightly lower than the 50fg sensitivity of the LAMP detection method. And (3) detecting the simulated corn sample and the real corn sample by using the fluorescent RAA, wherein the detection result is similar to that of the reference national standard GB/T36840-2018: the quarantine identification method of the wilting disease in China in corn is consistent. The established fluorescent RAA detection method is rapid, sensitive and specific, and is suitable for rapidly detecting the wilting disease in the corn in a field laboratory.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
Fig. 1 is a flowchart of a method for constructing a fluorescence recombinase-mediated isothermal amplification detection kit for wilting virus in maize according to an embodiment of the present invention.
FIG. 2 is a graph showing the relative fluorescence intensity of forward primers 1F, 2F, 3F, 4F and 1R, 2R, 3R, 4R after 40 cycles.
FIG. 3 is a graph showing the relative fluorescence intensity of forward primers 2F, 5F, 6F, 7F and 4R, 5R, 6R, 7R after 40 cycles.
FIG. 4 is a graph showing the relative fluorescence intensity of forward primer 2F and 3R, 4R, 6R after 40 cycles.
FIG. 5 is a schematic diagram of the detection result of the 1F/1R fluorescence RAA sensitivity of the primer provided by the embodiment of the invention.
FIG. 6 is a schematic diagram of the detection result of the 2F/6R fluorescence RAA sensitivity of the primer provided by the embodiment of the invention.
FIG. 7 is a schematic diagram of a fluorescent RAA-specific assay provided in an embodiment of the present invention.
FIG. 8 is a schematic diagram of the position of the primers and probes and the multiple sequence analysis provided in the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a fluorescence recombinase mediated isothermal amplification detection kit for wilting disease in maize, and the invention is described in detail with reference to the accompanying drawings.
The fluorescence recombinase mediated isothermal amplification detection kit for wilting disease in maize provided by the embodiment of the invention comprises:
forward primer SEQ ID NO: 1: gttactggtgaatgtgagcacggggttgc, respectively;
reverse primer SEQ ID NO: 2: cacgtctgagctcgtcatcactgcgaagt, respectively;
and probe SEQ ID NO: 3: acgtgatgcggctcgacgccacggtgcact [ FAM-dT ] c [ THF ] [ dT-BHQ1] gccccagctgttcg-block.
Target sequence SEQ ID NO: 4 is as follows:
TGCTCGATCTTAACCGCGACCCCACGAGGAAGCGGCGCCGGCCACCGAGCGGGATGCGCTCGACGGCCGACGCCGCGGATGATGGGCGGGAGCCCTGGTCAGTTACTGGTGAATGTGAGCACGGGGTTGCTCGGAGCCTGAGTTCCCGCGACGTTCAGACCGACGTGCATGGTCTGTCCGGCCGGGATCTTGGCGGCCCAGCCATCACCGGTGCACGTGATGCGGCTCGACGCCACGGTGCACTTCATGCCCCAGCTGTTCGTGACAGCCGTGATTCCGGGGCTGTCCCAGGAGACCGTCCAACTCGACACCGCGGACTTCGCAGTGATGACGAGCTCAG ACGTGTAGCCCGTGGGCCACGATCCGTCCAGCTTGTA
as shown in fig. 1, the method for constructing the fluorescent recombinase-mediated isothermal amplification detection kit for wilting virus in maize according to the embodiment of the present invention includes:
s101, activating a strain by using an NA culture medium, collecting thalli by using an inoculating loop scraper, extracting bacterial DNA, verifying an extraction effect by using a nucleic acid tester, and storing at-20 ℃ for later use;
s102, designing and synthesizing a primer probe, and screening a primer and establishing a system; using sterilized ddH2O, diluting the determined DNA with the lowest detection concentration, performing fluorescence RAA detection of the optimal primer combination, and determining the sensitivity of the fluorescence RAA detection method;
s103, verifying the specificity of the fluorescent RAA detection method; and (5) verifying the detection effects of the simulated sample and the real sample to obtain the fluorescent recombinase-mediated isothermal amplification detection kit for the wilting disease in the state of corn.
In step S101, the strain activation provided in the embodiment of the present invention includes: activation was carried out at 28 ℃ for 48 h.
In step S102, the designing and synthesizing of the primer probe provided in the embodiment of the present invention includes:
analyzing the difference of cellulase gene sequences among C.microganiensis subspecies closely, and selecting a CelB sequence as a template to design a primer and a probe; designing primers Cmn1, Cmn2 to amplify target sequences with reference to Genbank sequence HE614873, aligning the Cmn1/Cmn2 sequence with the full database sequence using BLAST, determining if the primer combination recognizes the Cmn strain 100% and does not match other sequence data; if the primer combination identifies the Cmn strain 100% and is not matched with other sequence data, the Cmn DNA is used as a template, a target sequence is amplified by using a primer Cmn1/Cmn2, and a PCR product is subjected to gel cutting recovery through 1.5% agarose gel electrophoresis and is subjected to clone sequencing; with reference to the RAA principle, forward primers 1F, 2F, 3F, 4F, 5F, 6F, 7F, reverse primers 1R, 2R, 3R, 4R, 5R, 6R, 7R and probe P were designed.
The amplification provided by the embodiment of the invention comprises the following steps:
the reaction system is a 50-mu-L system and comprises: 2 × Taq PCR StarMix with Loading Dye 25. mu.L, 10. mu.M (cmn1/cmn2) 2. mu.L, DNA template 2. mu.L, ddH2O 19. mu.L;
the amplification condition is 94 ℃ for 3 min; 30s at 94 ℃, 30s at 62 ℃, 45s at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min.
In step S102, the primer screening provided in the embodiment of the present invention includes:
cmn DNA extraction to sterilize ddH2Performing 10-fold equal gradient dilution on O, randomly selecting a primer combination, performing fluorescent RAA detection by respectively taking Cmn DNAs with different concentrations as templates, and determining the lowest detection concentration of the fluorescent RAA to the Cmn; taking the DNA with the lowest detection concentration as a template, firstly combining the upstream primers 1F, 2F, 3F and 4F with the downstream primers 1R, 2R, 3R and 4R, carrying out fluorescence RAA detection, and screening out the forward primer, the reverse primer and the primer combination with the highest amplification efficiency; secondly, combining the screened forward primers 5F, 6F and 7F with the screened reverse primers 5R, 6R and 7R respectively, and exploring the primer combination with the highest amplification efficiency; finally, the screened primer combinations are comparatively analyzed, and the primer combinations with good stability and high amplification efficiency are screened out.
The gradient concentrations provided by the embodiment of the invention are respectively 26.1, 2.6 and 2.6 multiplied by 10-1、2.6×10-2、2.6×10-3、2.6×10-4、2.6×10-5、2.6×10-6ng/μL。
In step S102, the system establishment provided in the embodiment of the present invention includes:
reaction system: buffer ABuffer40.9 μ L, B Buffer 2.5 μ L, upstream primer and downstream primer each 2.0 μ L (10mM), fluorescent probe 0.6 μ L (10mM), DNA template 2.0 μ L;
amplification conditions: 30s at 39 ℃ for 40 cycles.
In step S103, the specificity of the method for verifying fluorescence RAA detection provided in the embodiment of the present invention includes:
extracting DNA of Cmm, Cmi, Pssy, Mt, Pss, Pan, Pag, Xo, Ba, Dz and Ecz, determining the concentration, taking 11 reference strain DNA as a template and Cmn DNA with different concentrations as a positive control, sterilizing ddH2And O is negative control, and the fluorescence RAA detection is carried out to verify the specificity of the fluorescence RAA detection method.
In step S103, the verification of the detection effect of the simulated sample and the real sample provided in the embodiment of the present invention includes:
using sterilized ddH2O, uniformly mixing the Cmn thalli collected by 1.2.1 to prepare a bacterial suspension; the concentration of the bacterial suspension is determined to be 5.7 multiplied by 10 by using a plate coating method5cfu/mL; screening a feeding sample with a negative test result by a quarantine identification method for wilting disease in China in corn, grinding the feeding sample into powder and dividing the powder into 4 parts, wherein each part is 10 g; adding the prepared bacterial suspension into a ground sample, and uniformly mixing to prepare a positive simulation sample; extracting the DNA of 4 positive simulation samples and 24 real corn samples by adopting a novel plant genome DNA extraction kit, and carrying out fluorescence RAA detection; meanwhile, the quarantine identification method of the wilting disease in the interior of corn is used for recheck and verification.
The technical effects of the present invention will be further described with reference to specific embodiments.
Example 1:
1 materials and methods
1.1 test materials
24 samples of feed corns entering the border of the Zhoushan port in 2018-2019 are collected, and each sample is 2kg and is reserved. The test strains are 12 strains in total, wherein the maize inner periphery wilting bacteria (Cmn) and maize bacterial wilt bacteria (Pantoea. stewartii, Pss) are ATCC standard strains which are numbered as ATCC 27794 and ATCC 29229 respectively and are donated by the Hangzhou customs integrated technology service center; pseudomonas syringae tomato pathogenic variety (Pseudomonas syringae pv. syringae, psyy), Microbacterium rubrum (Microbacterium testaceum, Mt), corynebacterium michiganensis subsp.35890, subspecies (clavibacterium microsps subsp.idisosus, Cmi), clavibacterium michiganensis (clavibacterium michiganensis subsp.miciganensis, cm), Xanthomonas (xantozae, Xo) were kept in the laboratory, and Pantoea berk (Burkholderia andagogonis, Ba), dicke zeae (Dickeya, Dz), europaea (winia chrysopharia var. zea, Ecz), Pantoea agglomerans (panzea ), Pantoea benthamella paraguajavanica (pangama technologies, panoxa.
Main apparatus and reagents: real-time fluorescent PCR instrument (applied biosystems, usa, ABI 7500); fluorescent RAA Kit (Hangzhou Mass-testing Biotechnology Co., Ltd., S002ZC), TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.3.0 (Bao bioengineering (Dalian) Co., Ltd., 9763), novel plant Genomic DNA Extraction Kit (GenBiotechnology technology Co., Ltd., DP320), and 2 XTaqPCR StarMix with Loading Dye (GenStar-Kangrunbio, A012).
1.2 methods
1.2.1DNA extraction and validation
The strain was activated by NA medium (28 ℃ C., 48 hours), and the cells were collected by an inoculating loop scraper. Extracting bacterial DNA according to the instruction of the bacterial genome DNA extraction kit, verifying the extraction effect by a nucleic acid tester (Nanodrop 2000), and storing at-20 ℃ for later use.
1.2.2CelB sequence screening, primer Probe design and Synthesis
The Cmn strain NCPPB2581 whole genome sequence (sequence number: HE614873) is downloaded from NCBI database, the cellulase gene sequence is screened out, corresponding bases 2525339nt-2525973nt are screened out, BLAST is utilized to analyze the sequence difference between the Cmn cellulase gene sequence and other C.microganiensis related subspecies (sequence information is shown in Table 1), and the CelB sequence is screened out.
TABLE 1 Gene sequences used in the experiments
Table 1Genome sequences of bacterial strains used in this study
a: performing Blast analysis by taking the Cmn strain NCPPB2581 cellulase gene sequence as a reference sequence;
a:Chose the cellular gene sequence of NCPPB2581 strain as reference sequence to Blast analysis
the Primer cmn1 was designed using Primer premier 5.0, targeting the entire genome sequence of strain NCPPB 2581: TGCTCGATCTTAACCGCGAC, respectively; cmn 2: TACAAGCTGGACGGATCGTG, corresponding to genomes 2525241nt and 2525598nt, respectively. Alignment of the Cmn1/Cmn2 sequences to the full database sequence using BLAST revealed: the matching rate of the primer combination with Cmn strain 7580(CP033722) HF4(CP033722)61-1(CP033722) NCPPB2581(HE614873) is 100%, the sizes of products are 377bp, and no matching phenomenon exists with other sequence data.
The target sequence was amplified using the Cmn DNA as a template and primers Cmn1/Cmn 2. The reaction system is a 50 μ L system: 2 XTaq PCR StarMix with Loading Dye 25. mu.L, cmn1/cmn2 (10. mu.M) each 2. mu.L, DNA template 2. mu.L, ddH2O 19. mu.L. Amplification conditions: 3min at 94 ℃; 30s at 94 ℃, 30s at 62 ℃, 45s at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min. The PCR product was recovered by 1.5% agarose gel electrophoresis gel tapping and subjected to cloning sequencing. The sequenced CelB sequence and c.microorganisnsis interspecies sequences (sequence information see table 1) were subjected to multiple alignments using MEGA6.06 to analyze the inter-sequence differences.
TABLE 2 fluorescent RAA primers, probes
Note: FAM: 6-carboxyfluorescein; BHQ: a fluorescence quenching group; THF: tetrahydrofuran (THF)
Note：FAM：6-Carboxyfluorescein；BHQ：Black hole quencher；THF:Tetrahydrofuran；
1.1.1 primer screening and System establishment
The fluorescence RAA detection adopts a kit to recommend a reaction system and a reaction program. Reaction system: buffer A Buffer 40.9. mu.L, B Buffer 2.5. mu.L, forward primer and reverse primer each 2.0. mu.L (10mM), fluorescent probe 0.6. mu.L (10mM), DNA template 2.0. mu.L. Amplification conditions: 30s at 39 ℃ for 40 cycles.
Cmn DNA (26.1 ng/. mu.L) was extracted according to method 1.2.1 to sterilize ddH2Performing 10-fold equal gradient dilution on O, wherein the gradient concentration is 26.1, 2.6 and 2.6 multiplied by 10 respectively-1、2.6×10-2、2.6×10-3、2.6×10-4、2.6×10-5、2.6×10- 6ng/. mu.L. Randomly selecting a primer combination, respectively taking Cmn DNA with different concentrations as templates, carrying out fluorescent RAA detection, and preliminarily exploring the lowest detection concentration of the fluorescent RAA to the Cmn.
Taking the DNA with the lowest detection concentration as a template, firstly combining the upstream primers 1F, 2F, 3F and 4F with the downstream primers 1R, 2R, 3R and 4R, carrying out fluorescence RAA detection, and screening out the forward primer, the reverse primer and the primer combination with the highest amplification efficiency; secondly, combining the screened forward primers, 5F, 6F and 7F with the screened reverse primers, 5R, 6R and 7R respectively, and exploring the primer combination with the highest amplification efficiency; finally, the screened primer combinations are comparatively analyzed, and the primer combinations with good stability and high amplification efficiency are screened out.
1.1.2 measurement of sensitivity and verification of specificity
Using sterilized ddH2And O, diluting the preliminarily explored DNA with the lowest detection concentration, performing fluorescence RAA detection of the optimal primer combination, and determining the sensitivity of the fluorescence RAA detection method.
DNA of Cmm, Cmi, Pssy, Mt, Pss, Pan, Pag, Xo, Ba, Dz, Ecz was extracted according to method 1.2.1, using 11 reference strain DNAs as templates and Cmn DNAs (Cmn-1, Cmn-2) of different concentrations as positive controls, and sterilizing ddH2And O is negative control, and the fluorescence RAA detection is carried out to verify the specificity of the fluorescence RAA detection method.
1.1.3 simulation sample and real sample detection effect verification
Using sterilized ddH2And O, uniformly mixing the Cmn thalli collected at 1.2.1 to prepare a bacterial suspension. The concentration of the bacterial suspension is determined to be 5.7 multiplied by 10 by using a plate coating method5cfu/mL. Screening is carried out by GB/T36840-2018: a feeding sample (number 1-3103304) with negative detection result of the quarantine identification method of wilting disease in China in corn is ground into powder and divided into 4 parts, each of which is 10 g. Adding the prepared bacterial suspension into the groundGrinding the sample, and mixing uniformly to prepare a positive simulation sample. And (3) extracting the DNA of 4 positive simulation samples and 24 real corn samples by adopting a novel plant genome DNA extraction kit, and carrying out fluorescence RAA detection. Meanwhile, the GB/T36840-2018 detection method is used for rechecking and verification.
2 results and analysis
2.1 analysis of primer and Probe sequences
BLAST analysis results (table 1) using the Cmn strain NCPPB2581 cellulase gene sequence as a reference sequence show that: the consistency of the cellulase gene partial sequence (CelB sequence, 2525339nt-2525815nt) and the genome sequence of other subspecies of C.microganiensis is only about 70%, compared with the consistency of other cellulase gene sequences (2526816nt-2525973nt) and the genome sequence of the subspecies of C.microganiensis, the consistency is between 82.46-84.55%, the difference of the Cmn strain CelB sequence is larger, and meanwhile, the CelB sequence has high conservation among different Cmn strains, and the consistency is more than 95%. Therefore, a Cmn strain NCPPB2581 cellulase gene partial sequence CelB sequence is selected, and fluorescent RAA primers and probes are designed by taking corresponding bases 2525339nt-2525815nt as target sequences.
The primer cmn1/cmn2 is utilized to obtain a 377bp sequence through PCR amplification and clone sequencing. BLAST analysis of the sequences showed: the sequence has 100% consistency with the Cmn sequence NCPPB2581(HE614873), 61-1(CP033723), 7580(CP033722) and HF4(CP033721) and has only 70% consistency with C.microorganisensis approximate subspecies (Cmi, Cmm, Cmc, Cms). The result (fig. 8) of the comparative analysis of the sequencing target sequence and the c. microorganisnsis genome sequence in table 2 by MEGA6.06 shows that the CelB sequence (part) in the sequencing target sequence has high conservation in different strains Cmn, and has obvious gene locus difference with the closely related subspecies, and can be used as a specific sequence fragment for identifying Cmn.
Comparing the designed fluorescent RAA probe P sequence with the full database sequence through Primer-Blast, and analyzing to show that: the recognition rate and the coverage rate of the probe P on the Cmn strains NCPPB2581(HE614873), 61-1(CP033723), 7580(CP033722) and HF4(CP033721) are all 100%, and the probe P has no recognition phenomenon on other sequence data of a database; the designed forward primers 1F, 2F, 3F, 4F, 5F, 6F, 7F and reverse primers 1R, 2R, 3R, 4R, 5R, 6R, 7R combinations were aligned with the database sequences respectively, and the analysis results showed that all primer combinations recognized 100% of the Cmn strains NCPPB2581(HE614873), 61-1(CP033723), 7580(CP033722) and HF4(CP 033721). Example (c): primers 1F and 1R respectively correspond to strains NCPPB2581(HE614873) 2525335nt and 2525547nt, strains 7580(CP033722)2525691nt and 2525903nt, strains 61-1(CP033723)2525698nt and 2525910nt, strains HF4(CP033721)2526093nt and 2526305nt, the sizes of products are both 213bp, and no identification phenomenon exists on other sequence data; the primers 2F and 6R respectively correspond to strains NCPPB2581(HE614873) 2525342nt and 2525585nt, strains 7580(CP033722)2525696nt and 2525939nt, strains 61-1(CP033723)2525703nt and 2525946nt, strains HF4(CP033721)2526098nt and 2526341nt, the sizes of products are 244bp, and no recognition phenomenon exists on other sequence data.
2.2 fluorescent RAA System establishment
Selecting a primer combination 1F1R, and performing fluorescence RAA detection by using Cmn DNA with different concentrations respectively, wherein the result is as follows: the concentration is 26.1, 2.6 × 10-1、2.6×10-2、2.6×10-3、2.6×10-4The Cmn DNA of ng/. mu.L shows a typical amplification curve through fluorescent RAA detection, and the concentration is 2.6X 10-5There is no typical curve for Cmn at ng/. mu.L. Therefore, the minimum detection concentration of fluorescent RAA by the primer combination R1F1 was 2.6X 10-4ng/μL。
At a concentration of 2.6X 10-4The results of fluorescent RAA detection using ng/. mu.L of Cmn DNA as template and upstream primers 1F, 2F, 3F, 4F in combination with downstream primers R1, R2, R3, R4 (FIGS. 2 to 4) are shown: the amplification efficiency of the same reverse primer in combination with different forward primers was higher than that of the combination of primers 2F and 1F, 3F and 4F, for example: primer 2F1R fluorescence RAA 40 cycle relative fluorescence intensity Δ Rn was 339522.9, which was much greater than 1F1R (Δ Rn ═ 248390.3), 3F1R (Δ Rn ═ 153493.6), and 4F1R (Δ Rn ═ 293477.5), so: selecting 2F as an optimal forward primer; in the same forward primer and different reverse primer combinations, none of the definite reverse primers had the highest amplification efficiency in combination with the forward primer, examples: the most efficient combination with 1F is 1R (═ Δ Rn ═248390.3), the highest efficiency of combining with 2F is 3R (delta Rn-465121.7), the highest efficiency of combining with 3F is 4R (delta Rn-275858.6), the highest efficiency of combining with 4F is 3R (delta Rn-349036.5), but the amplification efficiency of the reverse primer 4R and different forward primers is the most stable, so 4R is selected as the best reverse primer; considering the amplification efficiency of the primer combination, the amplification efficiency of the 2F/3R (Delta Rn-465121.7) and the 2F/4R (Delta Rn-405603.0) primer combination is the highest.
2.3 sensitive and specific assays
Using sterilized ddH2O, Cmn DNA 2-fold equal-gradient dilution at a concentration of 2.6X 10-4 ng/. mu.L (261 fg/. mu.L) to a concentration of 130, 65 fg/. mu.L. Three concentrations of DNA were used as templates, each concentration was 5 replicates, and sterilized ddH was used2And O is used as a negative control, and the fluorescent RAA detection is carried out. The results show (fig. 6): the fluorescent RAA detection method can detect samples with the concentrations of 261 and 130 fg/muL, and 3 of 5 repetitions with the concentration of 65 fg/muL can detect only relative fluorescence intensity of 20000 after 40 repetitions, while the other 2 can not detect fluorescence signals with effective intensity, thus proving that the fluorescent RAA method can not effectively detect samples with the concentration of 65 fg/muL. Therefore, the sensitivity of the established fluorescent RAA detection method for the wilting disease in China is 130 fg/mu L, is improved by 2 times compared with the sensitivity of the fluorescent RAA combined by the primer 1F1R, and is slightly lower than the 50fg sensitivity of the LAMP detection method established in a laboratory.
Use of 2 closely related subspecies (Cmi, Cmm) and9 other strains (Pssy, Mt, Pss, Pan, Pag, Xo, Ba, Dz, Ecz) were sterilized ddH using Cmn samples Cmn-1 and Cmn-2 as positive controls, at DNA concentrations of 26.1, 1.3X 10-4 ng/. mu.L, respectively2And O is used as a negative control to verify the specificity of the fluorescent RAA detection system established by the combination of the primer 2F6R and the probe P. Results (fig. 7): the fluorescent RAA detected two Cmn samples Cmn-1, Cmn-2 with different DNA concentrations, and no other strains including 2 similar subspecies Cni, Cmm were detected. The fluorescent RAA detection system combining the primer 2F6R and the probe P can specifically detect Cmn.
In FIGS. 5 to 6, the concentrations of E0-E7 DNA were 26.1, 2.6, and 2.6X 10, respectively-1、2.6×10-2、2.6×10-3、2.6×10-4、2.6×10-5、2.6×10-6ng/. mu.L, N negative control. 1-11 in FIG. 7, maize bacterial wilt pathogen, Pseudomonas syringae tomato pathogenic variety, Microbacterium rubrum, Clavibacter michiganensis rog 35890, Clavibacter michiganensis, Pantoea agglomerans, Pantoea ananatis, Xanthomonas, Burkholderia, Dicksia zeae, Erwinia europaea; n: sterile water; cmn-1: cmn genome at a concentration of 26.1 ng/. mu.L; cmn-2: the concentration was 1.3X 10-4 ng/. mu.L Cmn genome.
2.4 simulation of sample and real sample detection effects
Extracting DNA from the prepared maize interior state wilting positive simulation sample (4 parts) and the prepared maize entrance situation sample (24 parts) by using a novel plant genome DNA extraction kit, performing fluorescence RAA detection, and simultaneously performing fluorescence RAA detection by using GB/T36840-: and (5) verifying a quarantine identification method of wilting bacteria in China in corn. The results show that 4 simulation samples are detected by both methods, 24 entry corn samples are not detected by both methods, and the detection rate is 100%. The newly established fluorescent RAA detection method for the wilting disease in the interior of the corn can be used for detecting corn seed samples.
3 compared with the LAMP detection method, the fluorescent RAA detection method for the wilting disease in China built in the corn has the advantages of constant temperature, sensitivity and specificity, is simpler and faster, only one pair of primers is needed for RAA, the amplification of a specific fragment can be realized within 20min, and the product is a single strip. By junctions with fluorescent probesIn addition, the RAA technology can realize dynamic monitoring of the amplification process and carry out quantitative detection. The invention tries to use template DNA 10 times gradient diluent to carry out fluorescence RAA quantitative detection research, and takes the concentration of template LOG as abscissa and relative fluorescence intensity delta Rn>A standard curve was established for the ordinate at 20000 cycles, showing: when the sample concentration is selected to be 26.1, 2.6X 10-1ng/. mu.l of data was fitted to a standard curve, which was found to be-3.5 x +21.792(R2 is 0.9932), but with decreasing concentration of the selected samples, the confidence of the fitted standard curve was R2<0.95, for analytical reasons or in relation to insufficient uniformity of dilution of the template gradient, it was necessary to prepare standard plasmids for fluorescent RAA quantitative detection studies.
The invention utilizes RAA amplification technology to detect the wilting disease in the corn, and designs forward primers 1F, 2F, 3F, 4F, 5F, 6F and 7F, reverse primers 1R, 2R, 3R, 4R, 5R, 6R and 7R and a probe P according to the RAA principle. The fluorescence RAA detection experiment is carried out by utilizing different primer combinations, and the following results are found: although the primer combinations attempted were all amplifiable, the difference in amplification efficiency was significant (fig. 2-4). For analytical reasons, or in connection with the RAA principle of primer binding to recombinase proteins as dimers, different primer sequences bind to recombinase proteins with different efficiencies. Therefore, it is necessary to further study the binding mechanism between the primer and the recombinase protein in the RAA to guide the primer design and improve the efficiency.
4 results
The invention designs primers and probes aiming at CelB sequence segments of the wilting bacteria in China, and establishes a fluorescent RAA detection method for the wilting bacteria in China through experiments such as primer screening, specificity verification, sensitivity determination and the like. The method has the advantages of quick reaction, high specificity and high sensitivity up to 130 fg/mu L, takes only 20min, and passes the detection verification of actual samples and simulated samples. The fluorescent RAA detection method is rapid, specific, sensitive and simple, meets the requirements of accurate and rapid field detection, and can provide a new idea for effectively monitoring the wilting germs in the corn.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Sequence listing
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Claims (10)
1. A fluorescence recombinase-mediated isothermal amplification detection kit for wilting disease in China, which is characterized by comprising:
forward primer SEQ ID NO: 1; reverse primer SEQ ID NO: 2; and probe SEQ ID NO: 3; the target sequence is SEQ ID NO: 4.
2. a method for constructing the fluorescent recombinase-mediated isothermal amplification detection kit for wilting disease in maize according to claim 1, wherein the method for constructing the fluorescent recombinase-mediated isothermal amplification detection kit for wilting disease in maize comprises:
activating a strain by using an NA culture medium, collecting thalli by using an inoculating loop scraper, extracting bacterial DNA, verifying the extraction effect by using a nucleic acid tester, and storing at-20 ℃ for later use;
designing and synthesizing a primer probe, and screening and establishing a system; using sterilized ddH2O, diluting the determined DNA with the lowest detection concentration, performing fluorescence RAA detection of the optimal primer combination, and determining the sensitivity of the fluorescence RAA detection method;
step three, verifying the specificity of the fluorescence RAA detection method; and (5) verifying the detection effects of the simulated sample and the real sample to obtain the fluorescent recombinase-mediated isothermal amplification detection kit for the wilting disease in the state of corn.
3. The method according to claim 2, wherein the activating the bacterial strain comprises: activation was carried out at 28 ℃ for 48 h.
4. The method for constructing a fluorescence recombinase-mediated isothermal amplification detection kit for wilting disease in maize according to claim 2, wherein the designing and synthesizing of the primer probe in step two comprises:
(1) analyzing the difference of cellulase gene sequences among C.microganiensis subspecies closely, and selecting a CelB sequence as a template to design a primer and a probe;
(2) designing primers Cmn1, Cmn2 to amplify target sequences with reference to Genbank sequence HE614873, aligning the Cmn1/Cmn2 sequence with the full database sequence using BLAST, determining if the primer combination recognizes the Cmn strain 100% and does not match other sequence data;
(3) if the primer combination identifies the Cmn strain 100% and is not matched with other sequence data, the Cmn DNA is used as a template, a target sequence is amplified by using a primer Cmn1/Cmn2, and a PCR product is subjected to gel cutting recovery through 1.5% agarose gel electrophoresis and is subjected to clone sequencing;
(4) with reference to the RAA principle, forward primers 1F, 2F, 3F, 4F, 5F, 6F, 7F, reverse primers 1R, 2R, 3R, 4R, 5R, 6R, 7R and probe P were designed.
5. The method for constructing the fluorescence recombinase-mediated isothermal amplification detection kit for wilting disease in maize according to claim 4, wherein in the step (3), the amplification comprises:
the reaction system is a 50-mu-L system and comprises: 2 × Taq PCR StarMix with Loading Dye 25. mu.L, 10. mu.M (cmn1/cmn2) 2. mu.L, DNA template 2. mu.L, ddH2O 19μL；
The amplification condition is 94 ℃ for 3 min; 30s at 94 ℃, 30s at 62 ℃, 45s at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min.
6. The method for constructing the fluorescence recombinase-mediated isothermal amplification detection kit for wilting disease in maize according to claim 2, wherein the primer screening in step two comprises:
cmn DNA extraction to sterilize ddH2Performing 10-fold equal gradient dilution on O, randomly selecting a primer combination, performing fluorescent RAA detection by respectively taking Cmn DNAs with different concentrations as templates, and determining the lowest detection concentration of the fluorescent RAA to the Cmn; taking the DNA with the lowest detection concentration as a template, firstly combining the upstream primers 1F, 2F, 3F and 4F with the downstream primers 1R, 2R, 3R and 4R, carrying out fluorescence RAA detection, and screening out the forward primer, the reverse primer and the primer combination with the highest amplification efficiency; secondly, combining the screened forward primers, 5F, 6F and 7F with the screened reverse primers, 5R, 6R and 7R respectively, and exploring the primer combination with the highest amplification efficiency; finally, the screened primer combinations are comparatively analyzed, and the primer combinations with good stability and high amplification efficiency are screened out.
7. The method of claim 6, wherein the gradient concentrations are 26.1, 2.6, and 2.6 × 10 respectively-1、2.6×10-2、2.6×10-3、2.6×10-4、2.6×10-5、2.6×10-6ng/μL。
8. The method for constructing a fluorescence recombinase-mediated isothermal amplification detection kit for wilting disease in maize according to claim 2, wherein the system establishment comprises:
reaction system: buffer ABuffer40.9 μ L, B Buffer 2.5 μ L, upstream primer and downstream primer each 2.0 μ L (10mM), fluorescent probe 0.6 μ L (10mM), DNA template 2.0 μ L;
amplification conditions: 30s at 39 ℃ for 40 cycles.
9. The method for constructing the fluorescence recombinase-mediated isothermal amplification detection kit for wilting disease in maize according to claim 2, wherein in step three, the verifying the specificity of the fluorescence RAA detection method comprises:
extracting DNA of Cmm, Cmi, Pssy, Mt, Pss, Pan, Pag, Xo, Ba, Dz and Ecz to obtain 11 ginseng strainsTest strain DNA as template, Cmn DNA of different concentrations as positive control, sterilized ddH2Performing fluorescent RAA detection by taking O as negative control, and verifying the specificity of the fluorescent RAA detection method;
in the third step, the verification of the detection effect of the simulated sample and the real sample comprises the following steps:
1) using sterilized ddH2O, uniformly mixing the Cmn thalli collected by 1.2.1 to prepare a bacterial suspension; the concentration of the bacterial suspension is determined to be 5.7 multiplied by 10 by using a plate coating method5cfu/mL；
2) Screening a feeding sample with a negative test result by a quarantine identification method for wilting disease in China in corn, grinding the feeding sample into powder and dividing the powder into 4 parts, wherein each part is 10 g;
3) adding the prepared bacterial suspension into a ground sample, and uniformly mixing to prepare a positive simulation sample;
4) extracting the DNA of 4 positive simulation samples and 24 real corn samples by adopting a novel plant genome DNA extraction kit, and carrying out fluorescence RAA detection; meanwhile, the quarantine identification method of the wilting disease in the interior of corn is used for recheck and verification.
10. Use of the fluorescent recombinase-mediated isothermal amplification detection kit for wilting disease in maize according to claim 1 in detection of wilting disease in maize.
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| CN106687604A (en) * | 2014-09-11 | 2017-05-17 | 阿格洛法士公司 | Methods for pathogen detection and disease management on meats, plants, or plant parts |
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