CN111471795A - Primer, probe, kit and method for RT-QPCR (reverse transcription-quantitative polymerase chain reaction) detection of fusarium avenaceum - Google Patents
Primer, probe, kit and method for RT-QPCR (reverse transcription-quantitative polymerase chain reaction) detection of fusarium avenaceum Download PDFInfo
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Abstract
The invention discloses a primer, a probe, a kit and a method for RT-QPCR detection of fusarium avenae, belonging to the technical field of biology. The nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3; the kit comprises the primer and the probe. The fluorescent quantitative PCR detection method comprises the following steps: extracting total DNA of a sample to be detected; preparing a reaction system; diluting the template in a gradient manner to prepare a standard curve sample and a positive control sample; performing fluorescent quantitative PCR amplification on a sample to be detected, a standard curve sample, a positive control sample and a negative control sample by using a primer and a probe; and drawing a standard curve and calculating a result. The primer, the probe and the kit have high specificity and good sensitivity, and can be used for quickly and accurately detecting the fusarium avenae.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer, a probe, a kit and a method for real-time fluorescent quantitative PCR detection of fusarium avenae.
Background
Fusarium avenae (Fusarium avenaceum) is a kind of worldwide distributed fungus, and can infect grain crops such as wheat, oat, soybean and the like and medicinal flowers such as lily, chrysanthemum and the like. Fusarium avenaceum often causes a plurality of disease symptoms of root rot, stem base rot, flower rot and the like of plants, and the number of infected host plants is more than 100. Fusarium avenae often infects a vascular bundle system of a host plant, damages a dredging tissue of the plant, generates toxin in a metabolic process of plant growth and development to damage crops, causes plant wilting death, and affects yield and quality. Fusarium avenaceum is one of the most difficult important diseases to control in production. For many years, the polymorphism of the strain is researched according to tests such as morphological characteristics, pathogenicity and nutrient fusion groups, and the methods are time-consuming and labor-consuming and cannot accurately reveal the evolutionary relationship of the species.
In the prior art, although the oat sickle is detected by adopting multiple PCR (polymerase chain reaction) with good specificity and high accuracy, the PCR result needs to be analyzed by agarose electrophoresis, and the influence factors are more. With the continuous development of molecular biology technology, RT-QPCR (real-time fluorescence quantitative PCR) is widely applied to the detection of strains at present. Compared with the conventional isolated culture method, the method has the characteristics of short time consumption, simple and convenient operation and good specificity, the result can be directly observed, and the pollution caused in the operation process can be effectively avoided. However, Fusarium avenaceum has high homology with other Fusarium species, such as Fusarium solani, Fusarium triloba and Fusarium oxysporum. Therefore, the PCR detection method for fusarium avenaceum is provided, has high specificity and high accuracy, and becomes a problem to be solved by the technical personnel in the field.
Disclosure of Invention
One of the purposes of the invention is to provide a primer for RT-QPCR detection of fusarium avenaceum, which has good specificity and high accuracy.
The invention also aims to provide a probe for RT-QPCR detection of fusarium avenaceum.
The invention also aims to provide a kit for RT-QPCR detection of fusarium avenaceum.
The fourth purpose of the invention is to provide a method for detecting the RT-QPCR of the fusarium avenaceum.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
according to the invention, a bioinformatics comparison analysis is carried out on the complete sequence of the Fusarium avenae 18SrRNA gene in an NCBI database, a conservative fragment sequence suitable for designing a Primer and a probe is selected as a target, and a Primer express3 software, a Primer Premier 5 software and an Oligo 7 software are further applied to design a plurality of groups of real-time fluorescence quantitative PCR primers and probes, and a group of fluorescence quantitative PCR primers and probes for detecting the Fusarium avenae are finally determined through preliminary screening of tests.
The primer for RT-QPCR detection of fusarium avenaceum comprises an upstream primer and a downstream primer, the nucleotide sequences of which are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2,
an upstream primer: 5'-ATCGATGAAGAACGCAGCAA-3', respectively;
a downstream primer: 5'-CGATCCCCAACACCAAACCC-3' are provided.
The nucleotide sequence of the probe for detecting the RT-QPCR of the fusarium avenaceum is shown as SEQ ID NO. 3: 5'-CCTGTTCGAGCGTCATTTCAACCCT-3', respectively; preferably, the fluorescent reporter group marked at the 5 'end of the probe is VIC, and the fluorescent quencher group marked at the 3' end of the probe is BHQ.
The 18SrRNA gene is widely present in fusarium avenae and has high conservation. The invention adopts fusarium avenaceum 18SrRNA gene as a target sequence to synthesize a primer and a probe.
The invention relates to a primer and probe combination for real-time fluorescent quantitative PCR detection of fusarium avenae, which comprises the following components:
an upstream primer: 5'-ATCGATGAAGAACGCAGCAA-3', respectively;
a downstream primer: 5'-CGATCCCCAACACCAAACCC-3', respectively;
and (3) probe: 5'-CCTGTTCGAGCGTCATTTCAACCCT-3' are provided.
The kit for RT-QPCR detection of fusarium avenae comprises the primer and the probe.
In the technical scheme of the invention, the kit also comprises a QPCR template, wherein the QPCR template is shown as SEQ ID NO. 4; preferably, the template is in the form of a plasmid.
The nucleotide sequence synthesized by the corresponding plasmid of the primer and probe amplification fragment is shown as SEQ ID NO. 5, and specifically comprises the following steps:
5’-AACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACAT TGCGCCCGCTGGTATTCCGGCGGGCATGCCTGTTCGAGCGTCATTTCAACC CTCAAGCCCCCGGGTTTGGTGTTGGGGATCGGCTCTGCCTTCTGGCGGTGC CGCCCCC-3’。
in embodiments of the invention, the kit further comprises a negative sample; preferably, the negative control sample is ddH2O。
In the embodiment of the invention, the premix is also included, and preferably, the premix is 2 × Probe Mix.
The invention relates to a reaction system for RT-QPCR detection of fusarium avenaceum, which comprises the following components:
an upstream primer: 5'-ATCGATGAAGAACGCAGCAA-3', respectively;
a downstream primer: 5'-CGATCCCCAACACCAAACCC-3', respectively;
and (3) probe: 5'-CCTGTTCGAGCGTCATTTCAACCCT-3', respectively;
template: as shown in SEQ ID NO. 4;
preferably, the kit further comprises a negative control sample, and further preferably, the negative control sample is ddH2O。
The RT-QPCR detection method for detecting the plant fusarium avenaceum comprises the following steps of:
preferably, in the step 3, the concentration of the standard curve sample prepared by the gradient dilution is 3.1 × 10 respectively9copies/μL、3.1×108copies/μL、3.1×107copies/μL、3.1×106copies/μL、 3.1×105copies/μL、3.1×104copies/μL、3.1×103copies/μL、3.1×102copies/μL 3.1×101copies/μL、3.1×10-1copies/μL、3.1×10-2copies/μL；
Preferably, the concentration of the positive control sample is 3.1 × 1010copies/μL。
In the examples of the present invention, the reaction conditions of PCR were: digestion of the contamination at 37 ℃ for 2min, pre-denaturation at 95 ℃ for 10min, at 95 ℃ for 15s and 60 ℃ for 1min and collection of the fluorescence signal for 40 cycles.
As an embodiment of the present invention, the PCR reaction system is:
in the technical scheme, the Fusarium avenaceum 18SrRNA gene is amplified by adopting a PCR technology, is connected into a plasmid vector PUC57 by utilizing a gene recombination technology to construct a recombinant plasmid PUC57-18SrRNA, is subjected to corresponding PCR identification and sequencing identification, and is finally quantitatively used as an 18SrRNA gene standard product of the method.
The preparation method of the 18SrRNA gene standard substance comprises the following steps:
s1, extracting fusarium avenaceum (standard strain) genome DNA to obtain a DNA sample which is used as a template for PCR amplification of 18SrRNA genes;
PCR amplification of S2.18SrRNA Gene fragment: selecting an amplification sequence, designing a PCR primer and a probe,
s3, carrying out PCR amplification by taking the DNA sample obtained in the step S1 as a template; then purifying the obtained PCR amplification product;
s4, connecting the purified PCR amplification product obtained in the step S3 with a plasmid vector PUC 57; then transforming the ligation product to obtain a colony transformed with the plasmid;
s5, selecting a monoclonal colony, inoculating the colony in a culture solution, and culturing; the plasmid preparation kit is used for extracting positive recombinant plasmid PUC57-18SrRNA for PCR identification and sequencing analysis of bacterial liquid.
Compared with the prior art, the invention has the following beneficial effects:
the invention selects fusarium avenae 18SrRNA gene containing highly conserved and specific sequence to construct recombinant plasmid PUC57-18SrRNA as standard substance; and further screening fluorescent quantitative PCR primers and probes for detecting fusarium avenae. The primer, the probe and the kit for the real-time fluorescent quantitative PCR detection of the fusarium avenae have high specificity and good sensitivity, and can be used for quickly and accurately detecting the fusarium avenae.
Drawings
FIG. 1 is a diagram showing the results of blast alignment in NCBI database of 18SrRNA gene amplification sequences.
FIG. 2 is a diagram showing the results of Primer-Blast comparison of upstream and downstream Primer sequences in NCBI.
FIG. 3 is a graph showing a standard curve of the standard.
FIG. 4 is a graph showing the results of the sensitivity test of the present invention, wherein a, b, c, d, e, f, g, h, i, j, and k are relative fluorescence curves of different plasmid concentrations, wherein a represents a plasmid concentration of 3.1 × 109copies/. mu. L, b represents plasmid concentration of 3.1 × 108copies/. mu. L, c represents plasmid concentration 3.1 × 107copies/. mu. L, d represents plasmid concentration of 3.1 × 106copies/. mu. L, e represents a plasmid concentration of 3.1 × 105copies/. mu. L, f represents plasmid concentration of 3.1 × 104copies/. mu. L, g represents a plasmid concentration of 3.1 × 103copies/. mu. L, h represents a plasmid concentration of 3.1 × 102copies/. mu. L, i represents a plasmid concentration of 3.1 × 101copies/. mu. L, j represents a plasmid concentration of 3.1 × 10-1copies/. mu. L, k represents plasmid concentration of 3.1 × 10-2copies/. mu. L and negative control.
FIG. 5 is a diagram showing the results of the specificity test of the present invention.
FIG. 6 is a gel image verification chart of the specificity experiment of the present invention.
Detailed Description
The present invention is further illustrated by the following examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make certain insubstantial modifications and adaptations of the present invention based on the above disclosure and still fall within the scope of the present invention.
Example 1
This example discloses a method for preparing the 18s rrna gene standard of the present invention.
To establish a real-time fluorescent quantitative PCR method, an external standard substance required by the method must be prepared, and the standard substance should contain a highly conserved and specific sequence to ensure high specificity of the reaction. The 18SrRNA gene is widely present in fusarium avenae and has high conservation. The invention adopts fusarium avenaceum 18SrRNA gene as a target sequence. In the embodiment, the fusarium avenaceum 18SrRNA gene is amplified mainly by adopting a PCR technology, is connected into a plasmid vector PUC57 by utilizing a gene recombination technology to construct a recombinant plasmid PUC57-18SrRNA, is subjected to corresponding PCR identification and sequencing identification, and is finally quantitatively used as a standard product of a method to be established, so that a foundation is laid for the next method and evaluation.
First, preparation of template DNA
The genomic DNA of fusarium avenaceum (standard strain) is extracted and used as a template for PCR amplification of the 18SrRNA gene. The fungus genome extraction kit produced by Beijing Baitach company is adopted for extraction, and the specific extraction method is as follows:
① adding 1ml of the bacterial suspension into a 1.5ml centrifuge tube, centrifuging at 8000r/min for 2 minutes, and removing the supernatant;
② adding preheated Buffer FP1 at 550 mu L65 ℃ and RNaseA at 4 mu L ℃, mixing the mixed bacterial liquid precipitates by violent vortex oscillation, putting the mixed bacterial liquid precipitates into a water bath at 65 ℃ for 1h after mixing, and performing violent vortex oscillation for 5-6 times in the period;
③ adding 130 mu L Buffer P2, mixing well, centrifuging at 12000rmp for 3 min;
④ carefully pipette the supernatant onto a column A, carefully avoid pipetting the interfacial material, centrifuge at 12000rmp for 1min, and collect the supernatant;
⑤ adding 1.5 times volume of Buffer P3, immediately and gently vortexing, and mixing well;
⑥ adding the mixture obtained in the previous step into an adsorption column AC, centrifuging at 12000rmp for 1min, and removing waste liquid in the collection tube;
⑦ adding 700 μ L rinsing liquid WB, centrifuging at 12000rmp for 1min, and discarding the waste liquid;
⑧ adding 500 μ L of rinsing liquid WB, centrifuging at 12000rmp for 1min, and discarding the waste liquid;
⑨ placing the adsorption column AC back into the empty collection tube, centrifuging at 12000rmp for 3-5 min;
⑩ the adsorption column AC is taken out and put into a clean centrifuge tube, 50 μ L elution buffer EB (preheated in 65-70 deg.C water bath) is added in the middle of the adsorption film, and the mixture is placed at room temperature for 3-5min and then centrifuged at 12000rmp for 1min to collect DNA.
PCR amplification of 18S rRNA gene fragment
1. Design and Synthesis of primers
The 18SrRNA identification is to identify the species of the fungi by utilizing a method for sequencing the 18SrRNA sequence of the fungi. Is a method for rapidly obtaining the fungal species information. The invention utilizes real-time fluorescent quantitative PCR to detect the expression level of the 18SrRNA gene of the fusarium avenaceum, and the significance and specificity of the 18SrRNA in the detection of the fusarium avenaceum are determined.
According to the invention, a bioinformatics comparison analysis is carried out on the complete sequence of the Fusarium avenae 18SrRNA gene in an NCBI database, a conservative fragment sequence suitable for designing a Primer and a probe is selected as a target, and a group of real-time fluorescence quantitative PCR primers and probes and a group of peripheral primers of related sequences are designed by applying Primer express3 software, Primer Premier 5 software and Beacon Designer 7 software.
The amplification sequence SEQ ID NO. 4 selected by the invention is shown as follows:
>FR682079.1Fusarium avenaceum genomic DNA containing 18S rRNA gene,ITS1,5.8S rRNA gene,ITS2 and 28S rRNA gene,isolate 1
5’-GNAGACCAACCCCTGGNNACATACCTTAATGTTGCCTCGGCGGATCAG CCCGCGCCCCGTAAAACGGGACGGCCCGCCAGAGGACCCAAACTCTAATG TTTCTTATTGTAACTTCTGAGTAAAACAAACAAATAAATCAAAACTTTCAA CAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGAT AAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCAC ATTGCGCCCGCTGGTATTCCGGCGGGCATGCCTGTTCGAGCGTCATTTCAA CCCTCAAGCCCCCGGGTTTGGTGTTGGGGATCGGCTCTGCCTTCTGGCGGT GCCGCCCCCGAAATACATTGGCGGTCTCGCTGCAGCCTCCATTGCGTAGT AGCTAACACCTCGCAACTGGAACGCGGCGCGGCCATGCCGTAAAACCCCA ACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAA-3’。
this sequence is shown in FIG. 1 by blast results against the NCBI database.
FIG. 1 shows the results of the blast alignment in the NCBI database.
As can be seen from fig. 1: the sequence has specificity to fusarium avenaceum.
The peripheral primer sequences are shown as SEQ ID NO. 6 and SEQ ID NO. 7, and specifically comprise:
an upstream primer: fusarium avenaceum-136F 5'-ATCAAAACTTTCAACAACGG-3';
a downstream primer: fusarium avenaceum-404R 5'-GCTACTACGCAATGGAGGC-3'.
The amplified fragment size was: 269 bp.
2. PCR reaction system and reaction conditions
The PCR amplification is carried out by taking DNA as a template and the peripheral primer Fusarium avenaceum-136F/Fusarium avenaceum-404R as an amplification primer and adopting the following system and reaction conditions.
And (3) PCR system:
wherein the primer adopts Fusarium avenaceum-136F/Fusarium avenaceum-404R, the Taq enzyme adopts Beijing Baitak Power Taq Plus DNA polymerase, and the PCR amplification instrument is an ASA-4800PCR instrument of Suzhou Baiyuan gene technology Limited.
Amplification procedure/reaction conditions:
pre-denaturation at 94 deg.C for 5min, pre-denaturation at 94 deg.C for 30s, denaturation at 50 deg.C for 30s, and denaturation at 72 deg.C for 1min for 30 cycles, and electrophoresis of 5 μ L amplification product at 72 deg.C for 10min to detect PCR product size, and purifying and recovering residual PCR amplification product with DNA gel recovery kit from Shanghai's chemical company.
Construction and transformation of recombinant plasmid PUC57-18SrRNA
1. And (3) connection reaction: the PCR amplification product obtained by the above purification was ligated with PUC57 (Biotechnology Co., Ltd., Wuhan King Kerui), and prepared using the following ligation system:
after the preparation was completed, the ligation reaction was carried out overnight at 16 ℃.
2. Transformation and PCR identification of PUC57-18SrRNA plasmid
① taking out the frozen DH5 α competent cells from an ultra-low temperature refrigerator at-70 ℃, and placing the cells on an ice box to naturally thaw the cells;
② adding the ligation product 10 μ L into DH5 α competent cells 100 μ L;
performing heat shock in water bath at ③ 42 deg.C for 90s, immediately cooling on ice for 30 min;
④ A1.5 ml EP tube was mixed with a pre-cooled liquid medium L B (containing no ampicillin) of 800. mu. L, and incubated at 37 ℃ with gentle shaking at 140rpm for 1 hour;
⑤ centrifuging the above culture solution at 8000rpm for 1min, discarding supernatant, coating the cell suspension absorption residue on L B plate containing 0.1ngAmp, standing with front side upward for 30min, and inverting the culture dish to culture in 37 deg.C incubator overnight after the bacteria solution is completely absorbed by the culture medium;
⑥ the next day, picking single colony from the plate, culturing in 100 μ L L B liquid medium (containing ampicillin) PCR tube under shaking at 37 deg.C for 2-3 hr, absorbing 2 μ L as template for PCR identification, adding the residual bacterial liquid into 20ml L B liquid medium for amplification;
⑦ the diluted bacterial liquid is amplified by Fusarium avenaceum-1F/Fusarium avenaceum-1R specific primer, PCR product adopts 1% agarose gel electrophoresis, positive transformant is identified by detecting PCR product size.
The sequence of the primer Fusarium avenaceum-1F/Fusarium avenaceum-1R is as follows:
an upstream primer; fusarium avenaceum-1F 5'-ATCGATGAAGAACGCAGCAA-3'
A downstream primer; fusarium avenaceum-1R 5'-CGATCCCCAACACCAAACCC-3'
The amplified fragment size was: 162 bp.
The results of Primer-Blast comparison of the upstream and downstream Primer sequences in NCBI are shown in FIG. 2
FIG. 2 shows the results of Primer-Blast alignment of upstream and downstream Primer sequences in NCBI.
As can be seen from fig. 2: the pair of primers has specificity to fusarium avenaceum.
The PCR reaction system is as follows:
amplification procedure/reaction conditions: pre-denaturation at 94 ℃ for 5 min; 30 cycles of 94 ℃ for 30s, 67 ℃ for 30s and 72 ℃ for 20 s; 5min at 72 ℃.
A plasmid preparation kit produced by Baitach company is adopted to extract positive recombinant plasmid PUC57-18SrRNA, the concentration and the purity are determined, meanwhile, a part of purified plasmid is sucked and sent to Shanghai bioengineering limited company for sequencing, and the gene sequence of the insert fragment is determined to be consistent with the target sequence.
Fourth, obtaining and quantifying standard substance
1. E.coli DH5 α 100 mu L containing the recombinant PUC57-18SrRNA obtained in the step three is taken to be transferred into L B liquid culture medium with the thickness of 5m L and is shaken overnight at the temperature of 37 ℃ and the rpm of 200;
2. transferring 1ml of overnight-cultured bacterial liquid into 10ml of L B liquid culture medium, performing enrichment culture at 200rpm for 2-3 hours, and extracting plasmids by using a plasmid preparation kit produced by Beijing Baitaike company;
3. the extracted plasmid was measured by an ultramicro UV-visible spectrophotometer (ND5000) of Baitach Biotechnology Ltd, Beijing, and measurement A was carried out260、A280According to A260/A280Judging the purity of the plasmid;
4. calculation of the concentration (copy number) of the plasmid PUC57-18SrRNA
(1) Molecular weight of the plasmid 2920bp × 660 (average molecular weight per base pair)
(2) The plasmid concentration was found to be 100 ng/. mu. L, plasmid purity A260/A280When real-time fluorescence quantitative PCR is performed, it is necessary to convert the unit to copies/. mu. L because the unit must be "copy number".
Plasmid copies/. mu. L ═ avogalois constant × moles of plasmid
Wherein the Avogastron constant is 6.02 × 1023copies/mol。
The plasmid thus extracted had a concentration copies/. mu. L ═ 100 × 10-9ng/μL×6.02×1023copies/mol÷(2920bp×660g/bp·mol)＝3.1×1010copies/μL
10 u L plasmid and 90 u L sterile water to get the concentration of 3.1 × 109Plasmid copies/. mu. L, which were diluted 10-fold to obtain a range of concentrations of plasmid, and stored at-20 ℃ until use.
Example 2
The embodiment discloses a fluorescent quantitative PCR kit, which comprises the following components:
The upstream primer with the final concentration of 10 mu M and the downstream primer with the final concentration of 10 mu M;
probe at a final concentration of 10. mu.M;
the recombinant plasmid PUC57-18SrRNA gene series concentration standard prepared in example 1 (series concentration of the series concentration standard: 3.1 × 10)9copies/μL、3.1×108copies/μL、3.1×107copies/μL、 3.1×106copies/μL、3.1×105copies/μL、3.1×104copies/μL、3.1×103copies/μL、 3.1×102copies/μL、3.1×101copies/μL、3.1×10-1copies/μL、3.1×10-2copies/μL)；
Positive control, concentration 3.1 × 1010copies/. mu. L of the recombinant plasmid pUC57-18SrRNA prepared in example 1;
ddH2O，ddH2o was used as a reagent and negative control.
An upstream primer: fusarium avenaceum-1F 5'-ATCGATGAAGAACGCAGCAA-3';
a downstream primer: fusarium avenaceum-1R 5'-CGATCCCCAACACCAAACCC-3';
and (3) probe: 18SrRNA 1-15 '-CCTGTTCGAGCGTCATTTCAACCCT-3'.
The fluorescent reporter group marked at the 5 'end of the probe is VIC, and the fluorescent quencher group marked at the 3' end of the probe is BHQ.
The nucleotide sequence synthesized by the corresponding plasmid of the primer and probe amplification fragment is shown as SEQ ID NO. 5, and specifically comprises the following steps:
5’-AACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGAT AAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACAT TGCGCCCGCTGGTATTCCGGCGGGCATGCCTGTTCGAGCGTCATTTCAACC CTCAAGCCCCCGGGTTTGGTGTTGGGGATCGGCTCTGCCTTCTGGCGGTGC CGCCCCC-3’。
the use method of the kit comprises the following steps:
1) extracting total DNA of a sample to be detected;
2) performing fluorescent quantitative PCR amplification on a sample to be detected, a standard sample with a series of concentrations, a positive control sample and a negative control sample by using the primers and the probes;
3) and drawing a standard curve, and calculating a result through the standard curve and the Ct value of the sample to be detected.
Example 3
This example discloses the use of the kit of example 2 to plot a standard curve for a standard.
Using 18SrRNA gene series concentration standard substance (series concentration of series concentration standard substance is 3.1 × 10)9copies/μL、3.1×108copies/μL、3.1×107copies/μL、3.1×106copies/μL、 3.1×105copies/μL、3.1×104copies/μL、3.1×103copies/μL、3.1×102copies/μL、 3.1×101copies/μL、3.1×10-1copies/μL、3.1×10-2copies/μ L) as a template, and performing fluorescent quantitative PCR amplification using primers and probes in the kit, while setting a positive control and a negative control.
Positive control, concentration 3.1 × 1010copies/. mu. L of the recombinant plasmid pUC57-18SrRNA prepared in example 1;
negative control: ddH2O。
The PCR reaction system is as follows:
reaction conditions are as follows: digestion of the contamination at 37 ℃ for 2min, pre-denaturation at 95 ℃ for 10min, at 95 ℃ for 15s and 60 ℃ for 1min and collection of the fluorescence signal for 40 cycles.
Method for obtaining standard curve: and taking the logarithm of the plasmid concentration of the standard substance as an abscissa and the ct value as an ordinate to obtain a standard curve. The original equation of the standard curve is Y ═ ax + b, the equation of the standard curve at this time is Y ═ -4.472x +49.93, and the standard curve graph is shown in the attached figure 3.
As can be seen from FIG. 3, the standard curve of the standard sample is smooth, and the correlation coefficient is high, specifically R2And (3) 0.988, which meets the requirement of real-time fluorescent quantitative PCR detection.
Example 4
This example discloses the performance test of the real-time fluorescent quantitative PCR kit of the invention
1. Sensitivity test
The plasmids prepared in example 1 were diluted 10-fold to obtain a series of concentrations of plasmids, and the concentration was 3.1 × 109copies/μL、3.1×108copies/μL、3.1×107copies/μL、3.1×106copies/μL、 3.1×105copies/μL、3.1×104copies/μL、3.1×103copies/μL、3.1×102copies/μL、 3.1×101copies/μL、3.1×10-1copies/μL、3.1×10-2copies/. mu. L and negative control served as templates.
The reaction system is as follows:
reaction conditions are as follows: digestion of the contamination at 37 ℃ for 2min, pre-denaturation at 95 ℃ for 10min, at 95 ℃ for 15s and 60 ℃ for 1min and collection of the fluorescence signal for 40 cycles.
The fluorescence curve obtained by processing the fluorescence signal detected by the instrument with software, and observing the signal of the fluorescence curve, the data show that when the plasmid concentration reaches 3.1 × 10-1Fluorescence signal is still present at copies/μ L, whenThe concentration of the plasmid reaches 3.1 × 10-2No fluorescence signal at copies/. mu. L, the sensitivity of the method of the invention is therefore 3.1 × 10- 1copies/μL。
FIG. 4 shows the results of the sensitivity test of the present invention, wherein a represents the plasmid concentrations of 3.1 × 109copies/. mu. L, b represents plasmid concentration of 3.1 × 108copies/. mu. L, c represents plasmid concentration 3.1 × 107copies/. mu. L, d represents plasmid concentration of 3.1 × 106copies/. mu. L, e represents a plasmid concentration of 3.1 × 105copies/. mu. L, f represents plasmid concentration of 3.1 × 104copies/. mu. L, g represents a plasmid concentration of 3.1 × 103copies/. mu. L, h represents a plasmid concentration of 3.1 × 102copies/. mu. L, i represents a plasmid concentration of 3.1 × 101copies/. mu. L, j represents a plasmid concentration of 3.1 × 10-1copies/. mu. L, k represents plasmid concentration of 3.1 × 10-2copies/. mu. L and negative control.
2. Experiment of specificity
In order to confirm the specificity of the invention for detecting fusarium avenaceum, other common clinical infection microorganism specificity experiments are selected, and the selected microorganisms comprise: fusarium graminearum, Alternaria alternata, Fusarium oxysporum, Fusarium sporotrichioides, Botrytis cinerea and Fusarium solani.
The specificity test includes a test using the genomic DNA of the above-mentioned sample as a template. The DNA extraction of the microorganisms adopts a DNA rapid extraction kit of Beijing Baitach biotechnology limited and adopts an AceQ U + Probe Master Mix real-time fluorescent quantitative PCR kit produced by Nanjing NuoZan biotechnology limited to carry out experiments, and the reaction system is as follows:
the primer and the probe are the primer and the probe in the embodiment 2 of the invention.
Reaction conditions are as follows: digestion of the contamination at 37 ℃ for 2min, pre-denaturation at 95 ℃ for 10min, at 95 ℃ for 15s, at 60 ℃ for 40s, at 72 ℃ for 20s and collection of the fluorescence signal for 40 cycles.
And processing the fluorescence signal detected by the instrument by software to obtain a fluorescence curve, observing the signal of the fluorescence curve, and analyzing the specificity. The results refer to fig. 5: the genome DNA is used as a template, only the fusarium avenae is detected to be positive, and the other microorganisms are negative, so that the invention has good specificity. The results are shown in Table 1 and FIG. 5.
TABLE 1
FIG. 5 is a diagram showing the results of the specificity test of the present invention. Wherein the standard amplification curve a is a fusarium avenae genome, and the standard amplification curve b represents that the standard amplification curve does not appear, and the standard amplification curve a is fusarium graminearum, alternaria alternata, fusarium oxysporum, fusarium sporotrichioides, botrytis cinerea and fusarium solani respectively.
3. Gel imaging verification chart for specificity experiment
In order to prove that the method has specificity for detecting the fusarium avenaceum, other common plant-infected microorganisms are selected for carrying out a specificity experiment. The selected microorganisms include: fusarium graminearum, Alternaria alternata, Fusarium oxysporum, Fusarium sporotrichioides, Botrytis cinerea and Fusarium solani.
And preparing a fluorescent quantitative result of a probe for detecting the fusarium avenae by using 1% agarose gel. And taking an amplification product of a specificity experiment as a template.
The results are shown in FIG. 6, in which lanes 1-8 are sequentially marked with 1: blank control (water), 2: positive control (Fusarium avenaceum), negative control (3: Alternaria alternata, 4: Fusarium graminearum, 5: Fusarium solani, 6: Fusarium oxysporum, 7: Fusarium sporotrichioides, 8: Botrytis cinerea) and D L2000.
The result shows that the method has specificity for detecting the fusarium avenaceum.
Example 5
The embodiment discloses a real-time fluorescent quantitative PCR detection method of fusarium avenaceum, which comprises the following steps:
the DNA of the sample to be tested and the 18SrRNA prepared in example 1 were used respectivelyGene series concentration standard substance (series concentration of series concentration standard substance: 3.1 × 10)9copies/μL、3.1×108copies/μL、 3.1×107copies/μL、3.1×106copies/μL、3.1×105copies/μL、3.1×104copies/μL、 3.1×103copies/μL、3.1×102copies/μL、3.1×101copies/μL、3.1×10-1copies/uL、 3.1×10-2copies/u L) as a template, performing fluorescent quantitative PCR amplification by using primers and probes in the kit, and setting a positive control and a negative control.
The PCR reaction system is as follows:
reaction conditions are as follows: reaction conditions are as follows: digestion of the contamination at 37 ℃ for 2min, pre-denaturation at 95 ℃ for 10min, at 95 ℃ for 15s and 60 ℃ for 1min and collection of the fluorescence signal for 40 cycles.
Drawing a standard curve, and carrying out rapid quantitative detection through the standard curve and the Ct value of the sample to be detected.
The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or changes made within the spirit and scope of the main design of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.
SEQUENCE LISTING
<110> Baiyuan Gene technology of Lanzhou Ltd
Primer, probe, kit and method for RT-QPCR (reverse transcription-quantitative polymerase chain reaction) detection of fusarium avenaceum
<130>20200417
<160>7
<170>PatentIn version 3.3
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<213> Artificial sequence
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cctgttcgag cgtcatttca accct 25
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<213> Artificial sequence
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<222>(2)..(2)
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gnagaccaac ccctggnnac ataccttaat gttgcctcgg cggatcagcc cgcgccccgt 60
aaaacgggac ggcccgccag aggacccaaa ctctaatgtt tcttattgta acttctgagt 120
aaaacaaaca aataaatcaa aactttcaac aacggatctc ttggttctgg catcgatgaa 180
gaacgcagca aaatgcgata agtaatgtga attgcagaat tcagtgaatc atcgaatctt 240
tgaacgcaca ttgcgcccgc tggtattccg gcgggcatgc ctgttcgagc gtcatttcaa 300
ccctcaagcc cccgggtttg gtgttgggga tcggctctgc cttctggcgg tgccgccccc 360
gaaatacatt ggcggtctcg ctgcagcctc cattgcgtag tagctaacac ctcgcaactg 420
gaacgcggcg cggccatgcc gtaaaacccc aacttctgaa tgttgacctc ggatcaggta 480
ggaatacccg ctgaacttaa 500
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aacggatctc ttggttctgg catcgatgaa gaacgcagca aaatgcgata agtaatgtga 60
attgcagaat tcagtgaatc atcgaatctt tgaacgcaca ttgcgcccgc tggtattccg 120
gcgggcatgc ctgttcgagc gtcatttcaa ccctcaagcc cccgggtttg gtgttgggga 180
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Claims (10)
1. The primer for RT-QPCR detection of fusarium avenae is characterized in that the primer comprises an upstream primer and a downstream primer, the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2,
an upstream primer: 5'-ATCGATGAAGAACGCAGCAA-3', respectively;
a downstream primer: 5'-CGATCCCCAACACCAAACCC-3' are provided.
2. The RT-QPCR detection probe for the fusarium avenaceum is characterized in that the nucleotide sequence of the probe is shown in SEQ ID NO: 3: 5'-CCTGTTCGAGCGTCATTTCAACCCT-3', respectively; preferably, the fluorescent reporter group marked at the 5 'end of the probe is VIC, and the fluorescent quencher group marked at the 3' end of the probe is BHQ.
3. Primer and probe combination that fusarium avenaceum's RT-QPCR detected, its characterized in that includes:
an upstream primer: 5'-ATCGATGAAGAACGCAGCAA-3', respectively;
a downstream primer: 5'-CGATCCCCAACACCAAACCC-3', respectively;
and (3) probe: 5'-CCTGTTCGAGCGTCATTTCAACCCT-3' are provided.
4. A kit for RT-QPCR detection of Fusarium avenaceum, comprising the primer of claim 1 and the probe of claim 2.
5. The kit according to claim 4, further comprising a QPCR template as shown in SEQ ID NO 4; preferably, the template is in the form of a plasmid.
6. The kit of claim 4 or 5, further comprising a negative sample; preferably, the negative control sample is ddH2O。
7. The kit according to claim 4 or 5, further comprising a premix, preferably the premix is 2 × Probe Mix.
8. A reaction system for RT-QPCR detection of fusarium avenaceum, which is characterized by comprising:
an upstream primer: 5'-ATCGATGAAGAACGCAGCAA-3', respectively;
a downstream primer: 5'-CGATCCCCAACACCAAACCC-3', respectively;
and (3) probe: 5'-CCTGTTCGAGCGTCATTTCAACCCT-3', respectively;
template: as shown in SEQ ID NO. 4;
preferably, the kit further comprises a negative control sample, and further preferably, the negative control sample is ddH2O。
9. An RT-QPCR detection method for detecting plant fusarium avenaceum is characterized by comprising the following steps:
step 1, extracting total DNA of a sample to be detected;
step 2. preparing a reaction system, wherein the reaction system is as described in claim 8;
step 3, diluting the template in a gradient manner to prepare a standard curve sample and a positive control sample;
step 4, performing fluorescent quantitative PCR amplification on a sample to be detected, a standard curve sample, a positive control sample and a negative control sample by using the primer and the probe;
step 5, drawing a standard curve, and calculating a result through the standard curve and the Ct value of the sample to be detected;
preferably, in the step 3, the concentration of the standard curve sample prepared by the gradient dilution is 3.1 × 10 respectively9copies/μL、3.1×108copies/μL、3.1×107copies/μL、3.1×106copies/μL、3.1×105copies/μL、3.1×104copies/μL、3.1×103copies/μL、3.1×102copies/μL、3.1×101copies/μL、3.1×10- 1copies/μL、3.1×10-2copies/μL；
Preferably, the concentration of the positive control sample is 3.1 × 1010copies/μL。
10. The method of claim 9, wherein the reaction conditions of the PCR are: digestion of the contamination at 37 ℃ for 2min, pre-denaturation at 95 ℃ for 10min, at 95 ℃ for 15s and 60 ℃ for 1min and collection of the fluorescence signal for 40 cycles.
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