阅读说明：本技术 一种检测SdhB-H278R点突变的含量的方法及其专用成套试剂 (Method for detecting content of SdhB-H278R point mutation and special reagent set thereof ) 是由 李宝聚 石延霞 孙炳学 谢学文 柴阿丽 于 2018-08-23 设计创作，主要内容包括：本发明公开了检测SdhB-H278R点突变的含量的方法及其专用成套试剂。方法包括如下步骤：分别以待测样本基因组DNA和SdhB-H278R点突变标准溶液为模板,分别采用特异引物对(由序列1和序列2所示的引物组成)和内参引物对(由序列3和序列4所示的引物组成)进行实时荧光定量PCR,依次得到CT1和CT2,进而得到ΔCT；ΔCT＝CT1-CT2；根据标准溶液中SdhB-H278R点突变的含量和相应的标准溶液ΔCT绘制标准曲线,将待测样本ΔCT代入标准曲线,得到待测样本基因组DNA中SdhB-H278R点突变的含量。该方法检测SdhB-H278R点突变的含量准确率高且特异性好,具有重要的应用价值。(The invention discloses a method for detecting the content of SdhB-H278R point mutation and a special reagent set thereof. The method comprises the following steps: respectively taking the genomic DNA of a sample to be detected and SdhB-H278R point mutation standard solution as templates, respectively adopting a specific primer pair (composed of primers shown in a sequence 1 and a sequence 2) and an internal reference primer pair (composed of primers shown in a sequence 3 and a sequence 4) to carry out real-time fluorescence quantitative PCR, and sequentially obtaining CT1 and CT2 so as to obtain delta CT; CT1-CT 2; and drawing a standard curve according to the SdhB-H278R point mutation content in the standard solution and the corresponding standard solution delta CT, and substituting the sample delta CT to be detected into the standard curve to obtain the SdhB-H278R point mutation content in the genomic DNA of the sample to be detected. The method has high content accuracy and good specificity for detecting SdhB-H278R point mutation, and has important application value.)

1. The kit comprises a specific primer pair, wherein the specific primer pair consists of a primer B-H278R-2F and a primer B-H278R-2R 14; the specific primer pair contains a specific DNA fragment A in a target sequence of the polyspora SdhB gene; the specific DNA fragment A is Y1) or Y2) as follows:

y1) is shown in the sequence 5 of the sequence table;

y2) is obtained by carrying out substitution and/or deletion and/or addition of one or more nucleotides on the sequence 5, and the DNA molecule has the same function as the sequence 5.

2. The kit of claim 1, wherein:

the primer B-H278R-2F is the following X1) or X2):

x1) single-stranded DNA molecule shown in sequence 1 in the sequence table;

x2) single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1;

the primer B-H278R-2R14 is the following X3) or X4):

x3) single-stranded DNA molecule shown in sequence 2 in the sequence table;

x4) is a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2.

3. The kit of claim 1 or 2, wherein: the kit also comprises an internal reference primer pair; the internal reference primer pair consists of a primer B-H278R-TY-F and a primer B-H278R-TY-R; the internal reference primer pair contains a specific DNA fragment B in a target sequence of the multi-major clavispora SdhB gene; the specific DNA segment B is a conserved segment of the SdhB gene of the polyspora, and does not generate any form of mutation.

4. The kit of claim 3, wherein:

the primer B-H278R-TY-F is Z1) or Z2) as follows:

z1) single-stranded DNA molecule shown in sequence 3 in the sequence table;

z2) single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3;

the primer B-H278R-TY-R is the following Z3) or Z4):

z3) single-stranded DNA molecule shown in sequence 4 in the sequence table;

z4) is a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and has the same function as the sequence 4.

5. The method for preparing the kit of parts according to any one of claims 1 to 4, wherein the primer B-H278R-2F and/or the primer B-H278R-2R14 and/or the primer B-H278R-TY-F and/or the primer B-H278R-TY-R in the kit of parts according to any one of claims 1 to 4 are packaged separately.

6. The use of the kit of any one of claims 1 to 4, which is at least one of (S1) - (S6):

(S1) detecting a SdhB-H278R point mutation;

(S2) preparing a product for detecting a SdhB-H278R point mutation;

(S3) evaluating boscalid resistance of corynebacterium polygamum;

(S4) preparing a product for evaluating boscalid resistance of corynebacterium polystachyum;

(S5) detecting the content of SdhB-H278R point mutation in the sample to be detected;

(S6) preparing a product for detecting the content of SdhB-H278R point mutation in the sample to be detected.

7. A product comprising a kit of parts according to any one of claims 1 to 4.

8. Use of the product of claim 7, at least one of (S1) or (S3) or (S5) as follows:

(S1) detecting a SdhB-H278R point mutation;

(S3) evaluating boscalid resistance of corynebacterium polygamum;

(S5) detecting the content of SdhB-H278R point mutation in the sample to be detected.

9. A method for detecting the content of SdhB-H278R point mutation in a sample to be detected comprises the following steps (a), (b) and (c):

the step (a) includes the steps of:

(a-1) carrying out real-time fluorescence quantitative PCR by using the genome DNA of a sample to be detected as a template and adopting the specific primer pair in claim 1 or 2 to obtain a sample to be detected CT 1;

(a-2) carrying out real-time fluorescence quantitative PCR by using the genomic DNA of a sample to be detected as a template and adopting the internal reference primer pair in claim 3 or 4 to obtain a sample to be detected CT 2;

(a-3) obtaining a sample delta CT to be detected; the sample to be detected delta CT is a sample to be detected CT 1-sample to be detected CT 2;

the step (b) comprises the steps of:

(b-1) carrying out real-time fluorescent quantitative PCR by using SdhB-H278R point mutation standard solution as a template and adopting the specific primer pair in claim 1 or 2 to obtain standard solution CT 1;

(b-2) carrying out real-time fluorescent quantitative PCR by using the SdhB-H278R point mutation standard solution as a template and adopting the internal reference primer pair in claim 3 or 4 to obtain a standard solution CT 2;

(b-3) obtaining a standard solution Delta CT; standard solution Δ CT ═ standard solution CT1 — standard solution CT 2;

the step (c): and (b) drawing a standard curve according to the SdhB-H278R point mutation content in the SdhB-H278R point mutation standard solution and the corresponding standard solution delta CT, and substituting the delta CT of the sample to be detected obtained in the step (a-3) into the standard curve to obtain the SdhB-H278R point mutation content in the genome DNA of the sample to be detected.

10. A method for detecting whether SdhB-H278R point mutation exists in genomic DNA of a sample to be detected is G1) or G2):

G1) using the genomic DNA of a sample to be tested as a template, carrying out PCR amplification by using the specific primer pair as described in claim 1 or 2, and then carrying out the following judgment: if the PCR amplification product contains a 244bp DNA fragment, SdhB-H278R point mutation exists or is suspected to exist in the genome DNA of the sample to be detected; if the PCR amplification product does not contain the DNA fragment of 244bp, the SdhB-H278R point mutation does not exist or is suspected to be absent in the genome DNA of the sample to be detected;

G2) using the genomic DNA of a sample to be tested as a template, carrying out PCR amplification by using the specific primer pair as described in claim 1 or 2, and then carrying out the following judgment: if the PCR amplification product contains a DNA fragment shown in a sequence 5 in the sequence table, SdhB-H278R point mutation exists or is suspected to exist in the genome DNA of the sample to be detected; if the PCR amplification product does not contain the DNA fragment shown in the sequence 5 in the sequence table, the SdhB-H278R point mutation does not exist or is suspected to be not exist in the genome DNA of the sample to be detected.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for detecting SdhB-H278R point mutation content and a special reagent set thereof.

Background

Corynebacterium polystachyum (corynespora cassicola) is an important plant pathogenic bacterium, is the earliest species found in corynespora and has the widest host range, and can infect most vegetables in cucurbitaceae, solanaceae, leguminosae and the like, economic crops such as horticultural flowers and rubber and grain crops such as soybeans and the like for about 300 species. In China, a plurality of provinces such as Beijing, Shandong, Liaoning, Hebei, Guangdong and Heilongjiang and the like and large-scale cucumber corymbose leaf spot disease occurs in Japan and Korea, the common field morbidity is 10% -25%, and can reach 60% -70% or even 100% in serious cases. Therefore, the cucumber corynespora leaf spot caused by the corynespora polystachya has become an important leaf disease in cucumbers.

Boscalid is an SDHIs fungicide developed by basf, germany, which can be used for controlling cucumber gray mold (caused by Botrytis cinerea) and cucumber clavulan leaf spot. Due to the long-term large-scale use of boscalid, both botrytis cinerea and corynespora polystachya have drug resistance to boscalid. Point mutations in succinate dehydrogenase B, C and the D subunit are the main contributors to resistance of most pathogenic bacteria to SDHIs-type bactericides. Wherein, the point mutations found on the pestalotiopsis polyspora for resisting boscalid mainly comprise SdhB-H278R/Y, SdhC-S73P, SdhD-S89P, SdhD-G109V and the like. The corynebacterium polymorpha exhibited four types of resistance, ultra-high resistance, medium resistance and low resistance to boscalid according to resistance index (RF). Research has shown that different point mutations on succinate dehydrogenase B, C and D subunit correspond to different resistance levels, and SdhB-H278Y (CAC-TAC) mutation mainly occurs to the ultra-high resistant strain; for the high-resistant strains, the SdhB-H278R (CAC-CGC) mutation mainly occurs; for the anti-strain, the resistance mutations included SdhC-S73P (TCG-CCG) mutation, SdhD-G109V (GGC-GTC) mutation, and SdhD-S89P (TCC-CCC) mutation.

When the inventor of the invention carries out boscalid resistance detection on cucumber corynebacterium sp samples collected in great Xingjiang areas, a resistant strain containing SdhB-H278R point mutation is found, which indicates that the resistant strain containing the point mutation appears in the field multi-dominant corynebacterium sp population. At present, no quantitative detection method for SdhB-H278R point mutation is established.

Disclosure of Invention

The invention aims to detect the content of SdhB-H278R point mutation.

The invention first protects the kit. The kit may include a specific primer pair; the specific primer pair can consist of a primer B-H278R-2F and a primer B-H278R-2R 14; the target sequence of the specific primer pair in the gene SdhB of the polyspora can contain a specific DNA segment A; the specific DNA fragment A can be Y1) or Y2) as follows:

y1) is shown in the sequence 5 of the sequence table;

y2) is obtained by carrying out substitution and/or deletion and/or addition of one or more nucleotides on the sequence 5, and the DNA molecule has the same function as the sequence 5.

The primer B-H278R-2F can be the following X1) or X2):

x1) single-stranded DNA molecule shown in sequence 1 in the sequence table;

x2) is a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1.

The primer B-H278R-2R14 is the following X3) or X4):

x3) single-stranded DNA molecule shown in sequence 2 in the sequence table;

x4) is a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2.

The kit may further comprise an internal reference primer pair; the internal reference primer pair can consist of a primer B-H278R-TY-F and a primer B-H278R-TY-R; the target sequence of the internal reference primer pair in the multi-major clavispora SdhB gene can contain a specific DNA segment B; the specific DNA segment B can be a conserved segment of the SdhB gene of the polyspora, and no mutation in any form occurs.

The primer B-H278R-TY-F can be the following Z1) or Z2):

z1) single-stranded DNA molecule shown in sequence 3 in the sequence table;

z2) is a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3.

The primer B-H278R-TY-R can be the following Z3) or Z4):

z3) single-stranded DNA molecule shown in sequence 4 in the sequence table;

z4) is a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and has the same function as the sequence 4.

Any of the kits described above may also include some conventional reagents for performing real-time fluorescent quantitative PCR and/or SdhB-H278R point mutation standard solutions.

The present invention also provides a method for preparing any one of the above kits, wherein the primer B-H278R-2F and/or the primer B-H278R-2R14 and/or the primer B-H278R-TY-F and/or the primer B-H278R-TY-R in any one of the above kits are packaged separately.

The invention also provides the use of any one of the kits described above, which may be at least one of (S1) - (S6):

(S1) detecting a SdhB-H278R point mutation;

(S2) preparing a product for detecting a SdhB-H278R point mutation;

(S3) evaluating boscalid resistance of corynebacterium polygamum;

(S4) preparing a product for evaluating boscalid resistance of corynebacterium polystachyum;

(S5) detecting the content of SdhB-H278R point mutation in the sample to be detected;

(S6) preparing a product for detecting the content of SdhB-H278R point mutation in the sample to be detected.

The invention also provides a product which may comprise a kit as described in any of the above.

The invention also protects the application of the product, which can be at least one of the following (S1), or (S3), or (S5):

(S1) detecting a SdhB-H278R point mutation;

(S3) evaluating boscalid resistance of corynebacterium polygamum;

(S5) detecting the content of SdhB-H278R point mutation in the sample to be detected.

The invention also provides a method for detecting the content of SdhB-H278R point mutation in a sample to be detected, which comprises the following steps (a), (b) and (c):

the step (a) includes the steps of:

(a-1) carrying out real-time fluorescence quantitative PCR by using the genome DNA of a sample to be detected as a template and adopting any one of the specific primers to obtain a sample to be detected CT 1;

(a-2) carrying out real-time fluorescence quantitative PCR by using the genome DNA of a sample to be detected as a template and adopting any one of the internal reference primer pairs to obtain a sample to be detected CT 2;

(a-3) obtaining a sample delta CT to be detected; the sample to be detected delta CT is a sample to be detected CT 1-sample to be detected CT 2;

the step (b) comprises the steps of:

(b-1) carrying out real-time fluorescent quantitative PCR by using SdhB-H278R point mutation standard solution as a template and adopting any one of the specific primer pairs to obtain standard solution CT 1;

(b-2) carrying out real-time fluorescent quantitative PCR by using SdhB-H278R point mutation standard solution as a template and adopting any one of the internal reference primer pairs to obtain standard solution CT 2;

(b-3) obtaining a standard solution Delta CT; standard solution Δ CT ═ standard solution CT1 — standard solution CT 2;

the step (c): and (b) drawing a standard curve according to the SdhB-H278R point mutation content in the SdhB-H278R point mutation standard solution and the corresponding standard solution delta CT, and substituting the delta CT of the sample to be detected obtained in the step (a-3) into the standard curve to obtain the SdhB-H278R point mutation content in the genome DNA of the sample to be detected.

Any one of the SdhB-H278R point mutation standard solutions can be a plurality of solutions with different SdhB-H278R point mutation contents. The SdhB-H278R point mutation standard solution can be prepared as follows: mixing the genomic DNA of the polysaccharomyces pluvialis strain only having SdhB-H278R point mutation with the genomic DNA of the polysaccharomyces pluvialis strain not having the point mutation according to different molar mass ratios to obtain mixed DNA. In one embodiment of the invention, the molar mass of the genomic DNA of the strain of corynebacterium polygamum in which the SdhB-H278R point mutation occurs in the mixed DNA is 6.25%, 12.5%, 25.0%, 50.0%, 75.0% or 100.0% of the molar mass of the mixed DNA.

The strain of the polysaccharomyces pluvialis with the SdhB-H278R point mutation can be specifically the strain of the polysaccharomyces pluvialis HG-R mentioned in the examples. SdhB gene of the polyspora spinosa HG-R strain has SdhB-H278R point mutation.

The non-mutated strain of Clavibacillus pluvialis may be specifically Clavibacillus pluvialis HG-H1 strain, Clavibacillus pluvialis HG-H2 strain, Clavibacillus pluvialis HG-H3 strain or Clavibacillus pluvialis HG-H4 strain mentioned in the examples. SdhB genes of the strains of the plectaria polystachya HG-H1, the strains of the plectaria polystachya HG-H2, the strains of the plectaria polystachya HG-H3 and the strains of the plectaria polystachya HG-H4 are all wild types.

The invention also provides a method for detecting whether the genomic DNA of the sample to be detected has SdhB-H278R point mutation, which can be G1) or G2):

G1) taking the genome DNA of a sample to be detected as a template, carrying out PCR amplification by adopting any one of the specific primer pairs, and then judging as follows: if the PCR amplification product contains a 244bp DNA fragment, SdhB-H278R point mutation exists or is suspected to exist in the genome DNA of the sample to be detected; if the PCR amplification product does not contain the DNA fragment of 244bp, the SdhB-H278R point mutation does not exist or is suspected to be absent in the genome DNA of the sample to be detected;

G2) taking the genome DNA of a sample to be detected as a template, carrying out PCR amplification by adopting any one of the specific primer pairs, and then judging as follows: if the PCR amplification product contains a DNA fragment shown in a sequence 5 in the sequence table, SdhB-H278R point mutation exists or is suspected to exist in the genome DNA of the sample to be detected; if the PCR amplification product does not contain the DNA fragment shown in the sequence 5 in the sequence table, the SdhB-H278R point mutation does not exist or is suspected to be not exist in the genome DNA of the sample to be detected.

Any one of the above test samples can be r1) strain; r2) strain of Corynebacterium polymorpha; r3) Botrytis cinerea; r4) cladosporium cucumerinum; r5) Rhizoctonia solani; r6) Alternaria solani; r7) Staphylococcus solani; r8) Fusarium oxysporum; r9) sclerotinia sclerotiorum; r10) Pseudoperonospora cubensis; r11) plant tissue; r12) plant leaves; r13) cucumber leaves.

In the above, if the SdhB-H278R point mutation exists, the sample to be tested has boscalid resistance. The higher the content of SdhB-H278R point mutation, the higher the proportion (i.e. the higher the frequency of appearance) of SdhB-H278R point mutation resistant population in the field. Therefore, the application of the pesticide can be guided according to the content of the SdhB-H278R point mutation. For example, if the content of SdhB-H278R point mutation is high, a bactericide other than boscalid needs to be used.

By adopting the method for detecting the content of the SdhB-H278R point mutation, the frequency and the dynamic change of the SdhB-H278R point mutation in the field can be accurately detected, the method has guiding significance for effective prevention and treatment of diseases and reasonable use of pesticides, and simultaneously provides a foundation for research on the dynamic change of different types of resistant populations under the screening pressure of medicaments. The invention has important application value.

Drawings

FIG. 1 is a schematic diagram of the position of a specific primer pair A on a gene SdhB of the polyspora.

FIG. 2 shows specificity experiments.

FIG. 3 is a standard curve of SdhB-H278R point mutation.

FIG. 4 shows melting curve analysis.

FIG. 5 shows melting curve analysis.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

2 XTaq PCR MasterMix is a product of Beijing Bomaide Gene technology, Inc. 2 XSuperRealPreMix Plus, 50 XROX Reference Dye and plant tissue DNA extraction kit are all products of Tiangen Biochemical technology (Beijing) Co., Ltd.