CN114574620B - Fusarium oxysporum cucumber specialized LAMP primer, detection kit and application - Google Patents

Abstract

The invention discloses a fusarium oxysporum cucumber specialized LAMP primer, a detection kit and application thereof, and belongs to the technical field of biology. The invention designs a group of fusarium oxysporum cucumber specialization LAMP primers based on the LAMP technology, and discloses a fusarium oxysporum cucumber specialization LAMP kit. The invention does not depend on any special instrument and equipment, can realize the on-site high-flux rapid detection, can judge the result by naked eyes, and has lower detection cost than fluorescent quantitative PCR. The invention can specifically identify fusarium oxysporum cucumber specialization in samples such as plants, soil, strains and the like, has high accuracy, short time consumption, simple operation and low cost, has important significance for diagnosing and preventing fusarium wilt of cucumber, and is easy for large-scale popularization of a base layer.

Description

Fusarium oxysporum cucumber specialized LAMP primer, detection kit and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a fusarium oxysporum cucumber specialized LAMP primer, a kit and application.

Background

Cucumber is a cucurbitaceae cucumber plant, and is an important vegetable crop in China. In recent years, sustainable development of cucumber industry is severely restricted by soil-borne diseases, especially cucumber fusarium wilt. The disease is caused by infection with fusarium oxysporum cucumber specialization (Fusarium oxysporum f.sp.cucumerinum). The pathogenic bacteria infect plants mainly through roots and are parasitic in rhizomes, causing necrosis of plant vascular bundles, resulting in cucumber wilting. Even without host, chlamydospores produced by the chlamydospores can survive in soil for a long time, and the chlamydospores have strong pathogenicity, high mortality and serious harm. For a long time, chemical pesticides are mainly used for preventing and treating cucumber fusarium wilt, and the chemical pesticides have very little effect for preventing and treating fusarium wilt in middle and later periods of disease. Therefore, the rapid detection of fusarium oxysporum cucumber specialization is particularly important in early stage, and provides basis for timely prevention and control of fusarium wilt of cucumber.

At present, the detection of pathogenic bacteria such as fusarium oxysporum and the like generally adopts a traditional morphological characteristic identification method, a conventional PCR method, a real-time fluorescent quantitative PCR method and the like. The traditional morphological characteristic identification method needs an experimenter to have the capability of accurately identifying different colony morphologies, and the colonies of different specialization types cannot be distinguished only by naked eyes. The conventional PCR method and the real-time fluorescent quantitative PCR method are adopted, expensive instruments and equipment such as an ultra-clean bench, a PCR instrument, a fluorescent quantitative PCR instrument and the like are required to be used, the operation flow is complex, and an experimenter is often required to have a certain experimental skill, so that the method is not suitable for basic level production or on-site rapid diagnosis.

Loop-mediated isothermal amplification (LAMP) is a novel gene diagnosis technique capable of amplifying nucleic acids in a short time (usually within 1 hour) under isothermal (60-65 ℃) conditions. Compared with the conventional PCR, the LAMP method does not need the processes of thermal denaturation of a template, temperature circulation, electrophoresis, ultraviolet observation and the like. The LAMP method is characterized in that at least four primers are designed for six regions on a target gene, amplification reaction is carried out by using strand displacement DNA polymerase under the condition of constant temperature, colored amplification products can be generated by the reaction, and whether the target gene exists or not can be judged by observing the existence or the depth of the color by naked eyes. LAMP has the characteristics of simplicity, rapidness and strong specificity. The technology can be comparable or even better than the PCR technology in indexes such as sensitivity, specificity, detection range and the like, does not depend on any special instrument and equipment to realize on-site high-flux rapid detection, and has detection cost far lower than fluorescent quantitative PCR.

Disclosure of Invention

The invention aims to: the technical problem to be solved by the invention is to provide a rapid, efficient, convenient, low-cost and easy-to-operate fusarium oxysporum cucumber specialized LAMP visual detection method.

The technical scheme is as follows: in order to solve the technical problems, the invention provides the following technical scheme:

fusarium oxysporum cucumber specialized LAMP primer group is characterized by comprising a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP and a downstream loop primer LB, wherein the primer sequences are as follows:

forward outer primer F3 (SEQ ID NO: 1): 5'-CGGATCTCTTGGTTCTGGC-3';

reverse outer primer B3 (SEQ ID NO: 2): 5'-TGGAAGCTCGACGTGACC-3';

forward inner primer FIP (SEQ ID NO: 3): 5'-GGCGCAATGTGCGTTCAAAGATTGAAGAACGCAGCAAAATGC-3';

reverse inner primer BIP (SEQ ID NO: 4): 5'-TGCCTGTTCGAGCGTCATTTCACGAATTAACGCGAGTCCCA-3';

downstream loop primer LB (SEQ ID NO: 5): 5'-ACCCTCAAGCACAGCTTGGT-3'.

Fusarium oxysporum cucumber specialization LAMP kit comprises the following reagents: 20 Xprimer mix (containing FIP and BIP 32. Mu. M, F3 and B3 4. Mu. M, LB 8. Mu.M), fusarium oxysporum cucumber specialised genomic DNA, 2 XLAMP MagicMix (containing DNA polymerase, blue DNA tracer dye, reaction buffer) and sterile ultra-pure water.

The application of the fusarium oxysporum cucumber specialization LAMP primer group in detecting fusarium oxysporum cucumber specialization comprises the following steps:

(1) Extracting genome DNA of a sample to be detected;

(2) LAMP reaction:

amplification reaction system: 1 μl of 20×primer mixture, 2 μl of the genomic DNA of the sample to be tested extracted in step (1), 2×LAMP MagicMix 10 μl, and sterilized ultrapure water to 20 μl, and using Fusarium oxysporum cucumber specialized genomic DNA as positive control and sterilized ultrapure water as negative control, the reaction procedure is: keeping the temperature at 65 ℃ for 50min;

(3) After the reaction was completed, the change in color was observed: if the reaction liquid is blue after the reaction is finished, the sample to be tested contains fusarium oxysporum cucumber specialization;

if the reaction liquid is colorless or light blue after the reaction is finished, the sample to be tested does not contain fusarium oxysporum cucumber specialization. Or performing agarose gel electrophoresis by using a PCR product, and if a specific fragment with the size of 226bp is obtained, the sample to be detected contains fusarium oxysporum cucumber specialization; if the specific fragment with the size of 226bp does not exist, the sample to be detected does not contain fusarium oxysporum cucumber specialization.

In order to verify the specificity of the fusarium oxysporum cucumber specialization primer group, 26 fusarium oxysporum cucumber specialization strains and 8 fusarium oxysporum cucumber specialization kindred strains are collected from the China center for agricultural microorganism strain preservation, the university of Nanjing agriculture, the university of northeast agriculture, the university of Nanjing, and the like as sample supply products. The invention detects fusarium oxysporum cucumber specialization, reacts for 50min at 65 ℃ in a water bath kettle, the result can be judged by observing the color change in the reaction tube with naked eyes, and only the color in the fusarium oxysporum cucumber specialization sample tube is blue, which is a positive result, and the designed primer group is proved to have specificity.

The beneficial effects are that: the invention has the following outstanding technical effects:

(1) The detection cost is low: the invention can realize the on-site high-flux rapid detection without any special instrument and equipment, and the result can be judged by naked eyes, and the detection cost is lower than that of a PCR method and the like;

(2) The operation is simple and quick: the invention gets rid of the complex operation flow such as the traditional morphological identification method and the PCR method, and solves the problems that an operator needs to have a certain experiment skill, an experiment sample needs to be strictly processed, time and labor are consumed, and the like. When the invention is applied to detection, the result can be judged by naked eyes only by processing for 60min under the constant temperature condition, and the invention is not limited by experimental conditions.

(3) High specificity: the invention designs five primers aiming at a specific gene fragment of the fusarium oxysporum cucumber specialization group, has very high specific recognition capability on the fusarium oxysporum cucumber specialization, has high sensitivity and quick combination, and can be used for quickly and efficiently detecting whether the fusarium oxysporum cucumber specialization exists in a sample.

Drawings

Fig. 1: LAMP amplification results are shown in the examples. ( In the figure, the number 1 shows a colorless or very light blue reaction, and the number 2 shows a sky blue reaction; the left image is before the reaction, the right image is after the reaction )

Fig. 2: the results of the specificity verification of the LAMP primer combinations are shown by natural light.

Fig. 3: the result diagram of the conventional PCR amplification agarose gel electrophoresis of the specific primer group (F3, B3 and FIP, BIP, LB) is shown, wherein M is Marker I (the electrophoresis bands of the Marker I are 1200bp, 900bp, 700bp, 500bp, 300bp and 100bp from top to bottom), N is a negative control, and 1-34 correspond to the following table 1.

Specific implementation steps

The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.

Example 1: specificity experiments of fusarium oxysporum cucumber specialization type LAMP reaction.

Lamp specific primer design:

extracting fusarium oxysporum cucumber specialized genome DNA, amplifying ITS sequence, and performing sequencing after TA cloning; obtaining a specific fragment with the size of 487bp through NCBI Blast sequence alignment; according to the fusarium oxysporum cucumber specialization specific fragment sequence, two groups of primers are designed, and the sequences are as follows:

a first set of primers:

F3-1：5’-CGGATCTCTTGGTTCTGGC-3’；

B3-1：5’-TGGAAGCTCGACGTGACC-3’；

FIP-1：5’-GGCGCAATGTGCGTTCAAAGATTGAAGAACGCAGCAAAATGC-3’；

BIP-1：5’-TGCCTGTTCGAGCGTCATTTCACGAATTAACGCGAGTCCCA-3’；

LB-1：5’-ACCCTCAAGCACAGCTTGGT-3’

second set of primers:

F3-2：5’-CAGTATTCTGGCGGGCATG-3’；

B3-2：5’-ACCTGATCCGAGGTCAACAT-3’；

FIP-2：5’-AGGAACGCGAATTAACGCGAGTGTTCGAGCGTCATTTCAACC-3’；

BIP-2：5’-CGGTCACGTCGAGCTTCCATAGAGTTGGGGTTTAACGGCG-3’；

LF-2：5’-AACACCAAGCTGTGCTTGAG-3’；

LB-2：5’-CGTAGTAGTAAAACCCTCGTTACTG-3’；

LAMP-specific primer set screening

Primer combination:

primer set 1 (without loop primer): f3-1, B3-1, FIP-1, BIP-1

Primer set 2 (containing loop primers): f3-1, B3-1, FIP-1, BIP-1, LB-1

Primer set 3 (without loop primer): f3-2, B3-2, FIP-2, BIP-2

Primer set 4 (containing loop primers): f3-2, B3-2, FIP-2, BIP-2, LB-2

Lamp reaction system construction:

the amplification reaction system comprises: 20 Xprimer mix 1. Mu.L, template DNA 2. Mu.L, 2 XSLAMP MagicMix 10. Mu.L, supplemented sterilized ultrapure water to 20. Mu.L, and Fusarium oxysporum cucumber specialized genomic DNA as positive control, sterilized ultrapure water as negative control.

Lamp reaction:

the LAMP reaction system is added into a reaction tube, and the amplification reaction procedure is as follows: and (3) reacting for 50min at 65 ℃ in the water bath kettle, and observing the color change in the tube after the reaction is finished.

5. Detection result:

the LAMP amplification reaction of the primer group 2 has specificity for fusarium oxysporum cucumber specialization, a blue positive reaction is formed in the fusarium oxysporum cucumber specialization reaction tube, a colorless or very light blue negative reaction is formed in the non-fusarium oxysporum cucumber specialization reaction tube, and the LAMP visual detection result is shown in the figure 2, wherein: 1-34 correspond to Table 1, 35 being a sterilized ultrapure water negative control. As can be seen from FIG. 2, the EP pipes numbered 1 to 26 exhibited blue positive reactions, and the EP pipes numbered 27 to 35 exhibited colorless reactions.

Table 1 names of strains corresponding to numbers 1 to 34 in FIGS. 2 and 3

Example 2: fusarium oxysporum cucumber specialization LAMP specific agarose gel electrophoresis verification experiments.

In order to further verify the specificity of the primer group 2 on the fusarium oxysporum cucumber specialization, the genomic DNA of 26 fusarium oxysporum cucumber specialization strains and 8 non-fusarium oxysporum cucumber specialization strains in table 1 are subjected to conventional PCR amplification, and the reaction system is as follows: 2. Mu.L of DNA template, 12.5. Mu.L of Taq Master Mix (2X), 1. Mu.L of primer Mix, sterilized ultrapure water to 25. Mu.L; the amplification conditions were: pre-denaturation at 95℃for 5 min; melting at 95 ℃ for 20s, annealing at 65 ℃ for 20s, extending at 72 ℃ for 30s, and 7min at 72 ℃ after 25 cycles; the amplification results were detected by agarose gel electrophoresis, and the detection results are shown in FIG. 3. In FIG. 3, M is Marker I,1-34 correspond to Table 1 above, and N is a sterilized ultrapure water control.

Sequence listing

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Claims (6)

1. Fusarium oxysporum cucumber specialized LAMP primer group is characterized by comprising the following primers:

F3：5’-CGGATCTCTTGGTTCTGGC-3’；

B3：5’-TGGAAGCTCGACGTGACC-3’；

FIP：5’- GGCGCAATGTGCGTTCAAAGATTGAAGAACGCAGCAAAATGC-3’；

BIP：5’- TGCCTGTTCGAGCGTCATTTCACGAATTAACGCGAGTCCCA-3’；

LB：5’- ACCCTCAAGCACAGCTTGGT-3’。

2. the application of the fusarium oxysporum cucumber specialization LAMP primer group of claim 1 in detection of fusarium oxysporum cucumber specialization.

3. The fusarium oxysporum cucumber specialized LAMP detection kit is characterized by comprising the following reagents: 20 Xprimer mixture, fusarium oxysporum cucumber specialised genomic DNA, 2 XSLAMP MagicMix and sterilized ultra-pure water;

the 20X primer mixture contains the fusarium oxysporum cucumber specialized LAMP primer group of claim 1.

4. The fusarium oxysporum cucumber specialized LAMP detection kit of claim 3, wherein the concentration of each primer in the 20 x primer mixture is as follows:

FIP 32 μM、BIP 32 μM、F3 4μM、B3 4μM、LB 8μM。

5. the fusarium oxysporum cucumber specialization LAMP detection kit of claim 3, wherein the 2 x LAMP MagicMix contains a DNA polymerase, a blue DNA tracer dye and a reaction buffer.

6. The application of the fusarium oxysporum cucumber specialization type LAMP detection kit according to claim 3 in detecting fusarium oxysporum cucumber specialization type, which is characterized by comprising the following steps:

(1) Extracting genome DNA of a sample to be detected;

(2) LAMP reaction:

amplification reaction system: the 20 Xprimer mixture of claim 3, wherein 1. Mu.L of the genomic DNA of the sample to be tested extracted in the step (1) is 2. Mu.L, 2 XLAMP MagicMix is 10. Mu.L, sterilized ultrapure water is supplemented to 20. Mu.L, and Fusarium oxysporum cucumber specialized genomic DNA is used as a positive control, sterilized ultrapure water is used as a negative control, and the reaction procedure is as follows: keeping the temperature at 65 ℃ for 50min;

(3) After the reaction was completed, the change in color was observed: if the reaction liquid is blue after the reaction is finished, the sample to be tested contains fusarium oxysporum cucumber specialization;

if the reaction liquid is colorless or light blue after the reaction is finished, the sample to be tested does not contain fusarium oxysporum cucumber specialization.