阅读说明：本技术 利用数字pcr检测番茄溃疡病菌的方法及其使用的成套试剂 (Method for detecting tomato canker pathogen by using digital PCR and kit reagent used by method ) 是由 田茜 张红梅 赵文军 周佩 于 2020-05-26 设计创作，主要内容包括：本发明公开了利用数字PCR检测番茄溃疡病菌的方法及其使用的成套试剂。本发明所提供的成套试剂由引物对Cmm和探针P组成；引物对Cmm由引物F和引物R组成；引物F、引物R和探针P的核苷酸序列依次如SEQ ID NO:1-SEQ ID NO:3所示。实验证明,采用本发明所提供的方法数字PCR检测番茄溃疡病菌,特异性好、灵敏度高且重复性好,还能够有效解决检测番茄溃疡病菌中出现的假阴性的结果。本发明为检测重要检疫性病害提供了一种新的可靠的检测手段,具有重要的应用价值。(The invention discloses a method for detecting tomato canker pathogen by using digital PCR and a kit used by the method. The kit provided by the invention consists of a primer pair Cmm and a probe P; the primer pair Cmm consists of a primer F and a primer R; the nucleotide sequences of the primer F, the primer R and the probe P are sequentially shown as SEQ ID NO. 1-SEQ ID NO. 3. Experiments prove that the method for detecting the tomato canker germ by adopting the digital PCR has the advantages of good specificity, high sensitivity and good repeatability, and can effectively solve the problem of false negative results in the detection of the tomato canker germ. The invention provides a new reliable detection means for detecting important epidemic diseases, and has important application value.)

1. The kit for detecting the tomato canker pathogen comprises a primer pair Cmm and a probe P; the primer pair Cmm consists of a primer F and a primer R;

the primer F is a1) or a 2):

a1) 1, a single-stranded DNA molecule shown in SEQ ID NO;

a2) a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID NO. 1 and has the same function as SEQ ID NO. 1;

the primer R is b1) or b2) as follows:

b1) a single-stranded DNA molecule shown as SEQ ID NO. 2;

b2) a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID NO. 2 and has the same function as SEQ ID NO. 2;

the probe P is c1) or c2) as follows:

c1) a single-stranded DNA molecule represented by SEQ ID NO. 3;

c2) 3 is a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO. 3 and has the same function as the sequence 3.

2. The kit of claim 1, wherein: the probe Cmm CP has a fluorescent label at its end.

3. The kit of claim 2, wherein: the probe Cmm CP has a FAM fluorescent label at the 5 'end and a TAMRA fluorescent label at the 3' end.

4. The primer pair Cmm in the kit of claim 1.

5. Use of a kit according to any one of claims 1 to 3, as d1) or d2) or d 3):

d1) preparing a kit for detecting or assisting in detecting the tomato canker germs;

d2) detecting or assisting to detect whether the sample to be detected contains or is suspected to contain the tomato canker pathogen;

d3) and identifying or assisting in identifying whether the bacteria to be detected are candidate tomato canker germs.

6. The primer pair Cmm of claim 4, which is d1) or d2) or d3) as follows:

d1) preparing a kit for detecting or assisting in detecting the tomato canker germs;

d2) detecting or assisting to detect whether the sample to be detected contains or is suspected to contain the tomato canker pathogen;

d3) and identifying or assisting in identifying whether the bacteria to be detected are candidate tomato canker germs.

7. Kit comprising a set of reagents according to any one of claims 1 to 3 or a primer pair Cmm according to claim 4.

8. Use of a kit according to claim 7, being d2) or d 3):

d2) detecting or assisting to detect whether the sample to be detected contains or is suspected to contain the tomato canker pathogen;

d3) and identifying or assisting in identifying whether the bacteria to be detected are candidate tomato canker germs.

9. A method for detecting whether a sample to be detected contains or is suspected to contain the tomato canker pathogen is A1) or A2):

A1) performing digital PCR using the total DNA of a sample to be tested as a template and the kit of any one of claims 1 to 3, and then performing the following evaluation: if the reagent set can realize digital PCR of the total DNA, the sample to be detected contains or is suspected to contain the tomato canker pathogen; if the reagent set can not realize the digital PCR of the total DNA, the sample to be detected does not contain or is suspected to contain the tomato canker pathogen;

A2) using the total DNA of the sample to be tested as a template, performing PCR amplification on Cmm by using the primer pair of claim 4, and then performing the following judgment: if the primer pair Cmm can realize the PCR amplification of the total DNA, the sample to be detected contains or is suspected to contain the tomato canker pathogen; and if the primer pair Cmm can not realize the PCR amplification of the total DNA, the sample to be detected does not contain or is suspected to contain the tomato canker pathogen.

10. A method for identifying whether a test bacterium is a candidate bacterial canker of tomato, B1) or B2):

B1) performing digital PCR with the genomic DNA of the bacteria to be tested or the bacterial liquid of the bacteria to be tested as a template and the kit of any one of claims 1 to 3, and then performing the following evaluation: if the reagent set can realize digital PCR on the genome DNA or the bacterial liquid, the bacteria to be detected are candidate tomato canker germs; if the complete set of reagents cannot realize the digital PCR of the genome DNA or the bacterial liquid, the bacteria to be detected are candidate non-tomato canker germs;

B2) using the genome DNA of the bacteria to be tested or the bacterial liquid of the bacteria to be tested as a template, performing PCR amplification on the Cmm by using the primer pair of claim 4, and then performing the following judgment: if the primer pair Cmm can realize PCR amplification on the genome DNA or the bacterial liquid, the bacteria to be detected are candidate tomato canker germs; and if the primer pair Cmm can not realize the PCR amplification of the genome DNA or the bacterial liquid, the bacteria to be detected are candidate non-tomato canker germs.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for detecting tomato canker pathogen by using digital PCR and a kit used by the method.

Background

Tomato canker pathogen, i.e., clavibacterium michiganensis subspecies (Cmm), can cause tomato canker, and infected plants can produce different symptoms due to different ages of host plants, susceptibility of varieties, virulence of tomato canker pathogen, and temperature and humidity of external environment. At present, the tomato canker pathogen has become a worldwide disease and is listed as an important epidemic disease by more than 20 countries including China. The tomato canker pathogen is a typical seed-borne disease, and a plant infected with the tomato canker pathogen can generate seeds or fruits infected with the pathogen, and the planting by using sterile seeds or the transplantation by using the sterile plant becomes an important method for preventing the spread of the tomato canker pathogen.

The detection method of the tomato canker pathogen comprises the traditional detection methods including a symptom observation method, a colony morphology observation method, a plant detection method, a seed detection method and a pathogenicity detection method, as well as a serological detection method and a molecular biological PCR detection method. The PCR detection method is the most commonly used technique at present, but the detection result of the seed sample with low bacteria quantity is not ideal, and false negative can be caused. Furthermore, with the discovery of new subspecies, the specificity of existing detection methods needs further confirmation to determine whether Cmm can be accurately distinguished from other subspecies, especially closely related non-pathogenic subspecies.

Digital PCR (Digital-PCR) is a novel quantitative molecular detection technology, and compared with real-time fluorescent quantitative PCR, the result value of the Digital PCR does not depend on the CT value any more. Digital PCR has been successfully applied to gene profiling, prenatal diagnosis, and cancer-related allele and mutation detection.

Disclosure of Invention

The invention aims to detect the bacterial canker of tomato.

The invention firstly protects a complete set of reagent for detecting the tomato canker pathogen, and the reagent can consist of a primer pair Cmm and a probe P; the primer pair Cmm can consist of a primer F and a primer R;

the primer F can be a1) or a2) as follows:

a1) 1, a single-stranded DNA molecule shown in SEQ ID NO;

a2) a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID NO. 1 and has the same function as SEQ ID NO. 1;

the primer R can be b1) or b2) as follows:

b1) a single-stranded DNA molecule shown as SEQ ID NO. 2;

b2) a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID NO. 2 and has the same function as SEQ ID NO. 2;

the probe P may be c1) or c2) as follows:

c1) a single-stranded DNA molecule represented by SEQ ID NO. 3;

c2) 3 is a single-stranded DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID NO. 3 and has the same function as the sequence 3.

The "substitution and/or deletion and/or addition by one or several nucleotides" in the a2), the b2), or the c2) may be a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

In the kit, the probe P may have a fluorescent label at its end.

In the kit, the probe P may have a FAM fluorescent label at the 5 'end and a TAMRA fluorescent label at the 3' end.

In the above kit, the molar ratio of the primer F, the primer R and the probe P may be specifically 1: 1: 1.

the primer pair Cmm in the kit also belongs to the protection scope of the invention.

In the primer pair Cmm, the molar ratio of the primer F to the primer R may be specifically 1: 1.

the invention also protects the application of any one of the above-mentioned kits, which can be d1) or d2) or d3) as follows:

d1) preparing a kit for detecting or assisting in detecting the tomato canker germs;

d2) detecting or assisting to detect whether the sample to be detected contains or is suspected to contain the tomato canker pathogen;

d3) and identifying or assisting in identifying whether the bacteria to be detected are candidate tomato canker germs.

The invention also protects the application of any one of the primer pairs Cmm, which is d1) or d2) or d 3):

d1) preparing a kit for detecting or assisting in detecting the tomato canker germs;

d2) detecting or assisting to detect whether the sample to be detected contains or is suspected to contain the tomato canker pathogen;

d3) and identifying or assisting in identifying whether the bacteria to be detected are candidate tomato canker germs.

The invention also provides a kit containing any one of the kit reagents or any one of the primer pairs Cmm.

The invention also protects a preparation method of the kit, and the primer F, the primer R and/or the probe P are respectively and independently packaged.

The invention also protects the application of the kit, namely d2) or d 3):

d2) detecting or assisting to detect whether the sample to be detected contains or is suspected to contain the tomato canker pathogen;

d3) and identifying or assisting in identifying whether the bacteria to be detected are candidate tomato canker germs.

The invention also protects a method for detecting whether the sample to be detected contains or is suspected to contain the tomato canker pathogen.

The method for detecting whether the sample to be detected contains or is suspected to contain the tomato canker pathogen, which is protected by the invention, can be a method A1), and comprises the following steps: taking the total DNA of a sample to be detected as a template, carrying out digital PCR by using any one of the above-mentioned complete sets of reagents, and then carrying out the following judgment: if the reagent set can realize digital PCR of the total DNA, the sample to be detected contains or is suspected to contain the tomato canker pathogen; if the reagent set cannot realize the digital PCR of the total DNA, the sample to be detected does not contain or is suspected to contain the tomato canker pathogen.

The method for detecting whether the sample to be detected contains or is suspected to contain the tomato canker pathogen, which is protected by the invention, can be a method A2), and comprises the following steps: taking the total DNA of a sample to be detected as a template, carrying out PCR amplification on the Cmm by using any one of the primers, and then carrying out the following judgment: if the primer pair Cmm can realize the PCR amplification of the total DNA, the sample to be detected contains or is suspected to contain the tomato canker pathogen; and if the primer pair Cmm can not realize the PCR amplification of the total DNA, the sample to be detected does not contain or is suspected to contain the tomato canker pathogen.

In any of the above methods, the sample to be tested may be a seed or a plant. The plant may specifically be a tomato plant or a tomato leaf. The seed may be a tomato seed.

When the sample to be detected is a plant, the total DNA of the sample to be detected can be the genome DNA of the plant to be detected.

When the sample to be detected is a seed, the total DNA of the sample to be detected can be the total DNA of the seed to be detected, the total DNA of the seed to be detected is obtained by firstly treating the seed, collecting the precipitate and then extracting the DNA from the precipitate, and the method for treating the seed comprises the steps of pouring the seed sample into a sterilized triangular flask, adding a proper amount of sterile water to completely cover the seed, soaking overnight at 4 ℃, taking 10m L leachate, centrifuging and collecting the precipitate.

The invention also protects a method for identifying whether the bacteria to be detected are candidate tomato canker pathogens.

The method for identifying whether the bacteria to be detected are candidate tomato canker pathogens or not, which is specifically the method B1), comprises the following steps: taking the genome DNA of the bacteria to be tested or the bacterial liquid of the bacteria to be tested as a template, carrying out digital PCR by using any one of the above complete sets of reagents, and then carrying out the following judgment: if the reagent set can realize digital PCR on the genome DNA or the bacterial liquid, the bacteria to be detected are candidate tomato canker germs; if the complete set of reagents can not realize the digital PCR of the genome DNA or the bacterial liquid, the bacteria to be detected are candidate non-tomato canker germs.

The method for identifying whether the bacteria to be detected are candidate tomato canker pathogens or not, which is specifically the method B2), comprises the following steps: taking the genome DNA of the bacteria to be detected or the bacterial liquid of the bacteria to be detected as a template, carrying out PCR amplification on the Cmm by using any one of the primers, and then carrying out the following judgment: if the primer pair Cmm can realize PCR amplification on the genome DNA or the bacterial liquid, the bacteria to be detected are candidate tomato canker germs; and if the primer pair Cmm can not realize the PCR amplification of the genome DNA or the bacterial liquid, the bacteria to be detected are candidate non-tomato canker germs.

In any of the above methods, the bacterial liquid of the bacteria to be tested may be obtained by inoculating the strain to be tested to an NB liquid culture medium and culturing.

In any of the above methods, the reaction system for performing digital PCR may be 20. mu. L, and comprises 10. mu. L2 × ddPCRTM supermix for probes (No dUTP), 1. mu. L aqueous solution of primer F, 1. mu. L aqueous solution of primer R, 0.5. mu. L aqueous solution of probe P, 1. mu. L template, and 6.5. mu. L double distilled water, wherein the concentrations of primer F, primer R, and probe P in the reaction system are all 10. mu.M.

In any of the above methods, the reaction procedure for performing digital PCR may be: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 10 s; annealing and extension at 60 ℃ for 45s, for 40 cycles, and curing the droplets at 98 ℃ for 10 min.

In any of the above methods, the reaction system for performing PCR amplification may be 20. mu. L, including 10. mu. L480probes master, 1 mu L primer F aqueous solution, 1 mu L primer R aqueous solution, 0.5 mu L probe P aqueous solution, 1 mu L template and 6.5 mu L double distilled water, wherein the concentration of the primer F, the primer R and the probe P in the reaction system is 10 mu M.

In any of the above methods, the reaction procedure of "performing PCR amplification" may be: 5min at 95 ℃; annealing at 95 ℃ for 10s and 60 ℃ for 45s, and 40 cycles.

Experiments prove that the method for detecting the tomato canker pathogen by adopting the digital PCR has higher specificity (only can amplify the tomato canker pathogen strains, and the amplification results of other 14 test strains are negative), high sensitivity, and the detection limit of real-time fluorescent quantitative PCR can reach 1.08 × 10³CFU/m L, detection limit of digital PCR of 10.8CFU/m L, digital PCR pair compared with real-time fluorescent quantitative PCRHigher reproducibility and higher detection rate at low copy number; the artificial simulation germ-carrying seeds and the natural seeds are detected, so that the method is not only quick, but also can accurately detect the low germ-carrying amount seed samples. Therefore, the method for detecting the tomato canker pathogen by using the digital PCR has the characteristics of good specificity, high sensitivity and repeatability, can effectively solve the problem of false negative results in the detection of the tomato canker pathogen, and provides a new reliable detection means for detecting important epidemic disease tomato canker pathogen. The invention has important application value.

Drawings

FIG. 1 shows the result of specificity detection.

FIG. 2 shows the results of the sensitivity detection by the digital PCR method.

FIG. 3 shows the results of the first step in example 6.

FIG. 4 shows the results of the detection in step two of example 6.

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 conventional biochemicals, unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The novel plant genome DNA extraction kit (centrifugal column type) is a product of Tiangen Biochemical technology (Beijing) Co., Ltd, and the product number is DP 320-02.480probes master, 2 × ddPCRTMsermix for probes (No dUTP), microdroplet oil, microdroplet card, gasket, and 96 well plate are all products of Bio-Rad, Inc., USA.

NB liquid culture medium, dissolving beef extract 3.0g, peptone 5.0g, yeast powder 0.5g and glucose 10.0g in appropriate amount of sterile water, adding sterile water to constant volume of 1000m L, adjusting pH to 7.0, and sterilizing at 121 deg.C for 20 min.

The strain names, strain numbers and sources of the 19 strains referred to in the examples below are detailed in table 1. Wherein 5 strains shown in sequence 1-sequence 5 are all Cmm; the 8 strains shown in the sequence 6-the sequence 13 are all clavibacterium with no Cmm; the 6 strains shown in the sequence 14-the sequence 19 can cause tomato diseases (non-tomato canker diseases).

TABLE 1

Note that ATCC is American type culture Collection, NCPPB is UK national center for the Collection of phytopathogenic species, ICMP is International center for the Collection of microorganisms from botanicals, and L MG is the university of root, Belgium microbiological laboratory.