CN1824798A - 16SrⅡGroup Phytoplasma PCR Detection Kit and Its Detection Method - Google Patents

Abstract

This invention relates to mycoplasma PCR detecting kit and its detecting method, it is specially used in 16SrII group mycoplasma detection. The kit includes PCR typic reaction reagent, mainly includes PCR reaction buffer solution, MgCl<2>,dNTPs,Taq DNA polyase, mycoplasma 16SrRNA gene general using primer and special primer of 16SrII group mycoplasma. The detection method includes PCR reaction and electrophoresis detection. 1500bp and 990bp two strip differential amplification stripes can be detected of the sample with 16SrII group mycoplasma, but there is only one 1500bp strip can be detected in the sample without 16SrII group mycoplasma. The using of this kit is convenient, and it has high specificity and detection sensitivity. It can directly detect mycoplasma disease sample and also can determine the mycoplasma that belongs to 16SrII.

Description

16SrII group phytoplasma PCR detection kit and its detection method

technical field

The invention relates to a phytoplasma PCR detection kit and a detection method thereof, belonging to the field of biotechnology.

Background technique

Phytoplasma (formerly known as Mycoplasma-like Organism, referred to as MLO) is a kind of prokaryotic organisms without cell walls that exist in plant sieve tube cells. Since phytoplasma was first discovered by Japanese scholar Yoji Doi in 1967, so far, there have been more than 300 plant phytoplasma diseases reported in the world. Because phytoplasma strictly parasitizes the phloem of host plants and cannot be artificially cultivated, its genetic essence, biochemical characteristics, nutritional requirements, etc. are unknown, and the development of detection and identification technology is also hindered. The early identification of phytoplasma was mainly carried out through biological characteristics, such as symptom characteristics, and the relationship with insect mediators. These methods are time-consuming and laborious, and the results are often not very reliable. In the 1980s, with the development and application of serology, molecular probes and PCR technology, a relatively simple, sensitive and reliable method was provided for the detection of phytoplasma. Phytoplasma can be identified and classified by sequence analysis of relatively conserved genes (16SrRNA gene or ribosomal protein gene) or RFLP analysis of amplified products during the evolution of phytoplasma, and the classification system of phytoplasma has also been established.

According to the RFLP analysis of phytoplasma 16SrRNA gene and ribosomal protein gene, phytoplasma has been divided into 14 definite groups, namely: 16SrI, 16SrII...16SrXIV. Among them, the 16SrII group was represented by peanut arbuscular disease, mainly including peanut arbuscular, sweet potato arbuscular, lime arbuscular, faba bean leaflet, sweet potato leaflet and cactus arbuscular, etc.

According to the phytoplasma 16SrRNA gene sequence, many specific primers for phytoplasma groups have been designed, including 16SrI group, 16SrIII group, 16SrV group, 16SrVI group, 16SrVII group, 16SrX group, etc. The 16SrRNA gene conserved region of the phytoplasma members of the 16SrII group was designed, and obviously the specific primers could not directly identify the phytoplasma of the 16SrII group.

Contents of the invention

The object of the present invention overcomes the deficiencies of the prior art, and provides a fast, accurate, sensitive, high specificity and convenient operation detection kit and application method specially used for 16SrII group phytoplasma.

The technical scheme of the present invention is: a PCR detection kit of 16SrII group phytoplasma, comprising the following components:

(1) Phytoplasma 16SrRNA gene universal primer sequence:

R16mF2: 5'-CATGCAAGTCGAACGGA-3'

R16mR1: 5'-CTTAACCCCAATCATCGAC-3'

(2) Specific primer sequences for 16SrII group phytoplasma:

R16(II)F1: 5'-AGTGGCGAACCATTTGTT-3';

R16(II)R1: 5'-CTCGAATAAACGAGTTAGG-3'

(3) Kit reaction system: containing 10×PCR reaction buffer, MgCl ₂ , dNTPs, and Taq DNA polymerase.

The final concentration of each reagent in the reaction system of the kit is: 1×PCR reaction buffer, 2.0mM MgCl ₂ , 0.2mMdNTPs, 0.6μM R16mF2/R1 primer (or R16(II)F1/R1) and 4-5 units of Taq DNA polymerase.

PCR detection method of 16SrII group phytoplasma:

(1) PCR reaction: After adding 0.1-1 μg of total DNA of susceptible plants to the above detection kit, add ultrapure water to make the total reaction volume 50 μl. Then carry out PCR reaction, the reaction parameters are 95°C pre-denaturation for 5min; 95°C denaturation for 30sec, 60°C annealing for 2min, 72°C extension for 3min, after 10 reaction cycles, add primer R16(II)F1/R1 and denature at 95°C for 30sec , annealed at 40°C for 2min, extended at 72°C for 3min, after 10 cycles, kept at 72°C for 10min, and stored in a refrigerator at 4°C.

(2) Electrophoresis detection: take 5 μl of the reaction product and electrophoresis in 0.7-1.0% agarose gel, stain with ethidium bromide and take pictures on the gel imaging system for observation: the electrophoresis detection results of samples containing 16SrII group phytoplasma can be observed Two specific amplified bands of 1500bp and 990bp were detected, but only the 1500bp amplified band could be observed in the electrophoresis test results of samples of non-16SrII phytoplasma.

Compared with the prior art, the 16SrII group phytoplasma PCR detection kit and detection method provided by the present invention have the following advantages and positive effects:

(1) It has high detection sensitivity. In the first PCR reaction, phytoplasma-specific large fragments can be amplified by using phytoplasma 16SrRNA gene universal primers for 10 cycles, and these products provide enough templates for the second amplification, which greatly improves the detection efficiency. sensitivity. After adding the specific primers of the 16SrII group, whether there is a specific amplification product of the 16SrII group can be detected by electrophoresis after 10 reaction cycles.

(2) It has high amplification specificity. Generally, during the PCR reaction, the higher the annealing temperature, the higher the specificity of the amplified product. In the method of using this kit, in the first amplification reaction using the phytoplasma universal primer (R16mF2/R1), in order to improve the specificity of the phytoplasma-specific large fragment, a higher annealing temperature (60°C) was adopted ; while in the second amplification reaction, a lower annealing temperature (40°C) was used, but still only a 990bp amplified fragment consistent with the primer design was amplified, which is enough to illustrate that the reaction kit Has a high specificity.

(3) The phytoplasma disease samples can be quickly detected, and at the same time, the phytoplasma belonging to the 16SrII group can be classified and identified. According to the 16SrDNA sequence comparison results of 6 kinds of 16SrII group and other 16Sr group phytoplasma strains, this kit designs a pair of specific primers for PCR detection of 16SrII group phytoplasma, and can simultaneously detect and identify 16SrII after a second PCR reaction Group phytoplasma.

Description of drawings

Figure 1 16SrII group phytoplasma-specific primer cycle PCR amplified bands

M: DNA molecular weight standard; 1: CK; 2-3 are Paulownia arbuscules and mulberry Verticillium dahliae, both are non-16SrII group phytoplasma samples; 4-5 are peanut arbuscular and cactus arbuscular branches, respectively, 16SrII group phytoplasma sample.

Detailed ways

1. Synthesis and preparation of universal primer sequence for phytoplasma 16SrRNA gene

Primers were synthesized with reference to the phytoplasma 16SrRNA gene universal primer pair R16mF2/R16mR1 sequence reported by Lee et al., and the amplified length was about 1500 bp. The primers were dissolved in an appropriate amount of sterile water to a final concentration of 10 μM.

2. Design, synthesis and preparation of 16SrII group phytoplasma PCR primers

According to the comparison results of the 16SrDNA sequences of six 16SrII group and other 16Sr group phytoplasma strains (Fig. 1), an upstream sequence homologous to the 16SrDNA segment 416-433 of the cactus arbuscular disease phytoplasma CWB strain was designed and synthesized. The primer R16(II)F1 and the downstream primer R16(II)R1 complementary to the 1387-1405 segment base sequence have an amplification length of 990bp. The primers were dissolved in an appropriate amount of sterile water to a final concentration of 10 μM.

416 433 1387 1405

↓ ↓ ↓ ↓ ↓ ↓

CWB AGTGGCGAACCATTTGTT……………CCTAACTCGTTTATTCGAG

FBP AGTGGCGAACCATTTGTT……………CCTAACTCGTTTATTCGAG

WBDL AGTGGTGAACCATTTGTT……………CCTAACTCGTTTA.TCGAG

PnWB AATGGCGAACCATTTGTT……………CCTAACTCGTTTATTCGAG

SUNHP AATGGCGAACCATTTGTT……………CCTAACTCGTTTATTCGAG

SPWB AATGGCGAACCATTTGTT……………CCTAACTCGTTTATTCGAG

WX AGT.G.GAAAAACTCCCT……………CCTAACTTCGCAAGA..AG

LY AGT.G.GAAAAACTATCT……………CCTAACTGCGTAAGC..AG

PPWB AGT.G.GAAAAACTATCT……………CCTAACTTCGTTAGA..AG

EY1 AGTGG.GAAAAACTATAT…………CTTAACTTCGCAAGA..AG

CP AGT.G.GAAAAACTATAT…………CTTAACTTCGCAAGA..AG

AshY AGT.G.GAAAAACTATCT…………CTTAACTTCGCAAGA..CG

LfWB AGT.G.GAAAAACTATCT…………CTTAATTTCGTAAGA..AA

SAY GAT.G.GAAAAATCATTC……………CCTAACTTCGCAAG..AAG

STOL GGT.G.GAAAAACCATTA……………CCTAACTTGAGCAATCAAG

AT GAT.G.GAAAAATCATTC……………CCTAACTTGCAA....AAG

3. Assembly of the PCR kit: The reagents for the PCR reaction are prepared according to the reaction system of the kit.

4. Detection method of 16SrII group phytoplasma PCR detection kit

(1) PCR reaction: After adding 0.1-1 μg of total DNA from susceptible plants to the above PCR detection kit, add ultrapure water to make the total reaction volume 50 μl. Then carry out PCR reaction, the reaction parameters are 95°C pre-denaturation for 5min; 95°C denaturation for 30sec, 60°C annealing for 2min, 72°C extension for 3min, after 10 reaction cycles, add primer R16(II)F1/R1 and denature at 95°C for 30sec , annealed at 40°C for 2min, extended at 72°C for 3min, and held at 72°C for 10min after 10 cycles.

(2) Electrophoresis detection: Mix 5 μl of the reaction product with 1 μl of 6-fold electrophoresis loading buffer, electrophoresis in 0.7-1.0% agarose gel, stain with ethidium bromide and take pictures on the gel imaging system for observation.

The experimental results are as follows:

Using the total DNA of 2 non-16SrII phytoplasmas such as Paulownia arbuscular disease and mulberry verticillium wilt as controls, the specificity of the kit was tested according to the method of use of the kit. The results showed that: Paulownia arbuscular disease, mulberry verticillium wilt PCR with the total DNA of 2 non-16SrII group phytoplasmas as templates only amplified a specific band of about 1.5kb, while the total DNA of 2 16SrII group phytoplasmas such as peanut arbuscules and cactus arbuscules as templates However, two mixed bands of about 1.5 kb and about less than 1.0 kb were amplified by PCR, and the band of about less than 1.0 kb was consistent with the primer design. It shows that the kit has high specificity.

Claims (3)

1, a kind of PCR detection kit and detection method thereof of 16SrII group pytoplasma, it is characterized in that: test kit comprises following composition:

(1) pytoplasma 16S rRNA gene universal primer sequence:

R16mF2：5’-CATGCAAGTCGAACGGA-3’

R16mR1：5’-CTTAACCCCAATCATCGAC-3’

(2) the special primer sequence of 16SrII group pytoplasma:

R16(II)F1：5’-AGTGGCGAACCATTTGTT-3’；

R16(II)R1：5’-CTCGAATAAACGAGTTAGG-3’

(3) test kit reaction system: contain 10 * PCR reaction buffer, MgCl ₂, dNTPs, Taq archaeal dna polymerase.

2, the PCR detection kit and the detection method thereof of 16SrII group pytoplasma according to claim 1, it is characterized in that: each reagent final concentration of described test kit reaction system is: 1 * PCR reaction buffer, 2.0mM MgCl ₂, 0.2mM dNTPs, the Taq archaeal dna polymerase of 0.6 μ M R16mF2/R1 (or R16 (II) F1/R1) primer and 4-5 unit.

3, the PCR detection kit and the method thereof of 16SrII group pytoplasma according to claim 1, its detection step is:

(1) PCR reaction: add the total DNA of 0.1-1 μ g disease plant in above-mentioned detection kit after, adding ultrapure water again, to make the reaction cumulative volume be 50 μ l, carries out the PCR reaction, and reaction parameter is for being 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30sec, 60 ℃ of annealing 2min, 72 ℃ are extended 3min, add primer R16 (II) F1/R1 after 10 reaction cycle and in 95 ℃ of sex change 30sec, 40 ℃ of annealing 2min, 72 ℃ are extended 3min, and 10 back 72 ℃ of insulation 10min of circulation preserve in 4 ℃ of refrigerators;

(2) electrophoresis detection: get 5 μ l reaction product electrophoresis in the sepharose of 0.7-1.0%, the observation of taking pictures on gel imaging system behind the ethidium bromide staining: the sample electrophoresis detected result that contains 16SrII group pytoplasma can be observed two specific amplified bands of 1500bp and 990bp, but not the sample electrophoresis detected result of 16SrII group pytoplasma can only be observed the amplified band of 1500bp.

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