WO2020173121A1

WO2020173121A1 - Double pcr detection method for sugarcane white leaf disease phytoplasma and xanthomonas albilineans and primer set thereof

Info

Publication number: WO2020173121A1
Application number: PCT/CN2019/115465
Authority: WO
Inventors: 张荣跃; 黄应昆; 李文凤; 王晓燕; 李婕; 单红丽; 仓晓燕; 尹炯; 罗志明
Original assignee: 云南省农业科学院甘蔗研究所
Priority date: 2019-02-27
Filing date: 2019-11-05
Publication date: 2020-09-03
Also published as: CN109680087B; CN109680087A

Abstract

Disclosed are a double PCR detection method for sugarcane white leaf disease phytoplasma and Xanthomonas albilineans and a primer set thereof. The present invention designs a pair of specific primers for the tuf gene of sugarcane white leaf disease phytoplasma and the rpoD gene of Xanthomonas albilineans, respectively, and by means of optimization, establishes a double PCR reaction system that is capable of simultaneously detecting the sugarcane white leaf disease phytoplasma and the Xanthomonas albilineans.

Description

A double PCR detection method for sugarcane white leaf disease phytoplasma and Xanthomonas albicans and its primer set Technical Field

The invention belongs to the technical field of plant protection, and specifically relates to the detection of sugarcane white leaf disease phytoplasma and Xanthomonas albicans by double PCR technology and specific primer sets used. Background technique

Sugarcane white leaf phytoplasma caused by Sugarcane white leaf phytoplasma and sugarcane white leaf phytoplasma caused by Xanthomonas albilineans (Ashby) Dowson are both devastating diseases of sugarcane deer. The sugar cane and sugar industry suffered huge economic losses. The main symptoms of sugarcane white leaf disease are leaf bleaching, increased tillers and dwarfing. The main symptoms of sugarcane white streak disease are white streaks on the leaves. Some tip leaves are albino, side buds germinate, and the germinated side bud leaves are also white. The symptoms of these two diseases are very similar, that is, leaf bleaching. Therefore, it is difficult to accurately distinguish the two diseases based on the symptoms alone, and the diagnosis must be confirmed by molecular testing.

At present, the method for detecting sugarcane white leaf disease phytoplasma is to perform nested PCR with universal primers (P1/P7 and R16F2n/R16R2) designed based on the 16S rRNA gene of phytoplasma. This method uses universal primers for phytoplasma, and the detection is accurate The detection rate is not high and the detection process is relatively cumbersome. Although the detection of Xanthomonas albicans uses specific primers XAF1/XAR1, the PCR reaction procedure used in this method is complicated and the reaction time is long. Since the symptoms of these two diseases are very similar, in order to detect and confirm the samples of suspected two diseases, the two pathogens must be detected separately and two PCR reactions must be performed. At present, there is no report on the method of detecting sugarcane white leaf disease phytoplasma using specific primers, and there is no double PCR method for detecting these two pathogens at the same time.

Both sugarcane white leaf disease and sugarcane white streak disease are quarantine diseases transmitted by seedlings. With the current frequency of introduction and seed adjustment at home and abroad, there is an urgent need for a more rapid and accurate method for detecting these two pathogens. Summary of the invention

In order to solve the problem of sugarcane white leaf disease caused by sugarcane white leaf disease phytoplasma and sugarcane white leaf disease caused by Xanthomonas albicans, the symptoms are very similar. At present, when the two diseases are diagnosed, two PCR reactions must be performed to detect the two diseases separately. The present invention provides a rapid, accurate and specific double PCR detection method for sugarcane white leaf disease phytoplasma and Xanthomonas albicans and its specificity. Primer set, kit.

The present invention provides a dual PCR specific primer set for detecting sugarcane white leaf disease phytoplasma and Xanthomonas albicans. It consists of a tuf gene specific primer for detecting sugarcane white leaf disease phytoplasma and a white-striped xanthoma The rpoD gene-specific primers of the spores are composed of tuf-SF primers and tuf-SR primers, and the target fragment length is 290 bp, the nucleotide sequence of the tuf-SF primer is shown in SEQ ID NO: 1, the nucleotide sequence of the tuf-SR primer is shown in SEQ ID NO: 2; the rpoD gene-specific primer is composed of rpoD- The SF primer and rpoD-SR primer consist of a target fragment length of 498 bp. The nucleotide sequence of the rpoD-SF primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the rpoD-SR primer is shown in SEQ ID NO: 4 shown.

The present invention provides a double PCR detection method for sugarcane white leaf disease phytoplasma and Xanthomonas albicans, including a double PCR reaction system and a double PCR reaction program. The primers used in the double PCR reaction system are the aforementioned one for detecting sugarcane white leaf disease. The double PCR specific primer set for leaf disease phytoplasma and Xanthomonas albicans. After the double PCR reaction is terminated, take the double PCR amplification product and perform agarose gel electrophoresis. If a single band with a size of 290 bp is amplified, then The tested sugarcane sample was infected with the sugarcane white leaf disease phytoplasma; if a single band with a size of 498 bp was amplified, the tested sugarcane sample was infected with Xanthomonas albicans; if it was amplified to both 290 bp and 498 bp In the two belts, the tested Ganlu samples were infected with the phytoplasma of Gansial white leaf disease and Xanthomonas albicans at the same time.

The described method for double PCR detection of sugarcane white leaf disease phytoplasma and Xanthomonas albicans, its double PCR reaction system: 25 pL double PCR reaction system, in which sterile ddH ₂ 0 12.7, 10 ^ PCR buffer 4.0 _[i L, 25 mmol/L MgCl ₂ 2.0 ^L, 10 mmol/L dNTPs 2.0 JIL, 20 _[i mol/L tuf-SF 1.0 _[i L, 20 ^irnol/L tuf-SR 1.0 _[i L, 20 pmol/L rpoD-SF 0.5 ^iL, 20 pmol/L rpoD-SR 0.5

5 U/^LL Taq DNA polymerase 0.3 ^LL, template

DNA lO ^L; its double PCR reaction program: 94 ^° (: pre-denaturation 3 1 ^11 _; 94 ^° C denaturation 30 s, 58 ^° C annealing 30 s, 72 ^° C extension 1 min, 35 cycles; the last 72 ^° C extends for 7 min.

The described method for double PCR detection of sugarcane white leaf disease phytoplasma and Xanthomonas albicans, when the double PCR amplified product is subjected to agarose gel electrophoresis, 5) _iL double PCR amplified product with 1.5% agar Glycogel electrophoresis.

The present invention provides a double PCR detection kit for sugarcane white leaf disease phytoplasma and Xanthomonas albicans, comprising: tuf gene-specific primers and rpoD gene-specific primers, and the tuf gene-specific primers are used for detecting sugarcane White leaf disease phytoplasma, the target fragment length is 290 bp, the tuf gene specific primer is composed of tuf-SF primer and tuf-SR primer, the nucleotide sequence of tuf-SF primer is shown in SEQ ID NO: 1, tuf- The nucleotide sequence of the SR primer is shown in SEQ ID NO: 2. The rpoD gene-specific primer is used to detect Xanthomonas albicans, the target fragment length is 498 bp, and the rpoD gene-specific primer is composed of rpoD-SF primer and rpoD-SR primer composition, the nucleotide sequence of rpoD-SF primer is shown in SEQ ID NO: 3, and the nucleotide sequence of rpoD-SR primer is shown in SEQ ID NO: 4.

The said double PCR detection kit for sugarcane white leaf disease phytoplasma and Xanthomonas albicans further includes a double PCR reaction solution, 24 pL of the double PCR reaction solution contains sterile ddH ₂ 0 12.7 ^L, 10xPCR buffer Solution 4.0, 25 mmol/L MgCl ₂ 2.0 ^L, 10 mmol/L dNTPs 2.0 JIL, 20 _[i mol/L tuf-SF 1.0 _[i L, 20 ^imol/L tuf-SR 1.0 _[i L, 20 _{[ i} mol/L rpoD-SF 0.5

20 ^mol/L rpoD-SR 0.5 5 U/ _[i L Taq DNA polymerase 0.3 _[ aL. When using this kit, add template DNA 1.0 to the 24 pL double PCR reaction solution. Compared with the prior art, the present invention has the following beneficial effects:

The elongation factor gene (tuf gene) of prokaryotes and the RNA polymerase c ⁷ <) factor gene (rpoD gene) are not as conserved as the 16S rRNA gene, and they are variable among different species. They are very suitable for designing primers for specific molecular detection and Identification, there is no report on the design of specific PCR primers for these two genes to detect sugarcane white leaf disease phytoplasma and Xanthomonas albicans.

In the prior art, in order to detect the two pathogens of sugarcane white leaf disease phytoplasma and Xanthomonas albicans, two PCR reactions must be carried out separately, and the primers used for detecting sugarcane white leaf disease phytoplasma are universal primers, which have low specificity. The process is labor-intensive and time-consuming. In contrast, the present invention has designed a pair of specific primers for sugarcane white leaf disease phytoplasma tucanine and Xanthomonas albicans rpoD gene respectively. After optimization of conditions, it is established that sugarcane can be detected simultaneously. Double PCR reaction system of white leaf disease phytoplasma and Xanthomonas albicans. The detection primers of the present invention are designed for genes with variability, so they are all specific primers with strong specificity and improve the accuracy of detection. At the same time, the dual PCR method established by the present invention can simultaneously detect sugarcane in one PCR reaction. White leaf disease phytoplasma and Xanthomonas albicans, and the PCR reaction time is only 1.5 hours, which greatly saves the detection time, improves the detection efficiency, and reduces the detection cost. After repeated verifications, stable results can be obtained. With the characteristics of rapidness, strong specificity, precision and stability, the present invention provides technical support for accurate and effective diagnosis of the two diseases, detection of virus-free seedlings, and introduction and quarantine, and is of great significance to the prevention and control of the spread of these two diseases .

SEQ ID NO: 1 in the sequence table shows the nucleotide sequence of the tuf-SF primer.

SEQ ID NO: 2 in the sequence table shows the nucleotide sequence of the tuf-SR primer.

SEQ ID NO: 3 in the sequence table shows the nucleotide sequence of the rpoD-SF primer.

SEQ ID NO: 4 in the sequence table shows the nucleotide sequence of the rpoD-SR primer. Description of the drawings

Figure 1 is a double PCR detection electrophoresis diagram of sugarcane white leaf disease phytoplasma and Xanthomonas albicans, where M is DNA Marker E, 1 is a sample mixed with sugarcane white leaf disease phytoplasma DNA and Xanthomonas albicans DNA. 2 is a sugarcane leaf sample infected with sugarcane white leaf disease phytoplasma, 3 is a sugarcane leaf sample infected with Xanthomonas albicans, 4 is a healthy sugarcane leaf sample,

5 is the specific implementation of ddH ₂ O _o

In order to better understand the present invention, the present invention will be further described below in conjunction with specific examples. The examples without special description are conventional methods.

Example

1. Primer design

According to the tuf gene sequence of the sugarcane white leaf disease phytoplasma, the tuf gene-specific primer was designed to detect the sugarcane white leaf disease phytoplasma. The expected amplified fragment size is 290 bp, the tuf gene-specific primer is composed of tuf-SF primer and tuf-SR primer, the target fragment length is 290 bp, and the nucleotide sequence of the tuf-SF primer is as SEQ ID NO: 1 and the nucleotide sequence of the tuf-SR primer are shown in SEQ ID NO: 2.

According to the rpoD gene sequence of Xanthomonas albicans, the rpoD gene-specific primers for detecting X. albicans are designed. The expected amplified fragment size is 498 bp. The rpoD gene-specific primers consist of ipoD-SF primers and rpoD- SR primer composition, the target fragment length is 498 bp, the nucleotide sequence of the rpoD-SF primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the rpoD-SR primer is shown in SEQ ID NO: 4.

2. DNA extraction

According to Wennan Dog “Zhang RY, Li WF, Huang YK, et al. Group 16SrXI phytoplasma strains, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D, are associated with sugar cane white leaf. International journal of systematic and evolutionary microbiology, 2016, 66(1): 487-491.” method to detect sugarcane leaves infected with the phytoplasma of sugarcane white leaf disease, according to the literature “Wang ZK, Comstock JC, Hatziloukas E, et al. Comparison of PCR, BIO- PCR, DIA, ELISA and isolation on semiselective medium for detection of Xanthomonas albilineans, the causal agent of leaf scald of sugarcane. Plant Pathology, 1999, 48(2): 245-252." The method detected infection with Xanthomonas albilineans Take 0.2 g of sugarcane leaves infected with sugarcane white leaf disease phytoplasma and Xanthomonas albicans respectively, and use a plant genomic DNA extraction kit (using Beijing Quanshijin Biotechnology Company’s Easy Pure plant Genomic DNA Kit) Take plant DNA extraction kit as an example) Extract the total DNA of leaves respectively, and the specific steps follow the instructions.

3. Double PCR detection

25 Double PCR reaction system, in which sterile ddH ₂ 0 12.7 pL, 10xPCR buffer 4.0 pL, 25 mmol/L

MgCl ₂ 2.0 pL, 10 mmol/L dNTPs 2.0 _(i L, 20 ^mol/L tuf-SF 1.0 pL, 20 ^mol/L tuf-SR 1.0 _(i L, 20 ^mol/L rpoD-SF 0.5

20 ^mol/L rpoD-SR 0.5

5 U/ _(i L Taq DNA polymerase 0.3 pL, template

DNA 1.0 ^L; its double PCR reaction program: 94 ^°( : pre-denaturation 3 1^11 _; 94 ^° C denaturation 30 s, 58 ^° C annealing 30 s, 72 ^° C extension 1 min, 35 cycles; the last 72 ^° C extends for 7 min.

4. Result judgment

Take 5 _{) i} L double PCR amplification products were detected by 1.5% agarose gel electrophoresis. The results are shown in Figure 1. The template was mixed with sugarcane white leaf disease phytoplasma DNA and Xanthomonas albicans DNA. Sex band, one is about 290 bp, and the other is 498 bp; the template only adds sugarcane white leaf disease phytoplasma DNA can amplify a single band of about 290 bp; template only adds Xanthomonas albicans DNA A single band of about 498 bp can be amplified by the, and no band has been amplified by the healthy Ganjian and water controls.

Claims

1. A dual PCR specific primer set for detecting sugarcane white leaf disease phytoplasma and Xanthomonas albicans, characterized in that the dual PCR specific primer set is used to detect sugarcane white leaf disease phytoplasma tuf gene The specific primer and the ipoD gene specific primer for detecting Xanthomonas albicans, the tuf gene specific primer is composed of tuf-SF primer and tuf-SR primer, the target fragment length is 290 bp, the tuf-SF primer The nucleotide sequence of the tuf-SR primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the tuf-SR primer is shown in SEQ ID NO: 2. The rpoD gene-specific primer is composed of rpoD-SF primer and rpoD-SR primer The target fragment length is 498 bp, the nucleotide sequence of the rpoD-SF primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the rpoD-SR primer is shown in SEQ ID NO: 4.

2. A double PCR detection method for sugarcane white leaf disease phytoplasma and Xanthomonas albicans, characterized in that: the primer used in the double PCR reaction system is the one described in claim 1 for detecting sugarcane white leaf disease phytoplasma And Xanthomonas albicans double PCR specific primer set, after the double PCR reaction is terminated, take the PCR amplified product and perform agarose gel electrophoresis. If a single band with a size of 290 bp is amplified, the tested sugarcane sample is infected If a single band with a size of 498 bp is amplified, the tested sugarcane sample is infected with Xanthomonas albicans; if two bands with a size of 290 bp and 498 bp are amplified at the same time, then The tested sugarcane samples were infected with both the sugarcane white leaf disease phytoplasma and Xanthomonas albicans.

3. A double PCR detection method for sugarcane white leaf disease phytoplasma and Xanthomonas albicans according to claim 2, characterized in that:

(1) Double PCR reaction system:

25 _(i L double PCR reaction system, in which sterile ddH ₂ 0 12.7 _(i L, 10xPCR buffer 4.0 _(i L, 25 mmol/L MgCl ₂ 2.0 nL, 10 mmol/L dNTPs 2.0 _(i L, 20 _{( i} mol/L tuf-SF 1.0 _(i L, 20 ^imol/L tuf-SR 1.0 _(i L, 20 [imol/L rpoD-SF 0.5

20 pmol/L rpoD-SR 0.5 pL, 5 U/[iL Taq DNA polymerase 0.3 [iL, template

DNA l .O _ji L;

(2) Double PCR reaction program:

Pre-denaturation at 94 ^° C for 3 minutes; denaturation at 94 ^° C for 30 s, annealing at 58 ^° C for 30 s, extension at 72 ^° C for 1 min, 35 cycles; finally, extension at 72 ^° C for 7 minutes.

4. The double PCR detection method for sugarcane white leaf disease phytoplasma and Xanthomonas albicans according to claim 2 or 3, characterized in that: the PCR amplification product is selected when performing agarose gel electrophoresis 5 |_iL PCR products were electrophoresed on a 1.5% agarose gel.

5. A dual PCR detection kit for sugarcane white leaf disease phytoplasma and Xanthomonas albicans, characterized in that the kit includes: tuf gene-specific primers and rpoD gene-specific primers, and the tuf gene is specific Sexual primers are used to detect sugarcane white leaf disease phytoplasma. The target fragment length is 290 bp. The tuf gene-specific primers consist of tuf-SF primers and tuf-SR primers. The nucleotide sequence of tuf-SF primers is shown in SEQ ID NO: As shown in 1, the nucleotide sequence of the tuf-SR primer is shown in SEQ ID NO: 2. The rpoD gene-specific primer is used to detect Xanthomonas albicans, the target fragment length is 498 bp, and the rpoD base Since the specific primer is composed of ipoD-SF primer and rpoD-SR primer, the nucleotide sequence of rpoD-SF primer is shown in SEQ ID NO: 3, and the nucleotide sequence of rpoD-SR primer is shown in SEQ ID NO: 4. Show.

6. A double PCR detection kit for sugarcane white leaf disease phytoplasma and Xanthomonas albicans according to claim 5, wherein the kit further comprises a double PCR reaction solution, 24 pL double PCR reaction There are sterile ddH ₂ 0 12.7 pL, 10xPCR buffer 4.0 pL, 25 mmol/L MgCl ₂ 2.0 pL, 10 mmol/L dNTPs 2.0 _(i L, 20 ^mol/L tuf-SF 1.0 _[i L, 20 _[j, mol/L tuf-SR 1.0 _[i L, 20 _[j, mol/L rpoD-SF 0.5 _[i L, 20 _[j, mol/L rpoD-SR 0.5 _[i L, 5 U/ _[i L Taq DNA polymerase 0.3 _(i L.