CN101985652B - High-throughput molecular detection of Fusarium graminearum on carbendazim medicament-resistant gene frequency - Google Patents

Abstract

The invention belongs to a high-throughput molecular detection method for the frequency of carbendazim-resistant genes of Fusarium graminearum, which can be used for monitoring the drug resistance of wheat scab and early warning of the epidemic of drug-resistant diseases. The invention is based on the research that more than 97% of the genotypes of Fusarium graminearum resistance to carbendazim belong to the 167 point mutation of β ₂ -tubulin gene, which is a high-throughput detection technology for drug resistance. The detection method is divided into three main steps, (1) extract the genomic DNA of the known sensitive and drug-resistant strains and the sample to be tested; (2) perform specific real-time quantitative PCR reaction to establish a standard curve; (3) control standard The frequency of the drug resistance gene in the test sample is obtained from the curve. This method has the characteristics of high throughput, rapidity and accuracy. The detection sensitivity of drug resistance gene frequency can reach 1/10,000 to 1/100,000, and the accuracy rate can reach over 96%.

Description

A high-throughput molecular assay for the frequency of carbendazim-resistant genes in Fusarium graminearum

1. Technical field

The invention belongs to a high-throughput molecular detection method for the frequency of carbendazim-resistant genes of Fusarium graminearum (Fusarium graminearum), which can be used to monitor the development dynamics of benzimidazole fungicide-resistant Fusarium graminearum groups, and to carry out anti- Epidemic prediction and early warning of medicinal wheat head blight. the

2. Technical Background

Wheat scab is a worldwide disease caused by Fusarium graminearum Schwabe, which seriously affects the yield and quality of wheat. For a long time, benzimidazole fungicides or compound agents based on such agents have been used for chemical control. Benzimidazole fungicides are used in production as a class of high-efficiency, broad-spectrum systemic fungicides, which solve the environmental toxicity problem of protective fungicides and improve the ability of humans to control the disease. Benzimidazole fungicides include carbendazim, benomyl, thiabendazole, thiophanate, etc. These fungicides have the same antibacterial spectrum and antibacterial mechanism, they also have the same resistance mechanism, and there is a positive cross-resistance between them. Due to the highly specialized nature of such agents, single action site, and high frequency of application, many phytopathogenic fungal populations will develop drug-resistant dominant physiological races after many years of use, making chemical control completely ineffective. After several years of hard work, the fungicide laboratory of Nanjing Agricultural University found that the resistance of wheat scab to carbendazim was mainly caused by the mutation of Fusarium graminearum β2-tubulin gene (FGSG_06611.3). The codon mutation of the 167th amino acid encoded by the gene (mutated from phenylalanine Phe to tyrosine Tyr, and the base is changed from TTT to TAT, which is a single base mutation) caused the resistance of the bacterium to carbendazim. Type accounted for more than 97% of the field drug-resistant head blight population. the

Due to the large number of pathogenic bacteria in nature, when the proportion of drug-resistant individuals in the population is very low (1-5%), it will cause the prevalence of drug-resistant diseases, making chemical control failure. Traditional detection methods need to isolate and cultivate pathogenic bacteria, then culture them on drug-containing media, and then identify drug resistance according to the effect of drugs on mycelial growth. This method of measuring sensitivity to pharmaceuticals usually takes several weeks, and the workload is heavy, and the number of samples tested is limited; it is difficult to find when the frequency of drug resistance genes is below 1%. Therefore, traditional drug resistance monitoring methods can only verify whether the failure of chemical control is caused by drug resistance, and cannot give early warning. Early detection of the frequency of drug-resistant physiological races/drug resistance genes in pathogenic populations, early implementation of drug resistance control strategies and early warning are the most effective measures for drug resistance control. On the basis of research on the mechanism of drug resistance, according to the principle of gene point mutation, the application of conventional nucleic acid technology such as ASO-PCR technology to detect the resistance of pathogenic fungi to benzimidazole fungicides such as carbendazim can be quickly and accurately detected. However, a PCR reaction system can only detect the sensitivity of one bacterial strain to carbendazim fungicide, and the sensitivity of a single strain to carbendazim can only be detected by electrophoresis after the completion of the entire PCR reaction system. The real-time quantitative PCR (Real-time quantitative PCR) method has changed the reality that the detection of pathogenic bacteria's sensitivity to fungicides can only be performed on a single sample. It has (1) high-throughput detection, that is, in a reaction system, it can Sensitivity determination of all samples to be tested to fungicides; (2) the real-time performance of the detection results, the size of the drug-resistant strain subpopulation in the sample can be understood during the PCR reaction; (3) high efficiency, the classic plate method ( At least two weeks or more to get the results), conventional ASO-PCR and other techniques to determine the resistance of strains can only be a single determination of the sensitivity of fungicides, and this method is simultaneously determined for a large number of samples, the time-consuming is only 6 Within hours, we can accurately understand the occurrence, development, and prevalence of drug-resistant strain subgroups in that year, and effectively guide the use of drugs in that year. the

(4) Make it possible to detect low-frequency drug resistance genes at an early stage. the

The invention uses a real-time quantitative PCR method to detect the frequency of the drug-resistant gene of Fusarium graminearum to carbendazim, including the process of sample pretreatment, genomic DNA extraction, and real-time quantitative PCR amplification. the

3. Contents of the invention

Technical Problem The purpose of the present invention is to provide a rapid detection method for the frequency of the carbendazim-resistant gene of Fusarium graminearum. This technology uses Cycling probe Real time PCR for the first time, and optimizes the reaction conditions according to the genotype of resistant strains. Real-time quantitative PCR (Real-time quantitative PCR) can conduct large-scale, fast and simple drug resistance during the onset and early stages of wheat scab. The diagnosis and detection of sub-populations, the detection accuracy rate can reach more than 96%, which is of practical value for timely taking effective measures to control the epidemic of drug-resistant diseases in the current season. the

Technical Solution The gene detection of Fusarium graminearum resistance to carbendazim is based on the β ₂ -tubulin gene (FGSG_06611.3), which encodes a point mutation TTT(Phe)→TAT(Tyr) in the 167th amino acid codon.

A high-throughput molecular detection method for the frequency of carbendazim-resistant genes in Fusarium graminearum is divided into three steps: 

(1) Using the conventional phenol·chloroform·isoamyl alcohol method, extract the nuclear genomic DNA of known drug-resistant strains, sensitive strains and samples to be tested, respectively. the

(2) Use primers and designed specific Cycling probes to carry out real-time quantitative PCR reaction, and establish standard curves for the detection of drug-resistant strains and sensitive strains, respectively. the

The key to the high-throughput real-time detection technology of drug resistance gene frequency in Fusarium graminearum population is the specificity of the Cycling probe used in real _- time quantitative PCR. Bit by Phe (TTT) → Tyr (TAT).

①Primer pair for amplifying the β ₂ -tubulin gene fragment of Fusarium graminearum

β ₂ -tubulin gene upstream primer 5′AAGCCATTGATGTTGTTCG 3′

β ₂ -tubulin gene downstream primer 5′CATGACGGTAGAAAATCAGGTAG 3′

②Specific Cycling Probe

GG(Eclipse)-3’  P1 5'-(FAM)CATAACGGAA

GG(Eclipse)-3'

GG(Eclipse)-3’  P2 5'-(HEX)CATAACGGAA

GG(Eclipse)-3'

Real-time quantitative PCR (Real-time quantitative PCR) reaction system and its amplification procedure:

reaction system:

*DNA template: Add 1 μL each of sensitive and resistant strains. the

Amplification condition:

Pre-denaturation: 95°C 30s

↓ ↓

Denaturation: 95℃ 5s

Annealing: 55℃ 20s

Extension: 72℃ 31s

40 cycles

(3) The sample to be tested uses the above-mentioned system and conditions to carry out real-time quantitative PCR reaction, and compares the two standard curves that have been established to obtain the corresponding drug-resistant strains and sensitive strains and the number of drug-resistant strains in Fusarium graminearum proportion of the total. the

Beneficial effect The detection method of Fusarium graminearum anti-carbendazim subgroup of the present invention, compared with the prior art,

1. The real-time quantitative PCR (Real-time quantitative PCR) method has changed the detection of the sensitivity of pathogenic bacteria to fungicides and can only detect a single sample. Compared with conventional plate detection and ASO-PCR molecular detection, it has the following advantages and positive effects . the

(1) High-throughput detection, that is, in one reaction system, the sensitivity of all samples to be tested to fungicides can be determined;

(2) The real-time performance of the detection results, the size of the subpopulation of drug-resistant strains in the sample can be known during the PCR reaction;

(3) Detection efficiency. The classic plate method, conventional ASO-PCR and other techniques to detect drug-resistant strains can only measure the sensitivity of a single fungicide, while this method measures a large number of samples at the same time, which is time-consuming. It takes only 6 hours to accurately understand the occurrence, development, and prevalence of drug-resistant strain subgroups in that year, and effectively guide the use of drugs in that year. the

(4) Make it possible to detect low-frequency drug resistance genes at an early stage. the

(5) The present invention is the first in the world to detect the resistance subpopulation of Fusarium graminearum to the carbendazim fungicide by using Cycling probe real-time quantitative PCR technology. the

(6) Compared with the common SYBR GREEN I dye method, this technology can detect resistant and sensitive strains at the same time, without separate tubes, to achieve multiple detection. the

2. At present, research institutes at home and abroad use the mycelium growth method to detect the drug resistance of Fusarium graminearum, but this method involves sampling, isolation and cultivation, which takes 3 to 5 days and a large number of indoor drug sensitivity testing experiments need at least 6 days. longer. At present, there is no mature molecular detection technology for carbendazim-resistant wheat head blight. Real-time quantitative PCR can detect the resistance level of all samples to be tested to carbendazim in one reaction system, and the dynamic result of the reaction can be known during the progress of the PCR reaction. This method is high-throughput, fast, and cost-effective. This is of practical significance for timely understanding of the development of drug-resistant pathogen subgroups, timely and rational guidance of scientific medication, effective control of drug resistance, and reduction of costs and environmental pollution. the

3. About 4% of the 100 Fusarium graminearum strains tested were resistant to carbendazim, and the real-time quantitative PCR test result was consistent with the result (4.02%) measured by the traditional colony diameter method. the

4. Description of drawings

Figure 1 Amplification curve of sensitive probe (P2)

Figure 2 Amplification curve of resistance probe (P1)

Figure 3 Quantitative PCR Specific Detection Demonstration Diagram

Figure 4 PCR amplification conditions of primer pair β ₂ -tubulin gene upstream primer/β ₂ -tubulin gene downstream primer

The standard curve of Fig. 5 sensitive probe (P2)

The standard curve of Figure 6 resistance probe (P1)

5. Specific implementation

Embodiment 1Cycling probe fluorescent dye quantitative PCR system optimization

1.1 Optimization of the annealing temperature In order to ensure the stability and reliability of the detection, the experiment selected the CycleavePCR ^TM Core Kit DCY501 kit from Treasure Bioengineering (Dalian) Co., Ltd.

During the PCR reaction, the annealing temperature Tm value is an important guarantee for the specificity of the reaction. If the annealing temperature is too low, non-specific products will be produced. In the present invention, due to the strong specificity of the probe, the requirement for the primer is relatively low, and it only needs to ensure a sufficiently high reaction efficiency. Referring to the Tm value of the primers, gradient PCR was carried out from 50 to 60°C, combined with the annealing temperature recommended by the kit, the optimal annealing temperature for quantitative PCR was determined to be 55°C. the

1.2 Probe Concentration Optimization The purpose of this step is to determine the optimum probe concentration. The concentration of the probe will affect the level of the fluorescent signal. When the probe concentration is too low and the PCR product is excessive, the correct standard curve cannot be obtained. And in order to prevent the mutual interference of the two fluorophores in the same system, the concentration of the two probes has to be adjusted. In the experiment, the probe (5 μM) was added in increments of 0.1 μl from 0.1 μl to 2 μl to the 25 μl reaction system to determine the optimal probe concentration P1 (5 μM) 0.6 μl, P2 (5 μM) 1 μl. the

The sensitivity of embodiment 2Cycling probe fluorescent dye quantitative PCR

Standard products require high purity, uniformity and stability. In this experiment, the purified PCR product fragments were cloned into the vector as the standard. The copy number of the standard product of the sensitive strain ZF43 was 1.33×10 ⁶ to 1.33×10 ² , and the copy number of the standard product of the resistant strain ZF52 was 1.78×10 ⁶ to 1.78×10 ² , both of which were 10-fold serial dilutions. Amplify the standard product and obtain two sets of amplification curves (Fig. 1, Fig. 2). It can be seen that the minimum detection copy number of this technology is on the order of 10 ² , and the sensitivity is at least 10 to 100 times that of the ordinary PCR detection method. It is more sensitive than the general SYBR GREEN I dye method.

The specificity verification of embodiment 3Cycling probe fluorescent dye quantitative PCR

The sensitive strain ZF43 and the resistant strain ZF52 were used as templates for quantitative PCR detection respectively, and the reactions had good specificity. When the template is ZF43, under the HEX channel, the P2 probe has a signal, and the sensitive strain ZF43 is detected, while the P1 probe has no signal (Fig. 3A); at this time, both probes have no signal under the FAM channel. When the template was ZF52, the signal could be detected only in the FAM channel (Fig. 3B). the

The standard curve establishment of embodiment 4Cycling probe fluorescent dye quantitative PCR

Quantitative PCR reaction was carried out using the standard samples of the sensitive strain ZF43 and the resistant strain ZF52 as templates. Real-time quantitative PCR reaction system and conditions:

reaction system:

*DNA template: Add 1 μL each of sensitive and resistant strains. the

Amplification conditions (three-step method, Figure 4):

Pre-denaturation: 95°C 30s

↓ ↓

Denaturation: 95°C 5s

Annealing: 55℃ 20s

Extension: 72℃ 31s

40 cycles

After quantitative PCR amplification, five connected amplification curves with clear signals can be obtained respectively; at the same time, the water control is used as a negative control (NTC), and no fluorescent signal is detected. The standard curve equation obtained by the instrument after analyzing the results is ZF43: y=-3.5198x+44.861 R ² =0.9965 (Figure 1, Figure 5); ZF52: y=-3.6133x+45.889R ² =0.9991 (Figure 2, Figure 6).

Embodiment 5 Known anti/sensitivity strain concentration ratio template verification

Take known concentrations of sensitive strain ZF43 and resistant strain ZF52 mixed (mass ratio ZF43:ZF52=1.39), as a template for quantitative PCR reaction, to obtain the Ct value of the sample (Table 1), calculated by the standard curve equation ZF43, The copy numbers of ZF52 were 6371/μl and 4400/μl respectively, and the copy number ZF43:ZF52=1.44:1, the results were basically consistent with the known sample ratio. It shows that the technology can be used to detect and quantify wheat head blight and the ratio of resistant/susceptible strains is credible. the

Example 6 2009 Jiangsu Tongzhou field strain detection

The collected mature ascospores (mixture of 100 field strains) were divided into two parts, one of them was diluted to 900 spores/ml, and 100 μl was respectively applied to carbendazim-containing (2ppm) and drug-free PDA plates (PDA Streptomycin and pentachloronitrobenzene were added to the plate to inhibit bacteria and other fungi), and each was repeated 5 times. Observe the germination in 1 to 2 days, and calculate the resistance ratio. Another ascospore genomic DNA was extracted and detected by quantitative PCR technique. the

The germination situation was observed and recorded. The ascospores germinated on the drug-free plate were 70, 61., 49, 63, and 55 respectively, and the ascospores germinated on the drug-containing plate were 4, 3, 2, 1, and 2. It was calculated that the resistant strains accounted for 4.03% of the total strains. the

The quantitative PCR results are shown in Table 2. According to the calculation of the standard curve equation, the resistant strains accounted for about 4% of the total strains, which was basically consistent with the traditional biological assay results. the

Table 1 Sample Quantitative Test Results

Table1 Results of test by Real time PCR

Table 2 Quantitative detection results of field samples

Table 2 Results of test of field samples by Real time PCR

High-throughput molecular detection of carbendazim-resistant gene frequency in Fusarium graminearum.txt

SEQUENCE LISTING

<110> Nanjing Agricultural University

<120>High-throughput molecular detection of carbendazim-resistant gene frequency in Fusarium graminearum

<140>201010247003.7

<141>2010-08-06

<160>4

<210>1

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> The upstream primer Codon167F used to amplify the β2-tubulin gene

<400>1

aagccattga tgttgttgg

<210>2

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> The upstream primer Codon167R used to amplify the β2-tubulin gene

<400>2

catgacggta gaaatcaggt ag

<210>3

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> Specific Cycling Probe P1 for Amplifying β2-Tubulin Gene of Sensitive Strains

<400>3

cataacggaa tagg

<210>4

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> Specific Cycling Probe P2 for Amplifying β2-Tubulin Gene of Sensitive Strains

<400>4

cataacggaa aagg

Claims (1)

1. Fusarium graminearum (Fusarium graminearum) produces the real-time detection method of the drug resistance gene frequency of intermediate resistance level (middle resistance) to carbendazim, it is characterized in that the Cycling that adopts real-time quantitative PCR (Real-timequantitative PCR) technology The specificity of the probe was designed according to the 167th amino acid codon of the β ₂ -tubulin gene of the moderately resistant strain from TTT(Phe)→TAT(Tyr). ①Primer pair for amplifying the β ₂ -tubulin gene fragment of Fusarium graminearum β ₂ -tubulin gene upstream primer 5′AAGCCATTGATGTTGTTCG 3′ β ₂ -tubulin gene downstream primer 5′CATGACGGTAGAAAATCAGGTAG 3′ ②Specific cycling probe P1 5'-(FAM)CATAACGGAA

GG(Eclipse)-3' P2 5'-(HEX)CATAACGGAA

GG(Eclipse)-3, It is capable of high-throughput detection of the drug resistance gene frequency of the bacterial population in the disease sample that has a moderate level of resistance to benzimidazole fungicides.

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