Method for rapidly detecting pear fire blight viable bacteria PMAxx-qPCR and application of pear fire blight viable bacteria PMAxx-qPCR

Abstract

The invention relates to the technical field of agricultural biology, and particularly provides a method for detecting pear fire blight viable bacteria based on PMAxx-qPCR and application. By optimizing the treatment concentration and exposure time of PMAxx and combining a qPCR method, the pear fire blight viable bacteria are quantitatively detected. The method can effectively eliminate dead bacteria interference in a specific range, and is suitable for accurate quantification of the viable count in a bacterial liquid concentration range of 10 < 3 >-10 < 8 > cfu / mL. The detection method has the advantages of high specificity, high accuracy, high universality, high sensitivity and the like.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology and relates to a method for rapidly detecting viable cells of Erwinia amylovora by PMAxx-qPCR and its application. Background Art

[0002] Fire blight is a highly contagious plant disease caused by Erwinia amylovora, which mainly infects Rosaceae plants such as apples and pears, posing a major threat to the production safety of the global fruit tree industry. Since it was first discovered in the Hudson River Basin in New York State, USA in 1780, it has gradually spread to the west coast of the United States and has now spread to more than 40 countries around the world, including regions such as Europe, Asia, and the Middle East. The pathogen spreads rapidly and can invade host plants through wounds, insects, and stomata. After infection by the pathogen, it can cause necrosis of leaves, flowers, and fruits, and even lead to the death of the whole plant in severe cases. It is urgent to achieve efficient monitoring and precise prevention and control of fire blight through scientific means.

[0003] Early prevention and control of fire blight is crucial for reducing economic losses and ensuring the sustainable development of the fruit tree industry. Therefore, it is particularly crucial to develop an accurate, universal, efficient, and sensitive pathogen detection technology, especially for the early diagnosis of fire blight. Currently, the main domestic detection methods for Erwinia amylovora include traditional selective medium streaking isolation method, immunological detection method, and molecular biology detection method. However, these methods have certain limitations in practical applications. Specifically, they can not only amplify the DNA of viable cells but also amplify the DNA of dead cells, resulting in false positive results and affecting the accuracy and reliability of detection. Currently, for the detection of viable cells of fire blight, dye methods are mainly used, and these methods usually use the PEA29 plasmid as the target for detection. However, false positive results often occur in the application of dye method qPCR. In addition, existing studies have shown that not all Erwinia amylovora strains contain the PEA29 plasmid, which may lead to false negative results. Therefore, there is an urgent need to develop a more specific and accurate detection method to overcome the limitations of existing technologies.

[0004] Therefore, establishing a detection method for viable cells of fire blight is crucial for the early diagnosis and quarantine of the disease, formulating scientific prevention and control measures to effectively block the spread of fire blight, and thus playing a key role in preventing and controlling the disease, reducing economic losses, and promoting the research on disease epidemiology. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for counting viable cells of Erwinia amylovora based on PMAxx-qPCR technology to achieve accurate counting of viable cells and overcome the false positives of conventional methods.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for counting viable cells of Erwinia amylovora based on PMAxx-qPCR technology, comprising the following steps:

[0008] (1) After completing the surface disinfection treatment of the sample, at the junction of the diseased area and healthy tissue, cut a small piece of tissue and shred it; place the shredded tissue in 1 mL of sterile water, let it stand for 30 min to fully release the pathogens; remove the supernatant by centrifugation, and resuspend the precipitate in 500 μL of sterilized water to obtain the target template for PMAxx-qPCR;

[0009] (2) Pretreat the sample suspension with PMAxx: Under light-shielded conditions, add modified propidium monoazide to the sample suspension, incubate in the dark wrapped with tin foil for 15 min, and then place it on ice and photolyze for 8 min with the lid open;

[0010] (3) Taq-man probe real-time fluorescence quantitative PCR detection: Using the sample suspension treated with PMAxx as the template, perform qPCR reaction;

[0011] The amplification primers are:

[0012] EaF1: 5'-TGAACGATATGTTAGGCGGTTCGC-3';

[0013] EaR1: 5'-GGTCGCTGGAGTCTGAGGT-3';

[0014] The Taq-man probe is:

[0015] 5'-FAM-AACACGCTGGGCTCGAAAGGCGGCAACAATAC-TAMAR-3';

[0016] (4) Quantitative analysis of results: According to the standard curve obtained by real-time fluorescence quantification, substitute the Ct value of the measured sample into the standard curve to obtain the viable cell count of Erwinia amylovora. When the Ct value > 35, it is judged as negative; when the Ct value ≤ 35, it is judged as positive. T value into the standard curve, to obtain the viable cell count of Erwinia amylovora. When the Ct T value > 35, it is judged as negative, and when the Ct T value ≤ 35, it is determined as positive.

[0017] Preferably, the specific operation of step (1) is as follows:

[0018] S1 Sample pretreatment: After performing surface disinfection treatment on the sample, at the junction of the diseased and healthy tissues, cut a small piece of the sample, and then use scissors to shred it;

[0019] Preparation of S2 bacterial solution: Put the treated sample into 1 mL of sterile water and let it stand for 30 min to fully release the pathogens into the water, thus forming a bacterial solution;

[0020] S3 Centrifugation treatment: Centrifuge the bacterial solution at a speed of 12000 r / min for 15 min, remove the supernatant, and retain the precipitate;

[0021] S4 Template preparation: Add 500 μL of sterilized water to the precipitate to suspend the precipitate and obtain the target template for the subsequent PMAxx-qPCR reaction.

[0022] Preferably, the final concentration of PMAxx in step (2) is 15 μmol / L. The specific operation of step (2) is: In a light-proof environment, add modified propidium monoazide with a concentration of 15 μmol / L to the sample suspension, then tightly wrap it with tin foil and incubate it for 15 min under dark conditions; After the incubation, open the lid of the bacterial solution and place it on the ice surface to ensure that the distance between the bacterial solution and the 590W incandescent lamp is kept within the range of 10 to 15 cm, and perform an 8-min exposure treatment.

[0023] Since halogen tungsten lamps will generate relatively high heat during long-term use, therefore, using a high-power incandescent lamp as an alternative light source has a better effect. The high-power incandescent lamp can provide a stable light source and generate relatively low heat during long-term operation.

[0024] Preferably, the system of the qPCR reaction in step (3) is: 2×Probe qPCR Mix 10 μL, 0.4 μL each of 10 μmmol / L upstream and downstream primers, 0.8 μL of probe, 0.2 μL of 50×ROX DyeⅡ, 2 μL of template, and 6.2 μL of ultrapure water.

[0025] Preferably, the program of the qPCR reaction in step (3) is: Pre-denaturation at 95°C for 30 s; 95°C for 5 s, 60°C for 34 s (collect fluorescence signal), for a total of 40 cycles.

[0026] Preferably, in step (3), sterilized ultrapure water is used as a template as a blank control, and the live and dead bacterial suspensions of Erwinia amylovora treated in step (2) are used as templates as positive and negative controls.

[0027] Preferably, the standard curve of step (4) is Y = -3.3074X + 42.704, where Y represents the CT value, X represents the logarithm of the bacterial solution concentration, and R 2 = 0.999, Eff = 99.99%.

[0028] The present invention also provides an application of the described method for rapid detection of viable cells of Erwinia amylovora by PMAxx-qPCR, and the method is applied to the qualitative and quantitative detection of viable cells of Erwinia amylovora in diseased fruit tree branches, leaves, flowers and fruits of fire blight of pear.

[0029] Advantages of the present invention:

[0030] The present invention has established a quantitative detection technology for viable cells of Erwinia amylovora based on PMAxx-qPCR. This technology is fast, accurate and sensitive, solves the problem that qPCR cannot accurately identify and quantitatively analyze only viable pathogenic bacteria, and provides a basis for formulating effective prevention and control strategies for fire blight of pear.

[0031] (1) High detection efficiency: This detection method does not require DNA extraction. The collected fruit tree branch, leaf and fruit samples are configured into a suspension, and after incubation with PMAxx and photolysis treatment, qPCR amplification is carried out to determine the result, which can be completed in only 2-3 hours.

[0032] (2) High accuracy: It overcomes the defect of the conventional detection method that can amplify the DNA of dead bacteria and cause false positive results, realizes the accurate counting of viable cells of Erwinia amylovora, and improves the detection accuracy and reliability.

[0033] (3) Strong specificity: It solves the problems of frequent false positives in the dye method qPCR targeting the PEA29 plasmid and false negatives caused by the fact that some Erwinia amylovora strains do not contain this plasmid. This method is more specific and accurate.

[0034] (4) High sensitivity: When using the bacterial suspension as a template, the detection limit is 10 3 cfu / mL.

[0035] (5) Wide application: It can be applied to the qualitative and quantitative detection of viable cells of Erwinia amylovora in diseased fruit tree branches, leaves, flowers and fruits of fire blight of pear, and has a wide range of applications. Description of the drawings

[0036] Figure 1 The following shows the amplification curves of specific detection of different strains of the target screened by this detection system: Figure 1 A in it represents the amplification curves of Erwinia amylovora strains from different regions and hosts; Figure 1 B-H in it respectively represent the amplification curves of the control strains of Pantoea agglomerans, Serratia marcescens, Bacillus subtilis, Erwinia pyrifoliae, Ralstonia solanacearum, Pseudomonas fluorescens and Clavibacter michiganensis subsp. michiganensis;

[0037] Figure 2 The following shows the standard curve of sensitivity detection of the target screened by this detection system;

[0038] Figure 3Shown is the optimization of PMAxx treatment conditions: Figure 3 In Figure 3 , A shows the effects of different concentrations of PMAxx on dead and live bacteria, Figure 3 and in Figure 3 , B shows the effects of different exposure times on amplification;

[0039] Figure 4 Shown is the standard curve for detecting viable cells of Erwinia amylovora by PMAxx-qPCR;

[0040] Figure 5 Shown is the scatter plot of the measured values and theoretical values of PMAxx-qPCR for different proportions of viable cells. Specific Embodiments

[0041] The following describes the specific embodiments of the present invention to facilitate those skilled in the art of this technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art of this technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions created using the concept of the present invention are within the scope of protection.

[0042] Instrumentation: The INFORS Ecotron shaking incubator was purchased from INFORS, Switzerland; the Memmert IN55 incubator was purchased from Memmert, Germany; the 590W incandescent lamp was purchased from Osram, Germany; the ABI 7500 fluorescence quantitative PCR instrument was purchased from Applied Biosystem, USA; The ultraviolet spectrophotometer was purchased from AnalytikJenaAG, Germany.

[0043] Test agents: Modified propidium monoazide (PMAxx) was purchased from Biotium, USA, and Probe qPCRmix was purchased from Takala.

[0044] Test strains: All test strains were isolated and preserved by the Crop Bacterial Diseases Group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0045] Example 1 Screening and Synthesis of Detection Targets

[0046] (1) Synthesis and screening of primers and probes: Based on 10 core pathogenic genes of Erwinia amylovora, the present invention screened candidate detection targets. Through the use of bioinformatics software such as DNAMAN and Primer Express, the core gene sequences were subjected to homology alignment and analysis to screen out target sequences with high specificity and conservation. Specific primers and probes were designed according to the screened target sequences and verified by PCR and qPCR respectively to ensure the specificity and sensitivity of the detection system. The primers and probes were synthesized by Shanghai Sangon Biological Engineering.

[0047] The amplification primers are as follows:

[0048] EaF1: 5'-TGAACGATATGTTAGGCGGTTCGC-3';

[0049] EaR1: 5'-GGTCGCTGGAGTCTGAGGT-3';

[0050] The Taq-man probe is as follows:

[0051] 5'-FAM-AACACGCTGGGCTCGAAAGGCGGCAACAATAC-TAMAR-3'.

[0052] (2) Detection of target specificity determination: Using 12 test strains of Erwinia amylovora isolated from different hosts and regions as the target strains, and the control strains are Pantoea agglomerans, Serratia marcescens, Bacillus subtilis, Erwinia pyrifoliae, Ralstonia solanacearum, Pseudomonas fluorescens, and Clavibacter michiganensis subsp. michiganensis isolated from the same habitat as Erwinia amylovora. PCR and qPCR were carried out respectively to verify their specificity.

[0053] (3) Detection of target sensitivity determination: The initial concentration of the bacterial suspension of Erwinia amylovora strain was adjusted to 10 8 cfu / mL, and serially diluted 10-fold to 10 2 cfu / mL. Using bacterial suspensions with different concentration gradients as templates, qPCR reactions were carried out to test the sensitivity of the designed probes and primers. The PCR reaction system was (20 μL): Probe qPCR Mix (2×) 10 μL, 10 μmmol / L upstream and downstream primers 0.4 μL each, probe 0.8 μL, ROX DyeⅡ (50×) 0.2 μL, template 2 μL, ultrapure water 6.2 μL. The reaction conditions were: pre-denaturation at 95°C for 30 s; 95°C for 5 s, 60°C for 34 s (collecting fluorescence signals), a total of 40 cycles. After amplification, with the logarithm of the bacterial concentration and DNA concentration as the abscissa and the C T value as the ordinate, a standard curve was established.

[0054] The specificity experiment proved that the primers and probes could only amplify Erwinia amylovora from different regions and hosts, but could not amplify the control bacteria; the sensitivity experiment proved that when the template concentration was not less than 10 3 cfu / mL, the C T values of all reactions were less than 35. The detection limit was 10 3 cfu / mL when using the bacterial suspension as the template.

[0055] Example 2 Establishment of PMAxx pretreatment system

[0056] (1) Pretreatment of PMAxx sample: PMAxx with a stock solution concentration of 20 mmol / L TM (PMAxxTM Dye, Biotum, USA) was diluted with ultrapure water before use to prepare a 1 mmol / L PMAxx working solution, and stored in the dark at -20 °C.

[0057] (2) Screening of the optimal concentration of PMAxx: Different concentrations of PMAxx dyes (0, 4, 8, 10, 15, 25 μmol / L) were added to 200 μL of bacterial suspensions containing 10 8 cfu / mL of live and dead bacteria, and gently shaken and mixed. Then, the samples were wrapped with tin foil and incubated in the dark for 15 min, and after incubation, ice bath treatment was carried out. The bacterial suspension was placed on ice with the lid open at a distance of 10 - 15 cm from a 590 W incandescent lamp and exposed for 8 min.

[0058] After the exposure, qPCR reaction was carried out using the treated bacterial suspension as a template. The qPCR reaction system was (20 μL): Probe qPCR Mix (2×) 10 μL, 10 μmmol / L upstream and downstream primers 0.4 μL each, probe 0.8 μL, ROX DyeⅡ (50×) 0.2 μL, template 2 μL, ultrapure water 6.2 μL. The reaction conditions were: pre-denaturation at 95 °C for 30 s; 95 °C for 5 s, 60 °C for 34 s (collecting fluorescence signals), a total of 40 cycles. After the amplification was completed, the logarithm of the bacterial suspension concentration was used as the abscissa, and the C T value was used as the ordinate to establish a standard curve. The obtained data was analyzed using GraphPad Prism10.

[0059] By adding different concentrations of PMAxx to the bacterial suspensions of Erwinia amylovora dead and live bacteria and observing the effect on the qPCR amplification results, it was found that when the final concentration of PMAxx reached 15 μmol / L, the qPCR amplification of dead cell DNA was effectively inhibited, and the amplification of live cell DNA was hardly affected. Further analysis showed that when the concentration of PMAxx was less than 15 μmol / L, the C T value of qPCR amplification of dead cell DNA gradually increased; when the concentration ≥ 15 μmol / L, the C T value of dead cell DNA amplification tended to be stable and no longer increased significantly. This concentration can effectively inhibit the amplification of dead cell DNA without significantly affecting the amplification of live cell DNA. Therefore, a PMAxx concentration of 15 μmol / L can be used as the optimal concentration for ideally distinguishing dead and live cells.

[0060] (3) Add 200 μL of a concentration of 10 8A bacterial suspension of dead and live bacteria at cfu / mL was added to a centrifuge tube. Subsequently, PMAxx dye was added to a final concentration of 15 μmol / L, which was the optimal concentration obtained through screening. The bacterial suspension was incubated in the dark for 15 min to ensure that the PMAxx dye fully bound to the DNA of dead bacterial cells and avoid interference with the DNA signal of live bacteria. After incubation, the bacterial suspension was placed in an ice bath, the tube lid was opened, and the sample was ensured to be approximately 10 - 15 cm away from a 590 W incandescent lamp and treated for different times of 0, 4, 8, 10, 15, and 25 min.

[0061] After the exposure ended, a qPCR reaction was performed using the treated bacterial suspension as a template. The qPCR reaction system was (20 μL): Probe qPCR Mix (2×) 10 μL, 0.4 μL each of 10 μmmol / L upstream and downstream primers, 0.8 μL of the probe, 0.2 μL of ROX DyeⅡ (50×), 2 μL of the template, and 6.2 μL of ultrapure water. The reaction conditions were: pre-denaturation at 95℃ for 30 s; 95℃ for 5 s, 60℃ for 34 s (collecting fluorescence signals), for a total of 40 cycles. After amplification, with the logarithm of the bacterial solution concentration as the abscissa and the C T value as the ordinate, a standard curve was established. The obtained data was analyzed using GraphPad Prism 10.

[0062] By treating the bacterial solution with different exposure times, it was observed that as the exposure time increased, the C T value in the amplification reaction gradually increased. This indicates that as the treatment time increased, the cross-linking effect of PMAxx with dead cell DNA was enhanced, resulting in an enhanced inhibitory effect on the amplification of dead cell DNA. Further experimental results showed that when the exposure time reached 8 min, the cross-linking effect reached saturation, and the inhibitory effect on the amplification of dead cell DNA was the most significant. After exceeding 8 min, the change in the C T value tended to be stable, indicating that extending the exposure time no longer significantly enhanced the inhibitory effect on dead cell DNA. In addition, the amplification of live cell DNA was not significantly affected, which may be because PMAxx cannot penetrate the live cell membrane to bind to DNA and undergoes photolysis under light conditions, thus not interfering with the amplification reaction. Therefore, based on the above experimental results, this study selected 8 min as the optimal exposure time to achieve the maximum inhibition of dead cell DNA.

[0063] Example 3 Establishment of the PMAxx-qPCR Standard Curve

[0064] The initial concentration of the Erwinia amylovora bacterial solution was adjusted to 10 8 cfu / mL and serially diluted 10-fold to 10 4cfu / mL. Using bacterial suspensions with different concentration gradients as templates, qPCR reactions were carried out. On the basis of optimizing the conditions of Example 2, bacterial suspensions of Erwinia amylovora at different concentrations were treated with PMAxx at a final concentration of 15 μmol / mL, and then exposed to an incandescent lamp at 590 W for 8 min for PMAxx-qPCR detection. Finally, a standard curve was plotted.

[0065] Taking the logarithm value of the concentration of Erwinia amylovora bacterial solution as the abscissa, and C T value as the ordinate, a regression equation was established. The regression equation was Y = -3.290X + 41.53, and the correlation coefficient R 2 = 0.999.

[0066] Example 4 Detection of different proportions of viable bacteria by PMAxx-qPCR

[0067] Dead bacteria and live bacteria were mixed in different proportions to obtain mixed samples with viable bacteria proportions of 0%, 10%, 20%, 50%, 80%, and 100% respectively. Each mixed sample was subjected to PMAxx-qPCR and qPCR detections respectively. Both PMAxx-qPCR and qPCR were carried out according to the above methods.

[0068] When detected by the direct qPCR method, it was found that no matter how the proportion of live cells changed, the C T value remained stable. However, after treatment with PMAxx, in the mixed samples of dead cells and live cells, as the proportion of live cells decreased, the C T value gradually increased, ranging from 18.75 to 28.10. When the proportion of live cells exceeded 10%, the PMAxx-qPCR detection results showed a good linear correlation with the proportion of live cells in the actual sample. The regression equation was: Y = 0.7856X + 1.5567, and R 2 = 0.992. The above results indicate that the PMAxx-qPCR technology can effectively distinguish live cells and dead cells and selectively amplify the DNA of live cells. While the traditional qPCR method cannot distinguish between live and dead cells, resulting in a measured concentration higher than the actual live cell concentration.

[0069] Example 5 Application of the PMAxx-qPCR detection system

[0070] First, surface sterilize the sample to be tested. Subsequently, take the tissue block at the junction of diseased and healthy parts, cut it into pieces with scissors, and soak it in 1 mL of sterile water for 30 min to ensure that pathogens are fully released from the tissue into the aqueous phase to form a bacterial suspension. After that, centrifuge the sample at 12,000 r / min for 15 min and remove the supernatant. Add 500 μL of sterilized water to the precipitate to form a suspension. Using this suspension as a template, perform qPCR and PMAxx-qPCR reactions respectively according to the above methods. At the same time, use the dilution plate coating method to compare the detection results, so as to evaluate the applicability of this method in Erwinia amylovora samples.

[0071] The detection results show (Table 1): When performing qPCR detection on 5 samples, the CT value ranges from 19.79 to 34.75, while the C T value detected by using the PMAxx-qPCR technique ranges from 21.14 to 35.43, and the viable cell count ranges from 7.08×10 to 1.58×10 6 cfu / mL. The detection results of PMAxx-qPCR are highly consistent with those of the traditional plate coating method. In particular, the viable cell count of sample 1 is 2.24×10 2 cfu / mL. Nevertheless, no colonies of Erwinia amylovora were observed by the plate isolation method, suggesting that the bacteria in this sample may have entered the VBNC (viable but non-culturable) state. This phenomenon further verifies the advantage of the PMAxx-qPCR technique in specifically identifying and amplifying the DNA of living cells, while the plate coating method effectively corroborates the detection results of this technique.

[0072] Table 1 Detection results of viable cell counts of 5 samples

[0073]

[0074] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for rapid detection of viable cells of Erwinia amylovora by PMAxx-qPCR, characterized in that, It includes the following steps: (1) Preparation of the sample suspension, i.e., the reaction template: After surface disinfection of the sample, a small piece of tissue is excised at the junction of the diseased area and healthy tissue and minced; the minced tissue is placed in 1 mL of sterile water and left standing for 30 min to allow the pathogens to be fully released; the supernatant is removed by centrifugation, and the precipitate is resuspended in 500 μL of sterilized water to obtain the target template for PMAxx-qPCR. (2) Pretreatment of the sample suspension with PMAxx: Under light avoidance conditions, modified propidium monoazide is added to the sample suspension, wrapped with tinfoil and incubated in the dark for 15 min, then the lid is opened and photolyzed on ice for 8 min. (3) Taq-man probe real-time fluorescence quantitative PCR detection: Using the sample suspension treated with PMAxx as the template, qPCR reaction is carried out. The amplification primers are: EaF1: 5'-TGAACGATATGTTAGGCGGTTCGC-3'; EaR1: 5'-GGTCGCTGGAGTCTGAGGT-3'; The Taq-man probe is: 5'-FAM-AACACGCTGGGCTCGAAAGGCGGCAACAATAC-TAMAR-3'; (4) Quantitative analysis of results: According to the standard curve obtained by real-time fluorescence quantification, substitute the C value of the measured sample into the standard curve to obtain the viable count of Erwinia amylovora. When the C value > 35, it is judged as negative; when the C value ≤ 35, it is judged as positive. T value into the standard curve to obtain the viable count of Erwinia amylovora. When the C T value > 35, it is judged as negative, and when the C T value ≤ 35, it is judged as positive.

2. A method for rapid detection of viable cells of Erwinia amylovora by PMAxx-qPCR according to claim 1, characterized in that, The specific operation of step (1) is as follows: S1 Sample pretreatment: After surface disinfection of the sample, a small piece of sample is excised at the junction of the diseased and healthy tissues, and then minced with scissors. S2 Bacterial suspension preparation: The treated sample is placed in 1 mL of sterile water and left standing for 30 min to allow the pathogens to be fully released into the water, thus forming a bacterial suspension. S3 Centrifugation treatment: The bacterial suspension is centrifuged at 12000 r / min for 15 min to remove the supernatant and retain the precipitate. S4 Template preparation: 500 μL of sterilized water is added to the precipitate to resuspend the precipitate, obtaining the target template for subsequent PMAxx-qPCR reaction.

3. A method for rapid detection of viable cells of Erwinia amylovora by PMAxx-qPCR according to claim 1, characterized in that, The final concentration of PMAxx in step (2) is 15 μmol / L. The specific operation of step (2) is: In a light-avoiding environment, modified propidium monoazide with a concentration of 15 μmol / L is added to the sample suspension, and then immediately wrapped tightly with tinfoil and incubated in the dark for 15 min; after incubation, the lid of the bacterial suspension is opened and placed on the ice surface, ensuring that the distance between the bacterial suspension and the 590 W incandescent lamp is within the range of 10 to 15 cm, and exposure treatment is carried out for 8 min.

4. A method for rapid detection of viable cells of Erwinia amylovora by PMAxx-qPCR according to claim 1, characterized in that, The system of the qPCR reaction described in step (3) is: 2×Probe qPCRMix 10 μL, 10 μmmol / L upstream primer 0.4 μL, 10 μmmol / L downstream primer 0.4 μL, Taq-man probe 0.8 μL, 50×ROX DyeⅡ0.2 μL, template DNA or bacterial suspension 2 μL, ultrapure water 6.2 μL.

5. A method for rapid detection of viable cells of Erwinia amylovora by PMAxx-qPCR according to claim 1, characterized in that, The procedure of the qPCR reaction described in step (3) is as follows: pre-denaturation at 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 34 s; after amplification is completed, a standard curve is established with the logarithm of the bacterial solution concentration as the abscissa and the C T value as the ordinate.

6. A method for rapid detection of viable cells of Erwinia amylovora by PMAxx-qPCR according to claim 1, characterized in that, In step (3), sterilized ultrapure water is used as the template as the blank control, and the live bacterial suspension and dead bacterial suspension of Erwinia amylovora treated in step (2) are used as the templates as the positive control and negative control.

7. A method for rapid detection of viable cells of Erwinia amylovora by PMAxx-qPCR according to claim 1, characterized in that, The standard curve for step (4) is Y = -3.3074X + 42.704, where Y represents the C T value and X represents the logarithm of the bacterial liquid concentration, and R 2 = 0.999, Eff = 99.99%.

8. Use of a method for rapid detection of viable cells of Erwinia amylovora by PMAxx-qPCR according to any one of claims 1-7, characterized in that, The method is applied to the qualitative and quantitative detection of viable cells of Erwinia amylovora in the branches, leaves, flowers and fruits of pear branches infected with fire blight.

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