Method for detecting viable watermelon fruit spot bacterial pathogens using PMA-PCR technology

Abstract

This invention provides a detection method for distinguishing between live and dead bacteria of the watermelon fruit spot bacterial disease fungus. [Solution] A detection method for distinguishing between live and dead bacteria based on PMA-PCR technology, comprising: a step of collecting and suspending bacteria in a sample to be detected, which involves suspending the sample in physiological saline to obtain a bacterial suspension and heating it in a 100°C water bath for 10 minutes to prepare a dead bacterial suspension; a step of PMA treatment, which involves adding propidium monoazide (PMA) to the bacterial suspension; a step of blue light crosslinking, which involves irradiating the PMA-treated bacterial suspension with blue light to activate the PMA and covalently bind it to the dead bacterial DNA; a step of DNA extraction, which involves collecting bacterial cells by centrifugation and extracting DNA using a kit method; a PCR amplification step, which involves amplification using a specific primer pair; and a step of detecting the amplified product by agarose gel electrophoresis, which involves determining the result, which involves a step of detecting the product by agarose gel electrophoresis, wherein a target band appears in the live bacterial sample and no band appears in the dead bacterial sample.

Description

[Technical Field]

[0001] The present invention relates to the field of plant bacteria detection, and more particularly to PMA-PCR technology. Rus Squid fruit spot bacterial disease fungus Live bacteria Regarding the detection method. [Background technology]

[0002] Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, belongs to the phylum Proteobacteria, class Betaproteobacteria, order Burkholderiales, family Comamonadaceae, and genus Acidovorax. It is a quarantine disease that causes fatal damage to various cucurbit crops such as watermelon and cantaloupe. Seed contamination is the main source of infection, and in the early stages of the disease, small water-soaked spots appear on the leaves. In the later stages, brown to black necrotic spots form on the fruit surface, and in severe cases, it causes fruit rot and fall off, resulting in a significant reduction in yield and making it a major limiting factor for the cucurbit industry worldwide.

[0003] In China, since its first report in 1998, the disease has spread to major breeding areas such as Xinjiang, Hainan, Shandong, Fujian, Hunan, and Guangdong. In areas where the disease is severe, 80% of watermelons lose their commercial value, and in 2007, the bacterial bacterium that causes cucurbit fruit staining was registered in China's list of advanced plant quarantine pests.

[0004] Distinguishing between live and dead cells of the watermelon fruit spot bacterial pathogen is crucial at every stage of agriculture. Live cells are a vital component of disease spread and progression, and differentiating between live and dead cells improves the accuracy of disease detection, avoids false positive results due to residual dead cells, and helps to more accurately assess the actual risk of disease spread and the extent of harm. In the commercial breeding process, seed companies generally inactivate the watermelon fruit spot bacterial pathogen by sterilizing seeds with methods such as hydrochloric acid oxidation, sodium hypochlorite, or dry heat disinfection, based on positive results from conventional PCR. However, because conventional PCR cannot accurately distinguish between live and dead cells, residual dead DNA after treatment is still amplified and shows a positive signal. Without technical means to distinguish between live and dead cells, disinfected seeds that have actually lost their infectivity are treated as "contaminated," downgraded, or discarded, resulting in significant economic losses. Furthermore, the difficulty for farmers in obtaining "true zero-viable bacteria" seeds, coupled with the persistent risk of disease outbreaks in the fields, creates lasting problems for the industry.

[0005] In current industrial practices, conventional PCR technology has significant limitations. It cannot accurately distinguish between active and inactivated fruit spot disease fungi. Dead DNA fragments persist after disinfection treatment, leading to false positives and misdiagnosis. A direct consequence of misdiagnosis is excessive prevention and control strategies. If dead fungi constitute the majority of a sample and live fungi are extremely rare, it is still judged as high risk, and high-intensity chemical agents are forcibly applied. This not only leads to resource waste and increased production costs, but also pollutes the environment, accelerates the evolution of drug resistance in the disease fungi, and triggers a negative chain reaction. Conventional detection methods, such as seedling growth methods, can distinguish between live and dead fungi, but they have low sensitivity, long cycles, are affected by environmental conditions, and have low result reproducibility, making it difficult to meet the need for rapid detection. Therefore, accurately identifying live fungi is key to the sustainable and healthy development of the cucurbit industry. [Overview of the project]

[0006] In view of this, the present invention provides a method for detecting active watermelon fruit spot bacterial pathogens using PMA-PCR technology, and the combination of propidium monoazide (PMA) and PCR technology (PMA-PCR) offers a novel concept for detecting active pathogens. PMA-PCR technology is an improvement on standard PCR detection methods based on the concept that DNA-binding dyes and active cells have a complete cell membrane. By suppressing the amplification of dead bacteria with the nucleic acid dye PMA, the detectability of live cells is improved. By selecting PMA concentration, photodegradation time, bacterial concentration, etc., the most efficient and accurate PMA-PCR system for identifying active fruit spot bacterial pathogens in watermelon seeds is constructed. For a single sample, the process from nucleic acid extraction to result output can be completed within 3 hours, significantly shortening the detection cycle compared to conventional isolation culture (3d-5d), and achieving efficient, accurate, and specific detection of live bacteria. Providing technical support for early warning of crops, accurately determining the safety of seeds after disinfection, avoiding misdiagnosis losses due to non-infectious dead bacteria, and offering reliable technical support for seed quarantine and disease prevention and control, this has significant strategic importance for ensuring the safety of China's watermelon industry.

[0007] The present invention The sample is suspended in physiological saline to obtain a bacterial suspension. Step (1) involves collecting and suspending bacteria in the sample to be detected. Step (2) involves adding propidium monoazide (PMA) to the bacterial suspension, The bacterial suspension after PMA treatment is irradiated with blue light to activate the PMA and covalently bind it to the dead bacterial DNA in a blue light crosslinking step (3), The DNA extraction step (4) involves collecting bacterial cells by centrifugation and extracting DNA using a kit method, PCR amplification step (5) using a specific primer pair, A step of detecting the amplified product by agarose gel electrophoresis, Live bacteria are present in the sample. The target band appeared in the viable bacterial sample. No viable bacteria are present in the sample. In the dead bacterial sample, there is no band in the result determination step. 5 ) and, including, PMA-PCR technology Rus Squid fruit spot bacterial disease fungus Live bacteria A detection method is provided.

[0008] In a further technical method, in the PMA treatment step (2), the final concentration of PMA is 30 μM, and the light-shielding incubation time is 10 minutes.

[0009] In a further technical method, in the blue light crosslinking step (3), the bacterial suspension after PMA treatment is irradiated with blue light of a wavelength of 465±10 nm for 20 minutes to activate the PMA and covalently bind it to the dead bacterial DNA. The blue light irradiation is performed under ice bath conditions, and the light source is located 15 cm to 25 cm away from the sample.

[0010] In a further technical method, in the PCR amplification step (5), the sequence of the specific primer is: WFB1:5'-GACCAGCCACACTGGGAC-3', WFB2:5'-CTGCCGTACTCCAGCGAT-3'

[0011] In a further technical means, in the step of collecting and suspending bacteria in the sample to be detected (1), the concentration of the bacterial suspension is ≥ 10 3 The concentration is CFU / mL.

[0012] In a further technical method, in the blue light crosslinking step (3), the verification of dead bacteria is achieved by inactivating them in a 100°C water bath for 10 minutes, then seeding them on a plate and confirming that no colonies proliferate.

[0013] The PMA-PCR technology of the present invention Rus Squid fruit spot bacterial disease fungus Live bacteria The detection method has the following beneficial effects compared to conventional techniques.

[0014] This invention offers a novel concept for detecting active pathogens using a combination of propidium monoazide (PMA) and PCR technology (PMA-PCR). PMA-PCR technology is an improvement on standard PCR detection methods based on the concept that DNA-binding dyes and active cells have complete cell membranes. By suppressing the amplification of dead bacteria with the nucleic acid dye PMA, the detectability of live cells is improved. By selecting PMA concentration, photodegradation time, bacterial concentration, etc., the most efficient and accurate PMA-PCR system for identifying active fruit spot disease fungi in watermelon seeds is established. As can be seen from the test results, the working mass concentration of PMA is 30 μM, incubated for 5 minutes under light-shielding conditions, exposed for 20 minutes, and under these conditions for 10 4 The CFU / mL level suppressed DNA amplification of dead watermelon fruit spot bacterial pathogen without affecting the amplification of live bacteria. This indicates that the established PMA-PCR detection method can effectively distinguish between live and dead watermelon fruit spot bacterial pathogens.

[0015] The detection method using PMA-PCR technology established in this invention has strong specificity and high sensitivity, with a minimum detection limit of 10 3 The concentration is CFU / mL. This invention can accurately distinguish between live and dead bacteria in a sample, significantly reduce false positives, and eliminate "interference from dead bacteria." It can accurately determine whether seeds treated by seed companies have bacterial spot disease and whether they are infectious. It can provide strong evidence for resolving disputes related to bacterial spot disease, and has significant implications for the early diagnosis of watermelon spot disease, import and export quarantine, understanding the source of infection, transmission routes and epidemic rules of the pathogen, and formulating scientific and efficient prevention and control measures. [Brief explanation of the drawing]

[0016] The drawings, which constitute part of the present invention, provide a further understanding of the invention, and the exemplary embodiments and descriptions thereof are for interpretation purposes only, but do not unduly limit the invention.

[0017] [Figure 1]This is a diagram showing the results of adding different concentrations of PMA to a bacterial suspension of 104 CFU / mL in the present invention. [Figure 2] This is a diagram showing the results of selecting the optimal exposure time in the present invention. [Figure 3] This is a diagram showing the sensitivity test results of the detection method by the PMA-PCR technique in the present invention.

Mode for Carrying Out the Invention

[0018] In addition, the examples in the present invention and the features in the examples can be combined with each other as long as they do not conflict. Hereinafter, the present invention will be described in detail with reference to the drawings by giving examples.

[0019] As shown in FIGS. 1, FIG. 2, and FIG. 3, the present invention preferably relates to a method for detecting active bacterial pathogens on watermelon fruit stains by the PMA-PCR technique, and the method includes the following steps (1) to (6).

[0020] (1) Collection and suspension step of bacteria in the detection target sample For the seed sample, 100 mL of physiological saline was added to 10 g of crushed seeds, shaken at room temperature for 4 h, immersed overnight at 4°C, the immersion solution was centrifuged at 1000 rpm for 1 min to remove the precipitate, the supernatant was centrifuged at 10000 rpm for 10 min, the supernatant was discarded, and the precipitate was taken. Suspended to 1×10 4 CFU / mL, and 1 mL of the bacterial solution was placed in a centrifuge tube for use.

[0021] For the dead bacteria control, it was inactivated in a water bath at 100°C for 10 min, seeded on a plate, and it was verified that no colonies grew.

[0022] (2) PMA treatment step The PMA working solution (final concentration 30 μmol / L, PMA, Biotium, USA) was added to the above centrifuge tube, mixed well and uniformly, and then incubated at room temperature in the dark for 10 min.

[0023] (3) Blue light crosslinking step The sample tube was placed on ice, and a blue LED light source with a wavelength of 465nm to 475nm was used to activate PMA and covalently bind it to dead bacterial DNA. The light source was positioned 15cm away from the sample, and the sample was irradiated for 20 minutes (shaking gently once every 5 minutes) to crosslink the PMA with the dead bacterial DNA.

[0024] (4) DNA extraction step DNA was extracted using Vazyme's FastPure Bacteria DNA Isolation Mini Kit.

[0025] (5) PCR amplification step Reaction system: [Table 1] Primer sequence: WFB1:5'-GACCAGCCACACTGGGAC-3' WFB2:5'-CTGCCGTACTCCAGCGAT-3'(360bp) Amplification program: Preliminary denaturation: 94°C, 2 min Denaturation: 95°C, 20s Annealing: 55℃, 20s Stretching: 72℃, 35s Number of cycles: 35× Stretching: 72℃, 7min

[0026] (6) Result determination step This step involves detecting the amplified product by agarose gel electrophoresis. A target band appears in live bacterial samples, while no band is present in dead bacterial samples.

[0027] (Example 1) Regarding the optimization of the optimal PMA concentration, Number of plates: 1 x 10 4A bacterial suspension with a CFU / mL concentration was selected, and 10 parts of 1 mL of the bacterial suspension were used. Five parts of this suspension were placed in a 100°C water bath for 10 minutes to prepare a dead bacterial suspension. This was then seeded onto a KB plate and incubated for 72 hours. The bacteria were determined to be dead when no colonies grew.

[0028] 1 x 10 4 Different amounts of PMA were added to dead and live bacterial suspensions at CFU / mL, respectively, to achieve final concentrations of 0 μM, 10 μM, 20 μM, 30 μM, and 45 μM. These were incubated in the dark for 10 minutes, exposed to light for 20 minutes, and DNA was extracted before performing PCR. As shown in Figure 1, the bands gradually weakened with increasing PMA concentration. After the final PMA concentration reached 30 μM, the dead bacterial bands completely disappeared, and the brightness of the live bacterial bands matched that of the untreated group. Therefore, the specific bacterial suspension concentration (1 × 10) 4 In CFU / mL, it was confirmed that the minimum effective concentration required for PMA to completely suppress the amplification of dead bacterial DNA is 30 μM.

[0029] (Example 2) Regarding the optimization of the optimal exposure time for PMA, Number of plates: 1 x 10 4 A bacterial suspension with a CFU / ml concentration was selected, and eight 1 mL portions of the bacterial suspension were used. Four portions of this bacterial suspension were prepared as dead bacterial suspensions. PMA working solution was added and the suspensions were uniformly mixed to a final concentration of 30 μM. These were incubated in the dark for 10 minutes, followed by exposure treatment for 5 min, 15 min, 20 min, and 30 min, respectively, before PCR reaction. As shown in Figure 2, no bands were observed in the dead bacterial group, meaning that the shortest exposure time at which PMA completely suppressed dead bacterial DNA amplification was 20 min. Therefore, a PMA exposure time of 20 min was selected as the optimal exposure time for subsequent experiments.

[0030] (Example 3) Regarding the sensitive detection of watermelon fruit spot bacterial disease fungus by PMA-PCR, By the PMA-PCR method, 10 samples were obtained by continuous gradient dilution.7 The viable cells of the watermelon fruit blotch bacterial pathogen at CFU / mL were detected, the PMA working solution was added and uniformly mixed to a final concentration of 30 μM, incubated for 10 min in the dark, exposed for 20 min, and then subjected to PCR reaction. As can be seen from Figure 3, the lowest viable cell concentration, i.e., the lower detection limit, of the clearly visible band was 10 3 CFU / mL.

[0031] As described above, the detection method by the PMA-PCR technique established by the present invention can effectively distinguish between viable and dead cells of the watermelon fruit blotch bacterial pathogen. The optimal PMA concentration is 30 μM, the optimal exposure time is 20 min, and the lowest detection limit is 10 3 CFU / mL.

[0032] The above description is only a preferred embodiment of the present invention and does not limit the present invention. All modifications, equivalent substitutions, improvements, etc. made within the concept and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. The sample is suspended in physiological saline to obtain a bacterial suspension. Step (1) is to collect and suspend bacteria in the sample to be detected. Step (2) involves adding propidium monoazide (PMA) to the bacterial suspension, The bacterial suspension after PMA treatment is irradiated with blue light to activate the PMA and covalently bond it with dead bacterial DNA in a blue light crosslinking step (3), The DNA extraction step (4) involves collecting bacterial cells by centrifugation and extracting DNA using a kit method, PCR amplification step (5) in which amplification is performed using a specific primer pair, A step of detecting amplification products by agarose gel electrophoresis, comprising a result determination step (5) in which a target band appears in live bacterial samples where live bacteria are present in the sample, and no band appears in dead bacterial samples where live bacteria are not present in the sample. A method for detecting viable watermelon fruit spot bacterial disease fungi using PMA-PCR technology, characterized by the following features.

2. In the PMA treatment step (2) described above, the final concentration of PMA is 30 μM, and the light-shielding incubation time is 10 minutes. A method for detecting viable watermelon fruit spot bacterial disease fungi by PMA-PCR technology as described in feature 1.

3. In the blue light crosslinking step (3) described above, the bacterial suspension after PMA treatment is irradiated with blue light of a wavelength of 465 ± 10 nm for 20 minutes to activate the PMA and covalently bind it to the dead bacterial DNA. The blue light irradiation is performed under ice bath conditions, and the light source is located 15 cm to 25 cm away from the sample. A method for detecting viable watermelon fruit spot bacterial disease fungi by PMA-PCR technology as described in feature 1.

4. In the PCR amplification step (5) described above, the sequence of the specific primer is: WFB1:5'-GACCAGCCACACTGGGAC-3', WFB2:5'-CTGCCGTAACTCCCAGCGAT-3' A method for detecting viable watermelon fruit spot bacterial disease fungi by PMA-PCR technology as described in feature 1.

5. In the step (1) of collecting and suspending bacteria in the sample to be detected, the concentration of the bacterial suspension is ≥ 10 ３ It is CFU / mL. A method for detecting viable watermelon fruit spot bacterial disease fungi by PMA-PCR technology as described in feature 1.

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