Method for detecting viable bacteria of maize bacterial wilt germs based on PMA-qPCR

Abstract

The invention relates to the technical field of agricultural biology, and discloses a PMA-qPCR-based detection method for viable bacteria of maize bacterial wilt germs, and the method comprises the following steps: S1, preparing a sample, and obtaining a suspension of the sample to be detected; s2, PMA pretreatment: taking a proper amount of a PMA solution, adding the PMA solution into the suspension of the sample to be detected, fully mixing, incubating in a dark place, and performing exposure treatment; s4, designing a primer and a probe by taking the sample DNA extracted in the step S3 as a template, and carrying out PCR (Polymerase Chain Reaction) amplification reaction to obtain a real-time fluorescent quantitative PCR detection result. The PMA-qPCR method established by the invention can effectively distinguish viable bacteria and dead bacteria of the maize bacterial wilt, and has relatively strong specificity and relatively high sensitivity, and the lowest detection limits are 10 < 3 > CFU / mL.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR. Background Art

[0002] Corn bacterial wilt is a systemic bacterial infection disease caused by corn bacterial wilt fungus (Pantoea stewartiisubsp. stewartii, PSS), which is listed as a quarantine pest prohibited from entering my country. Traditional conventional PCR technology and real-time fluorescence PCR technology can amplify the DNA of dead bacteria while amplifying the DNA of live bacteria, and cannot distinguish between dead and live bacteria in the sample.

[0003] Propidium monoazide (PMA) is a photosensitive DNA dye that cannot penetrate the cell membrane with a complete structure. It can only covalently bind to the DNA of dead bacterial cells to produce a photo-activated reaction, inhibiting the PCR amplification of the fluorescent dye, thus overcoming the problem that traditional PCR cannot distinguish between live and dead bacteria. However, when the PMA treatment conditions are not optimized, the cross-linking between PMA and DNA is insufficient, resulting in not all DNA from dead cells being inhibited during the qPCR amplification stage, which can easily lead to high test values ​​and false positives. In addition, if the PMA concentration is not appropriate, it may also penetrate the cell membrane of live bacteria and bind to their DNA, resulting in false negative results and inaccurate test results.

[0004] Therefore, there is an urgent need to provide an efficient, sensitive and rapid detection method for corn bacterial wilt, so as to facilitate early diagnosis and quarantine of the disease, formulate scientific prevention and control measures in a timely manner, and effectively block the spread of corn bacterial wilt. Summary of the invention

[0005] Without solving the above technical problems, the present invention proposes a method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides a method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR, comprising the following steps:

[0008] S1. Prepare a sample to obtain a suspension of the sample to be tested;

[0009] S2. PMA pretreatment: add an appropriate amount of PMA solution to the suspension of the sample to be tested, mix thoroughly, incubate in the dark, and then expose to light;

[0010] S3. Sample DNA extraction:

[0011] S4, using the sample DNA extracted in step S3 as a template, designing primers and probes, performing PCR amplification reaction, and obtaining real-time fluorescence quantitative PCR detection results;

[0012] The primers are:

[0013] Upstream primer PS3F: 5'-CATTGCGTGCATTTTTTCGC-3',

[0014] Downstream primer PS3R: 5′-GTCAATCTTTTGTAAAGGTTTCCA-3′.

[0015] Furthermore, the nucleotide sequence of the fluorescent probe used in conjunction with the primer is:

[0016] Probe PS-3P:

[0017] 5'-FAM-CCTCTTTTAGAATCATATCGTTAAGATTTG-TAMARA-3'.

[0018] Furthermore, in step S2, the final concentration of PMA in the sample suspension is not less than 10 μg / mL.

[0019] Furthermore, in step S2, the light-proof incubation time is 10 to 20 minutes.

[0020] Furthermore, in step 2, the exposure treatment time is 10 to 20 minutes.

[0021] Furthermore, in step 3, the amplification reaction system at least includes the above-mentioned primers and template, and the above-mentioned probe.

[0022] Furthermore, the amplification reaction system is: 10 μL 2×Permix Ex Taq, 0.5 μL 10 μmol / L upstream primer, 0.5 uL 10 μmol / L downstream primer, 0.25 μL 10 μmol / L probe, 2 μL 1 ng / μL to 10 ng / μL template, and double distilled water is added to 20 μL.

[0023] Furthermore, the amplification reaction procedure is: 95°C for 1 min, 95°C for 5 s, 60°C for 40 s, and 40 cycles.

[0024] The present invention also provides an application of a method for detecting live bacteria of corn bacterial wilt pathogens based on PMA-qPCR, wherein the method is applied to the qualitative and quantitative detection of live bacteria of corn bacterial wilt pathogens in corn plants.

[0025] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0026] 1. The present invention proposes a method for detecting live bacteria of corn bacterial wilt pathogen using PMA-qPCR, and the test results show that when the working mass concentration of PMA is 10ug / mL, incubated for 10min under light-proof conditions and then exposed to light for 10min, the growth of corn bacterial wilt pathogen can be inhibited by 10 5 CFU / mL and 10 7 The DNA amplification of dead CFU / mL corn bacterial wilt fungi had no effect on the amplification of live bacteria, which indicated that the established PMA-qPCR method could effectively distinguish live and dead corn bacterial wilt fungi.

[0027] 2. The PMA-qPCR method for detecting live bacteria of corn bacterial wilt has strong specificity and high sensitivity, with a minimum detection limit of 10 3 CFU / mL. The actual sample detection further verified that this method can effectively distinguish between dead and live bacteria of corn bacterial wilt in actual samples; it is of great significance for the diagnosis and quarantine of diseases, the monitoring and early warning of epidemics, the study and mastery of the infection sources, transmission routes and epidemic patterns of pathogens, and the formulation of comprehensive prevention and control technical measures. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of adding different PMA concentrations to bacterial suspensions of different concentrations in the present invention (A: concentration of 1×10 7 Bacterial suspension, B: concentration is 1×10 5 of bacterial suspension);

[0029] Figure 2 The sensitivity test results of the real-time fluorescence quantitative PMA-PCR detection method established in the present invention (Figures 1-7 respectively represent the concentration of 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 CFU / mL bacterial suspension, NC is negative control);

[0030] Figure 3 The sensitivity test results of the real-time fluorescence PCR detection method of the present invention are shown in Figure 1-6 (1-6 represent the concentration of 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×103 CFU / mL bacterial suspension, NC as negative control);

[0031] Figure 4 The specific test result diagram of the real-time fluorescence quantitative PMA-PCR detection method established in the present invention (Note: 1-2 is PSS; 3-32 are other test strains (wherein, 3-8 is P.stewartii subsp.indologenes, 8-9 is P.stewartii, 10 is Acidovorax citrulli, 11 is Pectobacterium sp., 12 is Pectobacterium sp., 13 is Pseudomonas syringae pv.maculicola, 14-15 is Pseudomonas syringaepv.phaseolicola, 16-19 is Pseudomonas syringae pv.pisi, 20-22 is Xanthomonas axonopodis pv.dieffenbachiae, 23-26 is Xanthomonas euvesicatoria pv.allii, 26-27 is Pseudomonas syringae pv.tomato, 28 is Pseudomonas syringae pv.phaselicola, 29-30 are Erwinia amylovora, 31-32 are Dickeya chrysanthemi). DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0033] The present invention provides a method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR, comprising the following steps:

[0034] S1. Prepare samples to obtain suspension of samples to be tested: culture corn bacterial wilt pathogen (P. stewartii subsp stewartii) on NA solid medium at 28°C for 48 hours, take a single colony and culture it on LB liquid medium at 28°C for 12-24 hours, and adjust the concentration of bacterial suspension to 10 5 ~108 CFU / mL and reserve for use.

[0035] S2. PMA pretreatment: Take an appropriate amount of PMA solution and add it to the suspension of the sample to be tested, so that the final concentration of PMA in the sample suspension is not less than 10 μg / mL, mix thoroughly, incubate in the dark for 10 to 20 minutes, and then expose to light for 10 to 20 minutes.

[0036] S3. Sample DNA extraction: Sample DNA is extracted using existing technology, which will not be elaborated here.

[0037] S4, using the sample DNA extracted in step S3 as a template, designing primers and probes, performing PCR amplification reaction, and obtaining real-time fluorescence quantitative PCR detection results;

[0038] The primers are:

[0039] Upstream primer PS3F: 5'-CACCATTGCGTGCATTTTTTCGCCG-3',

[0040] Downstream primer PS3R: 5'-CTGTCAATCTTTTGTAAAGGTTTCCATCC-3',

[0041] The nucleotide sequence of the fluorescent probe used in conjunction with the above primers is:

[0042] Probe PS-3P: 5′-FAM-TTTCCTCTTTTAGAATCATATCGTTAAGA-TAMARA-3′.

[0043] The amplification reaction system is: 10μL 2×PermixEx Taq, 0.5μL 10μmol / L upstream primer, 0.5uL 10μmol / L downstream primer, 0.25μL 10μmol / L probe, 2μL 1ng / μL~10ng / μL template, and double distilled water to 20μL.

[0044] The amplification reaction program was: 95°C for 1 min, 95°C for 5 s, 60°C for 40 s, and 40 cycles.

[0045] Example:

[0046] 1. Establishment of a real-time fluorescence PCR detection method for corn bacterial wilt pathogen

[0047] The invention is designed according to the conservative sequence on the corn bacterial wilt pathogen genome sequence (NCBI accession number: CP046558.1), and obtains corn bacterial wilt pathogen (PSS) real-time fluorescence PCR primers and probes. The upstream primer PS3F (5'-CACCATTGCGTGCATTTTTTCGCC G-3'), the downstream primer PS3R (5'-CTGTCAATCTTTTGTAAAGGTTTCCATCC-3'), and the probe PS-3P (5'-FAM-TTTCCTCTTTTAGAATCATATCG TTAAGA-TAMARA-3'), and the primers and the probe are synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0048] The PCR reaction system and procedure are as follows:

[0049] Table 1 Real-time fluorescence PCR reaction system

[0050]

[0051] The PCR reaction program was: 95°C for 1 min; 95°C for 5 s, 60°C for 40 s, for 40 cycles.

[0052] Optimization of PMA treatment for corn bacterial wilt

[0053] 2.1PMA concentration optimization results

[0054] Select a plate count of 1×10 5 CFU / mL, 1×10 7 Take 5 portions of 1 mL of the bacterial suspension and heat them to death at 100°C for 10 min to prepare a dead bacterial suspension. Spread it on NA plates and culture for 36 h to confirm whether it is lethal.

[0055] For 1×10 5 CFU / mL, 1×10 7 Different amounts of PMA were added to the dead bacteria suspension and live bacteria suspension with a final concentration of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, and 40 μg / mL, respectively. The suspension was incubated in the dark for 20 min, exposed to light for 10 min, and DNA was extracted and qPCR reaction was performed. The results are shown in the figure. Figure 1As shown in the figure, under different dead bacteria concentrations, as the concentration of PMA working solution increases, the Ct value gradually increases, and the inhibitory amplification effect becomes more obvious. When the final concentration of PMA reaches 10μg / mL, the Ct value is greater than 35. Under different live bacteria concentrations, as the concentration of PMA working solution increases, the Ct value fluctuates slightly, and the difference is not obvious. When the final concentration of PMA is 10μg / mL, the amplification of live bacteria is not inhibited. Therefore, the final concentration of PMA of 10μg / mL is selected as the optimal concentration for subsequent experiments.

[0056] 2.2 Selection of PMA exposure time

[0057] Select a plate count of 1×10 7 CFU / mL of dead bacteria suspension, take 4 portions of 1 ml of dead bacteria suspension, add PMA working solution and mix well to make the final concentration of 10 μg / mL, incubate in the dark for 20 minutes, and expose to light for 5 minutes, 10 minutes, 15 minutes, and 20 minutes respectively, and then perform PMA-qPCR.

[0058] Table 2PMA exposure time results

[0059]

[0060] The results in Table 2 show that when the exposure time is 10 min, the Ct value is greater than 35, that is, exposure for 10 min can completely dissolve the added PMA, so the exposure time is selected to be 10 min.

[0061] 2.3 Selection of PMA light-proof incubation time

[0062] Select a plate count of 1×10 5 CFU / mL of dead bacteria suspension, take 4 portions of 1mL of dead bacteria suspension, add PMA working solution and mix well to make the final concentration of 10μg / mL, incubate in the dark for 5min, 10min, 15min, 20min, then expose to light for 10min, and perform PMA-qPCR.

[0063] Table 3PMA light-proof incubation time results

[0064]

[0065] The results in Table 3 show that when the incubation time is 10 min away from light, the Ct value is greater than 35, which can fully inhibit the amplification of dead bacterial DNA. Therefore, the incubation time is selected to be 10 min away from light.

[0066] The optimal PMA treatment scheme was determined by testing the factors affecting the concentration, dark incubation and exposure time of PMA. 10 μg / mL was selected as the PMA working concentration, incubated for 10 minutes under dark conditions, and the exposure time was 10 minutes. Under this condition, the DNA amplification of dead bacteria of corn bacterial wilt can be inhibited without affecting the amplification of live bacteria.

[0067] 3. Comparison of sensitivity of PMA-qPCR and real-time fluorescence PCR for corn bacterial wilt pathogen

[0068] PMA-qPCR method was used to detect 10 8 CFU / mL of corn bacterial wilt pathogens were continuously diluted, and a PMA-untreated control group was set up, i.e., qPCR reaction. Figure 2 , Figure 3 It can be seen that the detection sensitivity of PMA-qPCR and qPCR methods is 10 3 CFU / mL, indicating that PMA has almost no effect on the proliferation of living cells.

[0069] 4. Specificity analysis of PMA-qPCR for corn bacterial wilt pathogen

[0070] Real-time PMA-qPCR detection was performed using DNA from 5 strains of P. stewartii subsp. Indologenes (Psi), a closely related species of corn bacterial wilt pathogen, and 25 other strains as templates. The results showed that (see Figure 4 )PMA-qPCR detection method has good specificity and only amplifies positive curves for target bacteria.

[0071] 5. Results of real-time fluorescence PCR and PMA-qPCR detection of mixed solutions with different dead / live bacterial ratios

[0072] PCR and PMA-qPCR were used to detect mixed bacterial solutions with different live bacterial ratios, and the test results are shown in Table 4.

[0073] Table 4 Test results of mixed solutions with different dead / live bacteria ratios

[0074]

[0075] Note: UD means not measured (Ct value ≥ 40).

[0076] From the results in Table 4, we can see that as the amount of live bacteria added decreases, the Ct value gradually increases, and the detection limit can reach 1×10 3CFU / mL. When the mixture without PMA treatment was amplified by qPCR, no matter how the amount of live bacteria added changed, the Ct value remained at a stable level without significant change. This shows that the DNA amplification of live bacteria was not significantly affected after PMA treatment. PMA-qPCR can effectively distinguish between dead and live cells in bacterial suspension, while qPCR cannot distinguish between dead and live cells, and the test results will overestimate the amount of live bacteria in the bacterial suspension.

[0077] 6. False Positive Verification of PMA-qPCR Detection

[0078] In order to verify whether the PMA-qPCR detection method would produce false positives, the dead bacteria suspension was set to 8 concentration groups and tested by qPCR, PMA-qPCR and plate culture counting method, see Table 5.

[0079] Table 5 False positive verification of PMA-qPCR detection

[0080]

[0081] Note: UD means not detected (Ct value ≥ 40); - means that corn bacterial wilt pathogens could not be isolated on the plate.

[0082] The results in Table 5 show that the results of PMA-qPCR and plate culture counting method are the same, both negative, which further verifies the reliability of PMA-qPCR; and the presence of bacteria can still be detected by qPCR.

[0083] VII. PMA-qPCR detection of actual samples

[0084] By comparing PMA-qPCR, qPCR and plate separation methods, 20 actual samples (5 positive, 5 positive heat-killed, and 10 healthy samples) were tested, see Table 6.

[0085] Table 6 Actual sample test results

[0086]

[0087] Note: UD means not detected (Ct value ≥ 40); + means that the plate can separate the bacterial wilt of corn; - means that the plate cannot separate the bacterial wilt of corn.

[0088] The results in Table 6 show that: the test results of 10 healthy samples (numbered 11 to 20) were all negative by the three detection methods; the test results of 5 positive samples (numbered 6 to 10) were all positive by the three detection methods; the test results of 5 positive heat-killed samples (numbered 1 to 5) were consistent with those of PMA-qPCR and plate culture counting method, all of which were negative, while the qPCR test results were positive, which could not effectively distinguish between dead and living cells.

[0089] In summary, the PMA-qPCR method established in the present invention can effectively distinguish between live and dead bacteria of corn bacterial wilt, and actual sample detection further verifies that the method can effectively distinguish between dead and live bacteria of corn bacterial wilt in actual samples. The PMA-qPCR method established in the present invention has strong specificity and high sensitivity, and its minimum detection limit is 10 3 CFU / mL.

[0090] The above-described embodiments only express the preferred implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR, characterized in that: The steps include: S1. Prepare a sample to obtain a suspension of the sample to be tested; S2. PMA pretreatment: add an appropriate amount of PMA solution to the suspension of the sample to be tested, mix thoroughly, incubate in the dark, and then expose to light; S3. Sample DNA extraction: S4, using the sample DNA extracted in step S3 as a template, designing primers and probes, performing PCR amplification reaction, and obtaining real-time fluorescence quantitative PCR detection results; The primers are: Upstream primer PS3F: 5'-CATTGCGTGCATTTTTTCGC-3', Downstream primer PS3R: 5′-GTCAATCTTTTGTAAAGGTTTCCA-3′.

2. The method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR according to claim 1, characterized in that: The nucleotide sequence of the fluorescent probe used in conjunction with the primer is: Probe PS-3P: 5'-FAM-CCTCTTTTAGAATCATATCGTTAAGATTTG-TAMARA-3'.

3. The method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR according to claim 2, characterized in that: In step S2, the final concentration of PMA in the sample suspension is not less than 10 μg / mL.

4. The method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR according to claim 2, characterized in that: In step S2, the light-proof incubation time is 10 to 20 minutes.

5. The method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR according to claim 2, characterized in that: In step 2, the exposure treatment time is 10 to 20 minutes.

6. The method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR according to claim 2, characterized in that: In step 3, the amplification reaction system at least includes the primers and template described in claim 1, and the probe described in claim 2.

7. The method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR according to claim 7, characterized in that: The amplification reaction system is: 10 μL 2×Permix Ex Taq, 0.5 μL 10 μmol / L upstream primer, 0.5 uL 10 μmol / L downstream primer, 0.25 μL 10 μmol / L probe, 2 μL 1-10 ng / μL template, and double distilled water is added to 20 μL.

8. The method for detecting live bacteria of corn bacterial wilt based on PMA-qPCR according to claim 2, characterized in that: The procedure of the amplification reaction is: 95°C for 1 min, 95°C for 5 s, 60°C for 40 s, and 40 cycles.

9. The use of the PMA-qPCR-based method for detecting live bacteria of corn bacterial wilt according to claims 1-8, characterized in that: The method is applied to the qualitative and quantitative detection of live bacteria of corn bacterial wilt in corn plants.

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