Establishment and application of PMA-qPCR method for viable bacteria of fusarium wilt in corn

Abstract

The invention relates to the technical field of agricultural biology, and discloses establishment and application of a PMA-qPCR method for viable bacteria of wilt blight in corn, and the method comprises the following steps: S1, preparing a to-be-detected sample suspension; s2, PMA dyeing treatment of the sample suspension: under a dark condition, adding a proper amount of PMA into the sample suspension until the final concentration of PMA in the sample suspension is 10 [mu] g / mL, fully mixing, oscillating and incubating for at least 5 min, and exposing for at least 10 min; s3, DNA of the sample suspension in the step S2 is extracted as a template, a primer and a probe are designed, PCR amplification reaction is carried out, and a real-time fluorescent quantitative PCR detection result is obtained. The PMA-qPCR method established by the invention can effectively distinguish viable bacteria and dead bacteria of the fusarium wilt in corn, 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 the establishment and application of a live bacteria PMA-qPCR method for corn wilt pathogen. Background Art

[0002] Clavibacter michiganensis subsp. nebraskensis (Cmn), also known as Clavibacter michiganensis subsp. nebraska, belongs to the Firmicutes, Firmicutes, and Clavibacter. Under natural conditions, the pathogen only harms corn, and in severe cases, the yield loss is as high as 50%. The pathogen is a vascular destructive bacterium that can be spread over long distances with seeds and other reproductive bodies, and the pathogen can survive in seeds for at least 1 year. Cmn is mainly spread over long distances through seed transmission, and can parasitize on a variety of plants such as Echinochloa, Setaria, and Sorghum. It has been reported in the literature that Cmn is suitable for colonization in most corn-growing areas. Therefore, the risk of Cmn being introduced into major corn-growing / importing countries along with corn is increasing. Establishing a rapid and accurate detection method is of great significance to biosafety and the sustainable development of the corn industry.

[0003] In recent years, studies have reported that PMA is combined with PCR technology for live bacteria detection. Propidium monoazide (PMA) is a specific active dye that irreversibly covalently binds to dead cell DNA under strong light, hindering PCR amplification of dead cell DNA. The DNA is extracted after pre-treating the bacterial suspension with PMA, and then amplified by qPCR. The Ct value after qPCR amplification is the Ct value of live cells, eliminating the interference of dead cells.

[0004] At present, a variety of Cmn molecular detection methods have been reported, including conventional PCR, nested PCR, real-time fluorescence PCR, etc. However, these methods cannot distinguish between dead and live bacteria, and false positives are prone to occur during the detection process. Therefore, it is urgent to establish a method for rapid detection of live Cmn bacteria in corn samples. Summary of the invention

[0005] The present invention takes the rapid detection of live bacteria of corn wilt pathogen as the starting point, uses the selective characteristics of PMA for live / dead cells, and combines real-time fluorescence qPCR to detect Cmn. The optimal concentration and exposure time of PMA acting on Cmn are explored, and then the live bacteria of Cmn are detected in corn seeds in the field, aiming to establish a method for rapid, sensitive, specific and quantitative detection of Cmn.

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

[0007] The present invention provides the establishment of a live PMA-qPCR method for corn wilt pathogen, comprising the following steps:

[0008] S1, preparing a suspension of a sample to be tested;

[0009] S2. PMA staining of sample suspension: Under light-proof conditions, add an appropriate amount of PMA to the sample suspension to make the final concentration of PMA in the sample suspension 10 μg / mL, mix thoroughly, incubate with shaking for at least 5 minutes, and then expose to light for at least 10 minutes;

[0010] S3, extracting the DNA of the sample suspension in step S2 as a template, designing primers and probes, performing PCR amplification reaction, and obtaining real-time fluorescence quantitative PCR detection results.

[0011] Furthermore, in step S2, the exposure process lasts for 10 to 20 minutes.

[0012] Further, in step S3, 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.

[0013] Further, the primer:

[0014] Upstream primer Cmn-F11: 5'-TGCACCTTCATCACGACATGG-3',

[0015] Downstream primer Cmn-R11: 5′-ACGATGATCATGCTGGCAATG-3′.

[0016] Furthermore, the nucleotide sequence of the fluorescent probe used in conjunction with the above primers is:

[0017] Probe Cmn-P11:

[0018] 5'-FAM-TGTTCGGTCTCGTCATCGCACGGCA-TAMRA-3'.

[0019] Furthermore, the amplification reaction procedure is: 95°C for 1 min, 95°C for 5 s, 60°C for 40 s, and 40 cycles.

[0020] The present invention also provides an application of a PMA-qPCR method for live bacteria of corn wilt pathogen, which is applied to the qualitative and quantitative detection of live bacteria of corn wilt pathogen in corn seeds.

[0021] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0022] 1. The present invention proposes a method for detecting live bacteria of corn wilt pathogen using PMA-qPCR, and the test results show that when the working mass concentration of PMA is 10ug / mL, incubated for 5 to 20min under light-proof conditions and then exposed to light for 10min, the growth of 10 5 The DNA amplification of dead fungi of corn bacterial wilt at CFU / mL had no effect on the amplification of live fungi, which indicated that the established PMA-qPCR method could effectively distinguish live and dead fungi of corn bacterial wilt.

[0023] 2. PMA-qPCR detection of live bacteria of corn wilt pathogen has strong specificity and high sensitivity, with a minimum detection limit of 10 3 The PMA-qPCR method was used to detect 15 batches of corn wilt pathogen-positive samples, and the results showed that this method can accurately reflect the actual sample infection situation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the results of the effects of different PMA concentrations on qPCR amplification in the present invention (A is a dead bacteria suspension, and B is a live bacteria suspension);

[0025] Figure 2 These are the results of the effects of different PMA exposure times on qPCR amplification in the present invention (A is a dead bacteria suspension, and B is a live bacteria suspension);

[0026] Figure 3 It is a comparison of the sensitivity of PMA-qPCR and real-time fluorescence PCR in the present invention (A is PMA-qPCR, B is real-time fluorescence PCR);

[0027] Figure 4It is the specificity verification of the PMA-qPCR detection method in the present invention (1-2 is corn wilt pathogen; 3-15 are other test strains (wherein, 3 is alfalfa bacterial wilt pathogen Clavibacter michiganensissubsp.insidiosus (Cmi), 4 is tomato ulcer pathogen Clavibacter michiganensissubsp.michiganensis (Cmm), 5 is potato ring rot pathogen Clavibacter michiganensissubsp.sepedonicus (Cms), 6 is corn bacterial wilt pathogen P.stewartii subsp.stewartii, 7 is P.stewartii subsp.indologenes, 8 is P.stewartii, 9 is melon fruit spot pathogen Acidovoraxcitrulli, 10 is crucifer bacterial black spot pathogen Pseudomonas syringae pv.maculicola, 11 is bean halo blight pathogen Pseudomonas syringae pv.phaseolicola, 12 is pea bacterial blight fungus Pseudomonas syringae pv.pisi, 13 is tomato bacterial leaf spot fungus Pseudomonas syringaepv.tomato, 14 is Erwinia amylovora, 15 is chrysanthemum rot fungus Dickeya chrysanthemi, and 16 is a blank control);

[0028] Figure 5 These are the results of PMA-qPCR and real-time fluorescence PCR in the present invention and the detection of different dead / live bacteria ratios. DETAILED DESCRIPTION

[0029] 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.

[0030] The present invention provides a method for establishing a live PMA-qPCR method for corn wilt pathogen, comprising the following steps:

[0031] S1. Prepare the sample suspension to be tested; after culturing the corn wilt pathogen 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 to 24 hours, adjust the concentration of the bacterial suspension to 10 5 ~10 8 CFU / mL for later use

[0032] S2. PMA staining of sample suspension: Under light-proof conditions, add an appropriate amount of PMA to the sample suspension to make the final concentration of PMA in the sample suspension 10 μg / mL, mix thoroughly, incubate with shaking for 5 to 20 minutes, and then expose to light for at least 10 minutes.

[0033] S3, extracting the DNA from the sample suspension in step S2 as a template, designing primers and probes, performing PCR amplification reaction, and obtaining real-time fluorescence quantitative PCR detection results;

[0034] The amplification reaction system was: 10 μL 2×PermixEx 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 was added to 20 μL.

[0035] Among them, the above primers:

[0036] Upstream primer Cmn-F11: 5'-TGCACCTTCATCACGACATGG-3',

[0037] Downstream primer Cmn-R11: 5′-ACGATGATCATGCTGGCAATG-3′.

[0038] The nucleotide sequence of the fluorescent probe used in combination with the above primers is: probe Cmn-P11: 5'-FAM-TGTTCGGTCTCGTCATCGCACGGCA-TAMRA-3'.

[0039] The amplification reaction program was: 95°C for 1 min, 95°C for 5 s, 60°C for 40 s, and 40 cycles.

[0040] Example:

[0041] 1. Establishment of a real-time fluorescence PCR detection method for corn wilt pathogen

[0042] Comparison of different detection primers and probes for corn wilt pathogen in the prior art shows that primer / probe Cmn-F11 / Cmn-R11 / Cmn-P11 has higher detection efficiency, which is better than the real-time fluorescence PCR primer / probe CMN-FP / CMN-RP-Cmn-P and Cmn3F / Cmn110R-cmn38p recommended in the current national standard "GB / T36840-2018 Quarantine and Identification Method for Corn Wilt Pathogen". Therefore, the present invention uses primer / probe Cmn-F11 / Cmn-R11 / Cmn-P11 (sequence shown in Table 1), which is synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0043] The PCR reaction system was: 10 μL 2×Permix Ex Taq (Probe qPCR), 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~10 ng / μL template, and double distilled water was added to 20 μL.

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

[0045] Table 1 Primer and probe sequences for real-time fluorescence PCR of corn wilt pathogen

[0046]

[0047] Optimization of PMA treatment for corn wilt pathogen

[0048] 2.1PMA concentration optimization results

[0049] Select a plate count of 1 × 10 5 cfu / mL dead bacteria suspension was treated with different PMA concentrations to make the final concentrations of 0μg / mL, 5μg / mL, 10μg / mL, 15μg / mL, and 20μg / mL, and then real-time fluorescence PCR reaction was performed. The results are shown in Figure 1 As shown in A, under different dead bacteria concentrations, as the concentration of PMA working solution increased, the Ct value gradually increased, and the inhibitory amplification effect became more obvious. When the final concentration of PMA was 10 μg / mL, the Ct value was the largest. As the PMA concentration continued to increase, the Ct value did not increase significantly and tended to be stable.

[0050] Select a plate count of 1 × 10 5 cfu / mL live bacterial suspension, different PMA concentrations were added to make the final concentrations of 0μg / mL, 5μg / mL, 10μg / mL, 15μg / mL, and 20μg / mL for treatment, and then real-time fluorescence PCR reaction was performed. The results are shown in Figure 1 As shown in Figure B, as the concentration of PMA working solution increases, the Ct value fluctuates slightly, but the change is not obvious. When the final concentration of PMA is 10 μg / mL, the amplification of live bacteria is not inhibited. Therefore, we choose a final concentration of PMA of 10 μg / mL as the optimal concentration for subsequent experiments.

[0051] 2.2 Selection of PMA exposure time

[0052] Select a plate count of 1×10 5 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 min, and perform real-time fluorescence PCR after exposure for 0 min, 5 min, 10 min, 15 min, and 20 min respectively. Figure 2 The results of A showed that when the exposure time was 10 min, the Ct value was the largest, indicating that PMA had the strongest inhibitory effect on the amplification of dead bacterial DNA at this time, so the exposure time was selected as 10 min.

[0053] Figure 2 The results of B showed that when the exposure time was gradually extended from 5 min to 20 min, PMA had no effect on the qPCR amplification of live cell DNA; however, when PMA was applied to dead cells, the qPCR amplification of dead cell DNA was completely inhibited when the exposure time was 10 min, and the Ct value was the largest and no longer changed (see Figure 2 A) Therefore, the optimal exposure time of PMA dye in the present invention is 10 min, and the optimal concentration of PMA is 10 μg / mL, which can completely inhibit 1×10 5 cfu / mL Cmn bacterial suspension, and had no significant effect on the amplification of live cell DNA.

[0054] The optimal PMA treatment scheme was determined by testing factors such as the concentration and exposure time that affect the effect of PMA. 10 μg / mL was selected as the PMA working concentration. After incubation for 5 to 20 minutes under light-proof conditions, the exposure time was 10 minutes. Under this condition, the DNA amplification of dead fungi of corn wilt pathogen can be inhibited without affecting the amplification of live fungi.

[0055] 3. Comparison of the sensitivity of PMA-qPCR and real-time fluorescence PCR for P. wilt in corn

[0056] PMA-qPCR method was used to detect 10 8 CFU / mL of corn wilt pathogens were diluted in a continuous gradient, and a PMA-untreated control group was set up, i.e., qPCR reaction. Figure 3 A. Figure 3 B shows that the detection sensitivity of PMA-qPCR and qPCR methods is 103 CFU / mL, indicating that PMA has almost no effect on the proliferation of living cells.

[0057] 4. Specificity analysis of PMA-qPCR of corn wilt pathogen

[0058] Real-time PMA-qPCR detection was performed using DNA from two closely related strains of corn wilt pathogens, Clavibacter michiganensis subsp. insidiosus (Cmi), tomato canker pathogen Clavibacter michiganensis subsp. michiganensis (Cmm), potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus (Cms), and 15 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.

[0059] 5. Results of real-time fluorescence PCR and PMA-qPCR detection of mixed solutions with different dead / live bacterial ratios

[0060] PMA-qPCR was used to detect the mixed bacterial solution with different live bacterial ratios. Cmn dead cells (1×10 5 cfu / mL) were mixed with live cells of different concentrations in equal volumes, and PMA with a final concentration of 10 μg / mL was added for qPCR amplification. As the proportion of live cells decreased, the Ct value showed a gradual increase, and the detection limit could reach 1×10 2 cfu / mL viable cells (see Figure 5 ), the Ct value of the mixed solution treated with PMA>35, indicating that the DNA of dead bacteria is inhibited by PMA and cannot be amplified. The mixed system of dead and living cells that has not been treated with PMA is directly amplified by qPCR. Regardless of how the proportion of living cells changes, the Ct value of its qPCR amplification always remains at a stable level without significant changes. Therefore, PMA-qPCR can effectively distinguish between dead and living cells in Cmn bacterial suspensions, and can selectively amplify live cell DNA, while traditional qPCR cannot distinguish between dead and living cells, and its detection results will cause a certain degree of overestimation of the number of live cells in the system. Therefore, the Ct value of fluorescent quantitative PCR can be used to quickly and quantitatively determine the number of live bacteria corresponding to the sample, reducing the possibility of false positive results.

[0061] 6. False Positive Verification of PMA-qPCR Detection

[0062] In order to verify whether the PMA-qPCR detection method would produce false positive results in the detection of Cmn dead cells, the dead cell bacterial suspension was set to 8 groups of different concentrations and tested by traditional fluorescence quantitative qPCR, PMA-qPCR and plate culture counting method (see Table 2).

[0063] Table 2 Comparison of different methods for detecting Cmn dead cells

[0064]

[0065] Note: NA has no fluorescent signal and the Ct value is ≥40.

[0066] The results in Table 2 show that when PMA-qPCR and plate culture counting methods were used to detect 8 groups of inactivated dead cells, the test results were all negative; while the presence of Cmn was detected by traditional fluorescence quantitative qPCR. Therefore, in the detection of Cmn dead cell DNA, traditional fluorescence quantitative qPCR cannot effectively distinguish between dead and living cells, while PMA-qPCR can only amplify the DNA of living cells without false positive amplification results; and the plate culture counting method was used to further verify the reliability of PMA-qPCR.

[0067] VII. Real-time fluorescence PMA-qPCR detection of actual samples

[0068] The PMA-qPCR established in this study was used to detect 5 corn seed samples positive for the maize wilt pathogen Cmn and 10 healthy corn seeds. The results are shown in Table 3.

[0069] Table 3 Real-time fluorescence PCR and PMA-qPCR detection results of actual samples

[0070]

[0071] Note: + The plate separation method can separate Cmn, which is positive; - The plate separation method cannot separate Cmn, which is negative.

[0072] The results in Table 3 show that the five samples carrying Cmn can all be detected with Cmn, and the Ct is between 18.26 and 32.21; the five samples carrying Cmn can be isolated by plate separation and culture, while the other 10 healthy samples did not have Cmn isolated, which verifies the accuracy of PMA-qPCR detection. At the same time, the 15 corn seed samples were heat-killed and tested by PMA-qPCR, and all the test results were negative. The traditional qPCR method can detect that all five samples are positive for Cmn (see Table 3). Therefore, PMA-qPCR can quickly and accurately detect the living state of Cmn in corn seed samples, thereby replacing the plate separation and culture method.

[0073] In summary, the PMA-qPCR detection method for live bacteria of corn wilt pathogen established in the present invention can completely inhibit the growth of 10 5 cfu / mL of DNA amplification of dead bacteria of corn wilt pathogen was not affected by the amplification of live bacteria. By comparing the sensitivity of real-time fluorescence PCR and PMA-qPCR, it was found that the difference in sensitivity between real-time fluorescence PCR and real-time fluorescence PCR was not significant. The quantitative method of PMA-qPCR was used to detect corn wilt pathogen, and the minimum detection rate was 10 3 The PMA-qPCR method was used to detect 15 batches of corn positive samples for the pathogen. The results showed that the method can accurately reflect the actual situation of the samples.

[0074] 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. Establishment of a live PMA-qPCR method for corn wilt pathogen, characterized in that: The steps include: S1, preparing a suspension of a sample to be tested; S2. PMA staining of sample suspension: Under light-proof conditions, add an appropriate amount of PMA to the sample suspension to make the final concentration of PMA in the sample suspension 10 μg / mL, mix thoroughly, incubate with shaking for at least 5 minutes, and then expose to light for at least 10 minutes; S3, extracting the DNA of the sample suspension in step S2 as a template, designing primers and probes, performing PCR amplification reaction, and obtaining real-time fluorescence quantitative PCR detection results.

2. The establishment of the live bacteria PMA-qPCR method for corn wilt pathogen according to claim 1, characterized in that: In step S2, the exposure process lasts for 10 to 20 minutes.

3. The establishment of the live bacteria PMA-qPCR method for corn wilt pathogen according to claim 1, characterized in that: In step S3, the amplification reaction system is: 10 μL 2×PermixEx 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.

4. The establishment of the live bacteria PMA-qPCR method for corn wilt pathogen according to claim 3, characterized in that: The primers: Upstream primer Cmn-F11: 5′-TGCACCTTCATCACGACATGG-3′, downstream primer Cmn-R11: 5′-ACGATGATCATGCTGGCAATG-3′.

5. The establishment of the live bacteria PMA-qPCR method for corn wilt pathogen according to claim 4, characterized in that: The nucleotide sequence of the fluorescent probe used in conjunction with the primer is: Probe Cmn-P11: 5'-FAM-TGTTCGGTCTCGTCATCGCACGGCA-TAMRA-3'.

6. The establishment of the live bacteria PMA-qPCR method for corn wilt pathogen according to claim 1, 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.

7. The use of the live bacteria PMA-qPCR method of corn wilt pathogen according to claims 1-6, characterized in that: The method is applied to the qualitative and quantitative detection of live bacteria of corn wilt pathogen in corn seeds.

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