Phaseolus vulgaris bacterial wilt pathogen activity detection method based on PMA-qPCR

Abstract

The invention discloses a PMA-qPCR-based activity detection method for Phaseolus vulgaris bacterial wilt germs, and belongs to the technical field of pathogen detection. According to the PMA-qPCR-based activity detection method for the bacterial wilt of phaseolus vulgaris disclosed by the invention, a group of high-specificity primers (CffFOR2 / CffREV4 / CF-P) are designed and are matched with an optimized PMA-qPCR detection system, so that live / dead bacterium mixtures in different proportions can be accurately distinguished, and the false positive rate is remarkably reduced in actual sample detection. The method not only provides a reliable technical means for bean seed health detection and plant quarantine, but also can be extensively applied to activity detection of other phytopathogens according to the technical principle and implementation scheme, and has important scientific value and wide application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of pathogenic bacteria detection, in particular to a bean bacterial wilt pathogen activity detection method based on PMA-qPCR. BACKGROUND

[0002] Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) is an important plant pathogen of legume crops, which seriously endangers the production of legume crops. Cff was first reported in the United States in 1922, and is now widely distributed in 13 countries and regions in South America, North America, Africa, Asia, Europe and Oceania. The main hosts of Cff are Phaseolus vulgaris, Vigna unguiculata, Vigna radiata, Glycine max and other legume crops, and barley (Hordeum vulgare), Brassica napus, Galinsoga parviflora and other crops and weeds are potential hosts of Cff. Cff can survive in seeds for 24 years under experimental conditions, in soil for up to 154 days, and in field debris for up to 8 months. Cff infection of legume crops can cause leaf wilting and seed discoloration, leading to yield reduction and causing serious economic losses. Cff has been listed as a quarantine harmful organism by 16 countries including China and 5 regional plant protection organizations.

[0003] Propidium monoazide bromide-quantitative polymerase chain reaction (PMA-qPCR) technology is a molecular biology method combining propidium monoazide (PMA) pretreatment and quantitative polymerase chain reaction (qPCR), and is widely used in the activity detection of pathogenic microorganisms. PMA is a dye that can penetrate the cell membrane of dead bacteria and bind to DNA. After light activation, PMA covalently binds to DNA, thereby inhibiting the amplification of dead bacterial DNA in subsequent qPCR. This property enables PMA-qPCR to selectively distinguish between live and dead bacteria, overcoming the limitations of traditional qPCR in distinguishing microbial activity. The core advantage of PMA-qPCR technology is its high sensitivity and specificity. By optimizing the PMA concentration, exposure time and light-avoiding incubation time, the amplification of dead bacterial DNA can be maximally inhibited while retaining the signal of live bacterial DNA. In addition, PMA-qPCR technology also shows high reliability in complex samples, such as pathogenic bacteria detection in food, environment and water samples. In the field of plant pathogen detection, PMA-qPCR technology shows great potential.

[0004] Currently, the detection methods of Cff mainly include traditional separation and culture method, antibody-based serological detection method and ordinary qPCR technology. However, these methods cannot distinguish between live bacteria and dead bacteria, which may lead to overestimation or underestimation of the detection results. The PMA-qPCR technology has not been reported in the detection of Cff.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide a PMA-qPCR-based Cff activity detection method for bean bacterial wilt. SUMMARY

[0006] Therefore, the present application provides a PMA-qPCR-based Cff activity detection method for bean bacterial wilt.

[0007] The present application is a PMA-qPCR-based rapid and accurate detection method for detecting the activity of Cff in legume seeds. By optimizing the key parameters such as PMA concentration, exposure time and light-free incubation time, the sensitivity, specificity and actual application effect of the technology are evaluated, and the ordinary qPCR is compared and analyzed. The establishment of the method will provide reliable technical support for the health detection of legume seeds, the epidemiological study of diseases and plant quarantine, and provide a reference for the activity detection of other plant pathogens.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] A PMA-qPCR-based Cff activity detection method for bean bacterial wilt, comprising the following steps:

[0010] (1) Preparation of the sample to be tested

[0011] Take 100g of the sample to be tested, add 300mL of sterilized physiological saline, soak at 4℃ overnight, filter with gauze, let the filtrate stand for 15min, centrifuge the supernatant at 10000r / min for 20min, suspend the precipitate with 1mL of PBS and transfer it into a 1.5ml centrifuge tube, add PMA to the suspension to make its final concentration 5μg / mL, incubate in the dark for 10min, and expose to light for 5min.

[0012] (2) DNA extraction and PCR amplification

[0013] Centrifuge the treated sample suspension at 12000r / min for 5min, extract the genomic DNA from the precipitate, and perform real-time fluorescent PCR reaction.

[0014] The primer and probe sequences are as follows:

[0015] Upstream primer CffFOR2: 5'-GTTATGACTGAACTTCACTCC-3'; SEQ ID NO. 1;

[0016] Downstream primer CffREV4: 5'-GATGTTCCCGGTGTTCAG-3'; SEQ ID NO. 2;

[0017] Primer probe CF-P: 5'-TCTCGGCCCGTCCGAATTTCCG-3'; SEQ ID NO. 3;

[0018] The primer probe is labeled with a fluorescence reporter group FAM at the 5' end and a quenching group TAMRA at the 3' end.

[0019] The real-time fluorescence PCR reaction system is 25 μL, including 12.5 μL 2x Premix Ex Taq, 1 μL of each of upstream and downstream primers (5 μmol / L) and probe (5 μmol / L), 1 μL template DNA, 0.5 μL 50x Rox Reference Dye, and adding sterile water to 25 μL. The reaction program is: 95℃ pre-denaturation for 10 s; 95℃ for 15 s, 60℃ for 1 min, 40 cycles.

[0020] (3) Result determination

[0021] When the sample to be tested has no fluorescence signal (Ct value ≥ 40), it is determined to be Cff negative;

[0022] When the sample to be tested has a typical amplification curve and the Ct value is ≤ 35, it is determined to be Cff positive;

[0023] When the sample to be tested has a typical amplification curve and the Ct value is less than 40 and greater than 35, it should be retested, if the Ct value of the retest is ≥ 40, it is determined to be Cff negative; if the Ct value of the retest is less than 40 and greater than 35, it is determined to be Cff positive.

[0024] The application provides a bean bacterial wilt bacterium (Cff) activity detection method based on combination of propidium monoazide (PMA) and real-time fluorescence quantitative PCR (qPCR), and the core innovation point is that the technology is applied to Cff live bacterium specific detection for the first time, and the key technical problem that traditional detection methods cannot distinguish live bacteria and dead bacteria is solved. By optimizing the key parameters of PMA treatment, the optimal combination of 5 μg / mL of the best PMA concentration, 5 minutes of exposure time and 10 minutes of light incubation time is determined, so that the DNA amplification of dead bacteria is completely inhibited while the high sensitivity (up to 10 3The application is particularly designed for a group of high-specificity primers (CffFOR2 / CffREV4 / CF-P) combined with an optimized PMA-qPCR detection system, which can accurately distinguish different proportions of live / dead bacteria mixture, and significantly reduce the false positive rate in actual sample detection. The method not only provides a reliable technical means for legume seed health detection and plant quarantine, but also can be applied to the activity detection of other plant pathogens based on the technical principle and implementation scheme, which has important scientific value and broad application prospect.

[0025] According to the above technical solution, compared with the prior art, the application provides a PMA-qPCR-based activity detection method for bacterial wilt of kidney bean, which has the following beneficial effects:

[0026] (1) High specificity to distinguish live bacteria from dead bacteria. By optimizing the concentration of propidium monoazide bromide (PMA), exposure time and light-avoiding incubation time, the amplification of dead bacteria DNA can be selectively inhibited, and only live bacteria signals can be detected, solving the problem that traditional qPCR cannot distinguish the activity of microorganisms.

[0027] (2) Optimized detection parameters ensure reliability. The optimal conditions of PMA-qPCR are determined, which ensures the sensitivity of live bacteria detection while completely inhibiting the interference of dead bacteria, and significantly improves the detection accuracy.

[0028] (3) The sensitivity meets the actual application requirements. The detection sensitivity of the bacterial wilt of kidney bean reaches 10 3 cfu / mL, which is slightly lower than that of ordinary qPCR, but its live bacteria specificity advantage is more valuable in actual application.

[0029] (4) Suitable for complex sample detection. In the live / dead bacteria mixture experiment, the Ct value of PMA-qPCR is negatively correlated with the proportion of live bacteria, while the result of ordinary qPCR shows no significant change, which proves that the method can accurately reflect the real content of live bacteria in the sample.

[0030] (5) The actual sample verification effect is significant. The detection of actual samples after heat treatment shows that PMA-qPCR almost completely inhibits the dead bacteria signal, while ordinary qPCR still detects all dead bacteria, highlighting its advantage in reducing false positives.

[0031] (6) The operation process is standardized. The complete standardized steps from bacterial suspension preparation, DNA extraction to PCR amplification are provided, which is convenient for popularization and use, and the combination of primers and probes ensures the high specificity of detection. In summary, through the innovative application of PMA-qPCR technology, the application realizes the high specificity, high accuracy and practicability of live bacteria detection, which has important value in the field of plant pathogen detection. BRIEF DESCRIPTION OF DRAWINGS

[0032] The technical solutions in the embodiments of the present application or the prior art will be described below more clearly with the help of the accompanying drawings needed in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description only represent some embodiments of the present application, and other drawings can be obtained by those of ordinary skill in the art without any creative effort on the basis of the provided drawings.

[0033] Figure 1 PMA-qPCR detection of mixed liquid of different proportions of live / dead bacteria;

[0034] Figure 2 PMA-qPCR and general qPCR sensitivity comparison;

[0035] Figure 3 Untreated sample detection results;

[0036] Figure 4 Inactivated sample detection results. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be described below more clearly with the help of the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments only represent some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without any creative effort belong to the protection scope of the present application.

[0038] Example 1: Selection of optimal PMA concentration

[0039] (1) Preparation of bacterial suspension

[0040] The Cff strain ATCC51876 was streaked on NA medium and cultured at 28°C for 48 h. A single characteristic colony was picked on NA liquid medium and cultured at 28°C for 36-48 h. The pure culture was extracted in 0.75% physiological saline to prepare a live bacterial suspension.

[0041] (2) Selection of bacterial suspension with a plate count of 1x10 5 cfu / mL and 1x10 6 cfu / mL. Five 1-ml bacterial suspensions were taken and heated at 100°C for 10 min to prepare dead bacterial suspensions. NA plates were inoculated and cultured for 36 h to confirm whether the bacteria were inactivated. Another five 1-ml live bacterial suspensions were taken for standby. For the bacterial suspension with a plate count of 1x10 5 cfu / mL and 1x10 6The bacterial suspension with cfu / mL was added with different amounts of PMA to make the final concentration 0 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL and 20 μg / mL respectively, and incubated in dark for 20 min, exposed to light for 20 min. After extracting DNA, real-time fluorescence PCR reaction was carried out by using CffFOR2 / CffREV4 / CF-P. The minimum concentration of PMA inhibiting DNA amplification of dead cells and the maximum concentration of PMA not affecting DNA amplification of living bacteria were determined. The results are shown in Table 1.

[0042] Table 1 Selection of optimal PMA concentration

[0043]

[0044] The results show that for Cff living bacteria with a concentration of 1×10 6 The Ct value of Cff living bacteria without adding PMA is 26.8, and the Ct value gradually increases (31.3 to >40) as the PMA concentration increases from 5 μg / mL to 20 μg / mL, indicating that PMA has an inhibitory effect on the DNA amplification of living bacteria. When the PMA concentration reaches 20 μg / mL, the Ct value of living bacteria is more than 40 (no amplification signal is detected), indicating that the DNA amplification of living bacteria is completely inhibited at this time. For Cff dead bacteria with the same concentration, the Ct value is 28.7 without adding PMA, and the Ct value is >40 when the PMA concentration is 5 μg / mL and above, indicating that 5 μg / mL of PMA can completely inhibit the DNA amplification of dead bacteria.

[0045] When the bacterial concentration is 1×10 5 The performance of Cff is similar to that of high-concentration bacteria. For Cff living bacteria, the Ct value is 33.4 when the PMA concentration is 5 μg / mL, and the Ct value increases to 37.5 when the PMA concentration is 20 μg / mL. The amplification of dead bacteria is also completely inhibited (Ct value >40) when the PMA concentration is 5 μg / mL and above.

[0046] In summary, when the bacterial concentration is 1×10 6 Therefore, 5 μg / mL is selected as the optimal concentration of PMA for detecting Cff by PMA-qPCR.

[0047] Example 2 Selection of optimal exposure time and optimal dark incubation time

[0048] The plate count is 1×10 6Take 4 1 ml dead bacteria suspension and add PMA working solution to make the final concentration 5 μg / mL, mix well, avoid light incubation for 20 min (choose 20 min to ensure complete incubation, and optimize later), then expose to light for 5 min, 10 min, 15 min, and 20 min respectively. After extracting DNA, real-time fluorescence PCR reaction is carried out using CffFOR2 / CffREV4 / CF-P. Take another bacteria solution and avoid light incubation for 5 min, 10 min, 15 min, and 20 min, then expose to light for 10 min, and then extract DNA for qPCR reaction. The results are shown in Table 2.

[0049] Table 2 Selection of optimal exposure time and optimal light-avoiding incubation time

[0050] Time min 5 10 15 20 Exposure treatment Ct value — — — — Light avoidance treatment Ct value 36.7 — — —

[0051] The results show that under the condition of PMA concentration of 5 μg / mL, the exposure time from 5 min to 20 min has no significant difference on the inhibition effect of dead bacteria DNA, and the Ct value of Cff is greater than 40. This shows that 5 min of exposure time is sufficient for PMA to fully bind to dead bacteria DNA and inhibit its amplification, so 5 min is selected as the optimal exposure time. In the light-avoiding experiment, a small amount of dead bacteria amplification (Ct value = 36.7) can still be detected when avoiding light incubation for 5 min, and when the incubation time is prolonged to 10 min and above, the dead bacteria signal is completely inhibited (Ct value > 40). Therefore, the optimal exposure and light-avoiding incubation time of Cff are determined as 5 min and 10 min respectively.

[0052] Establishment of PMA-qPCR:

[0053] (1) Preparation of the sample to be tested

[0054] Add PMA to the bacteria suspension to make the final concentration 5 μg / mL, avoid light incubation for 10 min, and expose to light for 5 min.

[0055] (2) DNA extraction and PCR amplification

[0056] Centrifuge the treated sample suspension at 12000 r / min for 5 min, extract the genomic DNA from the precipitate, and perform real-time fluorescence PCR reaction.

[0057] The primer and probe sequences are as follows:

[0058] Upstream primer CffFOR2: 5'-GTTATGACTGAACTTCACTCC-3'; SEQ ID NO. 1;

[0059] Downstream primer CffREV4: 5'-GATGTTCCCGGTGTTCAG-3'; SEQ ID NO. 2;

[0060] Primer probe CF-P: 5'-TCTCGGCCCGTCCGAATTTCCG-3'; SEQ ID NO. 3;

[0061] The primer probe is labeled with a fluorescence reporter group FAM at the 5' end and a quencher group TAMRA at the 3' end.

[0062] The real-time fluorescence PCR reaction system was 25 μL, including 12.5 μL 2x Premix Ex Taq, 1 μL of each of the upstream and downstream primers (5 μmol / L) and the probe (5 μmol / L), 1 μL of template DNA, 0.5 μL of 50x Rox Reference Dye, and sterile water added to 25 μL. The reaction program was: 95°C pre-denaturation for 10 s; 95°C for 15 s, 60°C for 1 min, 40 cycles.

[0063] (3) Result determination

[0064] When the sample to be tested has no fluorescence signal (Ct value ≥ 40), it is determined to be Cff negative;

[0065] When the sample to be tested has a typical amplification curve and the Ct value is ≤ 35, it is determined to be Cff positive;

[0066] When the sample to be tested has a typical amplification curve and the Ct value is less than 40 but greater than 35, it should be retested, if the Ct value of the retest is ≥ 40, it is determined to be Cff negative; if the Ct value of the retest is less than 40 but greater than 35, it is determined to be Cff positive.

[0067] Example 3 PMA-qPCR detection of different proportions of live / dead bacteria mixture

[0068] 1x10 6 cfu / mL concentration of bacteria suspension, heat-killed for 10 min at 100°C to prepare a dead bacteria suspension, mix the dead bacteria and live bacteria, so that the proportion of live bacteria in the mixture is 0%, 10%, 30%, 50%, 60%, 80%, and 100%, and detect them by the established PMA-qPCR, and also use ordinary qPCR reaction for comparison. The results are shown in Figure 1 .

[0069] The results show that PMA-qPCR can effectively distinguish between live bacteria and dead bacteria. As the proportion of live bacteria in the mixture increases from 0% to 100%, the Ct value of PMA-qPCR gradually decreases (Cff: 35.9→31.7), while the Ct value of ordinary qPCR does not change significantly (Cff: 29.0→27.1). This shows that PMA-qPCR can selectively detect live bacteria, and its Ct value is negatively correlated with the concentration of live bacteria, while ordinary qPCR cannot distinguish between live bacteria and dead bacteriaFigure 1 ).

[0070] Example 4 Comparison of PMA-qPCR and ordinary qPCR sensitivity

[0071] Take 0-10 8 cfu / mL 9 gradient concentration of viable bacteria suspension, each 1 mL, according to the determined PMA conditions for processing, after extracting DNA, real-time fluorescence PCR reaction, another set of no PMA control group, namely ordinary qPCR reaction. Results see Figure 2 .

[0072] The results show that the detection sensitivity of ordinary qPCR for Cff is 10 2 cfu / mL, higher than PMA-qPCR 10 3 cfu / mL( Figure 2 ). This shows that PMA treatment may slightly reduce the detection sensitivity, but its advantage is that it can specifically detect live bacteria and avoid dead bacteria interference.

[0073] Example 5 PMA-qPCR detection of actual samples

[0074] Ordinary qPCR detection: 5 Cff positive mung bean samples, each sample 100g added to 300mL sterilized physiological saline, 4℃ soaked overnight; gauze filtration, the filtrate was placed for 15min, 10000r / min centrifugation for 20min, 1mL PBS suspended the precipitate and transferred to 1.5ml centrifuge tube, 12000r / min centrifugation for 5min, the precipitate was extracted by DNA extraction kit.

[0075] PMA-qPCR detection: 5 Cff positive mung bean samples, each sample 100g added to 300mL sterilized physiological saline, 4℃ soaked overnight; gauze filtration, the filtrate was placed for 15min, 10000r / min centrifugation for 20min, 1mL PBS suspended the precipitate and transferred to 1.5ml centrifuge tube, PMA was added to the suspension to make its final concentration 5μg / mL, incubated for 10min in the dark, exposed for 5min; 12000r / min centrifugation for 5min, the precipitate was extracted by DNA extraction kit.

[0076] Take 1μL DNA and detect by PMA-qPCR and ordinary qPCR. Results see Figure 3 and Table 3.

[0077] Also detect by PMA-qPCR and ordinary qPCR according to the above method after 100℃ treatment for 10min of the 5 samples, compare the detection results of the two. Results see Figure 4 and Table 4.

[0078] The results show that both PMA-qPCR and regular qPCR can detect Cff in the positive samples without heat treatment. After heat treatment, regular qPCR can still detect dead bacteria signal in all samples, while PMA-qPCR only detects a weak signal (Ct value = 38.1) in one Cff sample, and no signal is detected in the rest of the samples. This shows that PMA-qPCR can significantly reduce the false positive signal of dead bacteria and improve the accuracy of the detection results.

[0079] Table 3 Detection results of untreated samples

[0080]

[0081] Table 4 Detection results of inactivated samples

[0082]

[0083] The foregoing description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting the activity of bean bacterial wilt pathogen based on PMA-qPCR, characterized in that: The following steps are involved: (1) Preparation of test samples Take 100g of the sample to be tested, add 300mL of sterile saline, and soak overnight at 4°C; filter through gauze, let the filtrate stand for 15 minutes, collect the supernatant and centrifuge at 10,000 rpm for 20 minutes, resuspend the precipitate in 1mL of PBS and transfer it to a 1.5ml centrifuge tube, add PMA to the suspension to a final concentration of 5μg / mL, incubate in the dark for 10 minutes, and expose to light for 5 minutes; (2) DNA extraction and PCR amplification The treated sample suspension was centrifuged at 12000 r / min for 5 min to precipitate and extract genomic DNA for real-time fluorescence PCR reaction; The primer and probe sequences are as follows: Upstream primer CffFOR2: 5′-GTTATGACTGAACTTCACTCC-3′; Downstream primer CffREV4: 5′-GATGTTCCCGGTGTTCAG-3′; Primer probe CF-P: 5′-TCTCGGCCCGTCCGAATTTCCG-3′; The 5' end of the primer probe is labeled with the fluorescent reporter gene FAM, and the 3' end is labeled with the quencher group TAMRA; (3) Result determination When the sample to be tested has no fluorescent signal (Ct value ≥ 40), it is judged as Cff negative; When the sample to be tested showed a typical amplification curve and the Ct value was ≤35, it was judged to be Cff positive; When the sample to be tested shows a typical amplification curve and the Ct value is less than 40 and greater than 35, the test should be retested. If the Ct value of the retest is ≥40, the Cff is judged to be negative; if the Ct value of the retest is less than 40 and greater than 35, the Cff is judged to be positive.

2. The method for detecting the activity of bean bacterial wilt pathogen based on PMA-qPCR according to claim 1, wherein: The real-time fluorescence PCR reaction system was as follows: 12.5 μL 2×Premix Ex Taq, 1 μL each of 5 μmol / L upstream and downstream primers and probes, 1 μL template DNA, 0.5 μL 50×Rox Reference Dye, and sterile water to 25 μL; the reaction procedure was as follows: pre-denaturation at 95°C for 10 s; 95°C for 15 s, 60°C for 1 min, and 40 cycles.

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