PMA-qPCR quantitative detection method for vinegar gas production deterioration pollution microorganism lactic acid bacteria Z-1 viable bacteria

Abstract

The invention discloses a PMA-qPCR (quantitative polymerase chain reaction) detection method for quantitative detection of a gas-producing vinegar pollution microorganism, namely viable bacteria of lactic acid bacteria Z-1, and provides an effective method for controlling the safety of vinegar products. According to the method, PMA is adopted to inhibit dead bacteria, recA genes of lactic acid bacteria Z-1 are adopted to design specific primers, and the PMA-qPCR detection method of the table vinegar pollution microorganism lactic acid bacteria Z-1 is established. The method is simple to operate, short in detection time, strong in specificity, high in standard recovery rate and low in detection limit, can be used for large-scale detection, and plays an important role in safety control of table vinegar products polluted by lactic acid bacteria Z-1.

Description

Technical Field

[0001] The invention relates to the field of food microorganism detection, and is a PMA-qPCR method for detecting live bacteria of lactic acid bacteria Z-1 in gas-producing vinegar. Background Art

[0002] In the open solid-state fermentation of vinegar, microorganisms in the environment can easily enter the fermentation system, causing the finished vinegar to deteriorate. Vinegar gas production and deterioration are mostly caused by microorganisms such as lactic acid bacteria and Bacillus, which seriously troubles production companies and brings a lot of losses to companies. Studies have reported that the growth and reproduction of lactic acid bacteria Z-1 (formerly known as lactic acid bacteria Z-1, later renamed Jinshan acetate Lactobacillus, Acetilactobacillus jinshanensis) in vinegar will cause vinegar to produce gas and deteriorate, and in severe cases, even sticky and foul smells. At present, vinegar production companies mainly use traditional separation detection for microbial detection in gas-producing vinegar, but there are literature reports that the contaminated microorganisms in vinegar may be in a live and uncultivable state or uncultivable microorganisms, so ordinary culture detection technology is difficult to detect or requires a long time of 7-14 days to detect.

[0003] The basic principle of PCR technology is highly similar to DNA replication. It is a molecular biology technology that uses specific primers to amplify specific DNA molecular fragments and detect the amplified products. Real-time quantitative PCR (q-PCR) adds fluorescent dyes or fluorescently labeled specific probes to ordinary PCR to label PCR products, and then continuously tracks the fluorescence intensity during the amplification process to achieve quantitative requirements.

[0004] Traditional PCR technology amplifies a certain DNA in the entire system. Since some dead microbial DNA is not completely degraded, this method cannot directly distinguish between live and dead bacteria. Propidium azide bromide (PMA) forms a covalently linked chemical modification with DNA molecules under strong natural light. The modified DNA cannot be amplified by PCR, and the dye cannot penetrate the cell membrane at all. Therefore, using PMA to treat the sample before q-PCR can effectively identify live bacteria in the sample.

[0005] At present, in the vinegar production process, there is an urgent need for an efficient and specific method that can effectively detect live lactic acid bacteria in vinegar for pre-delivery testing of finished vinegar. Therefore, it is very necessary to establish a PMA-qPCR quantitative detection method for gas-producing vinegar contamination microorganisms, lactic acid bacteria Z-1. Summary of the invention

[0006] The object of the present invention is to provide a PMA-qPCR detection method for lactic acid bacteria Z-1, a microorganism that causes vinegar gas production. The method can quantitatively detect live lactic acid bacteria Z-1 in vinegar, which is used for vinegar detection to ensure the quality of vinegar products. The lactic acid bacteria Z-1 (Lactobacillus sp. Z-1) in the patent is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation time is July 20, 2020, and the strain preservation number is CGMCC No. 20399.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] Design and verification of specific primers for lactic acid bacteria Z-1: The recA gene sequences of common bacteria in the vinegar production process were queried in NCBI, the recA gene of lactic acid bacteria Z-1 was determined to be specific, and specific primers were designed.

[0009] PMA-qPCR experimental steps: Take 500μL of lactic acid bacteria Z-1 bacterial liquid, centrifuge at 12000r / min for 1min to collect the precipitate, and wash it three times with 0.85% saline. Add 500μL of saline and vortex evenly, add 2μg / mL PMA solution, mix evenly and place in the dark for 10min. After the dark reaction is completed, place it on ice under 100W LED light for 10min, then wash it three times with 0.85% saline, extract DNA, and then perform q-PCR reaction.

[0010] The q-PCR reaction system in the above steps was: 1 μL each of DNA template, RA_F, and RA_R, 12.5 μL of SYBR Green MasterMix, and the remainder was ddH2O. The reaction procedure was 95°C pre-denaturation for 3 min; 95°C denaturation for 5 s, 53°C annealing for 10 s, and 72°C extension for 5 s, for 40 cycles.

[0011] Optimized PMA concentration: The optimized PMA concentration range is 0-25 μg / mL.

[0012] In this step, the lowest concentration of PMA was determined, and the highest concentration of PMA that did not affect the proliferation of live bacteria was also determined.

[0013] Optimize dark reaction time: The optimized dark reaction time range is 0 to 20 minutes.

[0014] Optimized illumination time: The optimized illumination time range is 0 to 20 minutes.

[0015] The standard curve regression equation of the lactic acid bacteria Z-1 is y=-3.3049x+44.6893, and the correlation coefficient R2 is 0.9975.

[0016] Verification of the accuracy of the detection method: Live bacteria and damaged bacterial liquid were mixed to prepare bacterial suspensions with different dead bacteria contents. They were processed according to the optimized detection method, and then DNA was extracted and q-PCR was performed. The inhibitory effect of this method on damaged bacteria was verified by using no PMA as a control.

[0017] Gas-producing vinegar spike recovery: 5.186, 6.186, 7.186, and 8.186 lg (CFU / mL) of lactic acid bacteria Z-1 were added to the gas-producing vinegar in a volume ratio of 1:1. The bacteria were treated according to the optimized detection method, and then DNA was extracted for q-PCR detection.

[0018] The present invention has the following advantages: a PMA-qPCR detection method is established for gas-producing vinegar contamination microorganisms, namely lactic acid bacteria Z-1; the method is simple to operate, and the detection result can be obtained within 3 hours; the method can be used in large quantities for the detection of vinegar before it leaves the factory, and an effective detection means is provided for the safety control of vinegar products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the nucleic acid electrophoresis diagram for verification of the specificity of primer RA.

[0020] Figure 2 The effect of different PMA concentrations on the PMA-qPCR amplification results of lactic acid bacteria Z-1.

[0021] Figure 3 It is the effect of dark reaction time on the PMA-qPCR amplification results of lactic acid bacteria Z-1.

[0022] Figure 4 The effect of light reaction time on the amplification results of lactic acid bacteria Z-1PMA-qPCR.

[0023] Figure 5 It is the standard curve regression equation for PMA-qPCR detection of lactic acid bacteria Z-1. DETAILED DESCRIPTION

[0024] Example 1 Design of specific primers for lactic acid bacteria Z-1.

[0025] The recA gene sequences of 17 common bacteria in the vinegar production process were queried in NCBI, and it was determined that the recA gene of lactic acid bacteria Z-1 was specific. In this patent, lactic acid bacteria Z-1 (Lactobacillus sp. Z-1) was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit time was July 20, 2020. The strain deposit number is CGMCC No. 20399. The specific primer RA was designed using the NCBI Primer-BLAST tool, and the sequences of primer RA were RA_F: 5'-CGACGCAGAAAACGCCTTAG-3', RA_R: 5'-ACCCTCGATTTCAGCTTGGG-3. The TIANamp Bacteria DNA Kit was used to extract the above 17 bacterial DNAs, and the PCR reaction was carried out using the above DNA as a template. The reaction system was 25 μL, including 1 μL each of DNA template, RA_F, and RA_R, 12.5 μL of 2×PCRTaq premix, and the remainder was ddH2O. The PCR reaction procedure was 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 56°C annealing for 60 s, and 72°C extension for 60 s, 30 cycles; 73°C extension for 5 min, and then 1.2% agarose gel was used for nucleic acid electrophoresis to determine the specificity of the primers.

[0026] Among them, 17 common bacteria in vinegar production process are Acetobacter ghanensis, Acetobacterpasteurianus, Acetobacter pomorum, Enterococcus durans, Enterococcus faecium, Enterococcus hirae, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactococcus lactis, Leuconostoccitreum, Pediococcuspentosaceus, Weissellahellenica, all of which are isolated from vinegar fermentation process. DNA extraction steps were performed according to the instructions of TIANamp Bacteria DNA Kit.

[0027] Depend on Figure 1It can be seen that only the second lane where lactic acid bacteria Z-1 is located has a single band of about 200 bp in length, indicating that primer RA is specific.

[0028] Example 2 Selection of the optimal PMA concentration.

[0029] (1) Determination of the minimum concentration of PMA for action.

[0030] Take 500 μL of boiling water for 5 minutes and the number of colonies is about 10 7 The damaged bacterial solution of lactic acid bacteria Z-1 with log (CFU / mL) was placed in a 1.5mL centrifuge tube, centrifuged at 12000r / min for 1min to collect the precipitate, washed 3 times with 0.85% saline, and then PMA was added to make the final mass concentration 0, 2.0, 5.0, 10, 15, 25μg / mL, mixed thoroughly, and reacted in the dark for 10min in the dark, and then the centrifuge tube was placed under a 100W LED lamp at 20cm and exposed for 15min (tube mouth facing up, placed on ice) for light reaction. Washed 3 times with 0.85% saline, then DNA was extracted, and q-PCR detection was performed to determine the minimum concentration of PMA to inhibit the amplification of dead cell DNA.

[0031] (2) Determination of the maximum concentration of PMA that does not affect the proliferation of viable bacteria.

[0032] Take 500 μL and the number of colonies is about 10 7 log(CFU / mL) of lactic acid bacteria Z-1 was placed in a 1.5mL centrifuge tube, centrifuged at 12000r / min for 1min to collect the precipitate, washed 3 times with 0.85% saline, and then PMA was added to make the final mass concentration 0, 2.0, 5.0, 10, 15, 25μg / mL, mixed thoroughly, incubated in the dark for 10min, and then the centrifuge tube was placed under a 100W LED lamp at 20cm and exposed for 15min (tube mouth facing up, placed on ice) for light reaction. Washed 3 times with 0.85% saline, then DNA was extracted and q-PCR was performed to determine the maximum concentration of PMA that did not affect the amplification of live bacterial DNA.

[0033] The q-PCR reaction system in the above and subsequent steps was: 1 μL each of DNA template, RA_F, and RA_R, 12.5 μL of SYBR Green Master Mix, and the rest was ddH2O.

[0034] The q-PCR reaction program in the above and subsequent steps was pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 s, annealing at 53°C for 10 s, and extension at 72°C for 5 s, for 40 cycles.

[0035] Depend on Figure 2It can be seen that when the amount of PMA added is between 2 and 10 μg / mL, the Cq value of live bacteria is not significantly different from that of the group without PMA addition, and the Cq value of dead bacteria is significantly greater than that of the group without PMA addition. When the amount of PMA added is greater than 10 μg / mL, the Cq value of live bacteria is significantly greater than that of the group without PMA addition, indicating that when the amount of PMA added is greater than 10 μg / mL, the proliferation of live bacteria will be inhibited. In summary, the optimal amount of PMA added is 2 μg / mL.

[0036] Example 3 Selection and determination of the optimal dark reaction time.

[0037] Take 500 μL and boil it in water for 5 min. The number of colonies is about 10 7 The damaged bacterial solution of lactic acid bacteria Z-1 with log (CFU / mL) was placed in a 1.5mL centrifuge tube, centrifuged at 12000r / min for 1min to collect the precipitate, washed 3 times with 0.85% saline, then added 2μg / mL PMA, mixed thoroughly, and reacted in the dark for 0, 5, 10, 15, and 20min in the dark, respectively, to allow PMA to fully react with the sample. Then lay the sample flat on ice to avoid excessive heating. Place the centrifuge tube under a 100W LED lamp at 20cm and expose it for 15min (tube mouth facing up, placed on ice) for light reaction. Wash 3 times with 0.85% saline, then extract DNA, perform q-PCR detection, and determine the optimal dark reaction time for PMA treatment.

[0038] Depend on Figure 3 It can be seen that as the dark reaction time increases, the Cq value increases. When the dark reaction time is 10-20 minutes, the Cq value reaches the maximum, which can inhibit the proliferation of dead bacteria to the greatest extent. Therefore, the optimal dark reaction time is 10 minutes.

[0039] Example 4 Selection and determination of the optimal light reaction time.

[0040] Take 500 μL and boil it in water for 5 min. The number of colonies is about 10 7 The damaged bacterial solution of lactic acid bacteria Z-1 with log (CFU / mL) was placed in a 1.5mL centrifuge tube, centrifuged at 12000r / min for 1min to collect the precipitate, washed 3 times with 0.85% saline, then added 2μg / mL PMA, mixed thoroughly, and reacted in the dark for 10min away from light to allow PMA to fully react with the sample. The sample was then placed flat on ice to avoid excessive heating. The centrifuge tube was placed under a 100W LED lamp at 20cm and exposed for 0, 5, 10, 15, and 20min (tube mouth facing up, placed on ice) for light reaction. It was washed 3 times with 0.85% saline, and then DNA was extracted and q-PCR was performed to determine the optimal light reaction time for PMA treatment.

[0041] Depend on Figure 4It can be seen that with the extension of light reaction time, the Cq value increases. When the light reaction time is 10-20 minutes, the Cq value reaches the highest and there is no significant difference, which can effectively inhibit the proliferation of dead bacteria. Therefore, the optimal light reaction time is selected as 10 minutes.

[0042] Example 5 Drawing of the standard curve.

[0043] Take the known lactic acid bacteria Z-1 bacterial solution as the standard solution and dilute it 10 times to 10 -8 , 500 μL of each gradient dilution was taken into a sterile 1.5 mL centrifuge tube, and the samples were processed according to the optimized PMA-qPCR method. Then DNA was extracted and q-PCR detection was performed. The logarithm of the standard sample concentration was used as the abscissa and the corresponding Cq value was used as the ordinate to draw the q-PCR standard curve. The total number of colonies of the unknown sample was calculated based on the regression equation and the Cq value of the PMA-qPCR detection.

[0044] The standard curve regression equation of the lactic acid bacteria Z-1 is y=-3.3049x+44.6893, and the correlation coefficient R 2 The linear range of detection of live lactic acid bacteria Z-1 was 9.96-2.96 lg (CFU / mL), and the minimum detection limit could reach 4 copies / reaction system.

[0045] Example 6 Verification of the dead bacteria inhibition effect.

[0046] Live lactic acid bacteria Z-1 and damaged bacterial liquid were mixed to prepare bacterial suspensions with dead bacteria contents of 0%, 0.1%, 1%, 10%, 30%, 50%, 70%, 90%, 99%, 99.9%, and 100%, respectively. The samples were processed according to the optimized PMA-qPCR method. DNA was then extracted and q-PCR was performed to verify the inhibitory effect of this method on damaged bacteria using the control without adding PMA.

[0047] Table 1 Total colony counts of different dead bacteria ratios determined by PMA-qPCR Table 1 shows the total colony counts at different dead bacteria ratios determined by PMA-qPCR. It can be seen that when the dead bacteria ratio is between 0% and 99%, the PMA-qPCR test results are basically consistent with the theoretical values. When the dead bacteria ratio is higher than 99.9% and the theoretical live bacteria count is lower than 4.63lg (CFU / mL), the difference between the lactic acid bacteria Z-1 in the sample detected by PMA-qPCR and the theoretical value is large. The results show that PMA can effectively inhibit 99% of the dead lactic acid bacteria Z-1, but its inhibitory ability is limited and cannot completely inhibit the proliferation of dead bacteria in the system.

[0048] Example 7 PMA-qPCR spike recovery rate for gas-producing vinegar.

[0049] Add 1:1 volume of 5.186, 6.186, 7.186, and 8.186 lg (CFU / mL) of lactic acid bacteria Z-1 bacterial solution to gas-producing vinegar (lactic acid bacteria Z-1 concentration of 5.927 lg (CFU / mL)), and treat the samples according to the optimized PMA-qPCR method. Then extract DNA, wash three times with 0.85% saline for q-PCR detection, and calculate the spiked recovery rate of lactic acid bacteria Z-1 samples.

[0050] The experimental results showed that the recovery rate of PMA-qPCR detection of lactic acid bacteria Z-1 in gas-producing vinegar was between 95% and 100%, indicating that this method can effectively and accurately detect lactic acid bacteria Z-1 in gas-producing vinegar.

[0051] The present invention constructs PMA-qPCR for the vinegar gas-producing microorganism, lactic acid bacteria Z-1: specific primers are constructed using the lactic acid bacteria Z-1 specific gene recA, 2 mg / mL PMA is added, followed by a dark reaction for 10 minutes, a light reaction for 10 minutes, and q-PCR is performed after DNA is extracted. This method can effectively inhibit 99% of the dead lactic acid bacteria Z-1 in the system, and the spike recovery rate in vinegar is 95% to 100%, indicating that this method can effectively determine the live lactic acid bacteria Z-1 in vinegar.

[0052] The above examples are merely descriptions of the embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may improve or modify the above descriptions, but such changes or modifications should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A PMA-qPCR method for detecting gas-producing vinegar contaminating microorganisms - Lactobacillus Z-1 ( Lactobacillus sp .) for quantitative detection of live bacteria, characterized in that, By adding PMA to inhibit dead bacteria in the test sample, specific primers were designed using the specific genes of lactic acid bacteria Z-1, and q-PCR reaction was performed on the vinegar-contaminating microorganism lactic acid bacteria Z-1, thereby achieving the purpose of quantitative detection of live bacteria.

2. The PMA-qPCR detection method according to claim 1 is used for quantitative detection of viable bacteria of gas-producing vinegar contamination microorganisms-lactic acid bacteria Z-1, characterized in that: The microbial lactic acid bacteria Z-1 is a difficult-to-culture acid-resistant microorganism, and its deposit registration number is CGMCC No. 20399.

3. The PMA-qPCR detection method according to claim 1 is used for quantitative detection of viable bacteria of gas-producing vinegar contamination microorganisms-lactic acid bacteria Z-1, characterized in that: By adding 2 mg / L PMA, dark reaction for 10 min, and then light reaction for 10 min, the lactic acid bacteria Z-1 in the test sample can be killed.

4. The PMA-qPCR detection method according to claim 1 is used for quantitative detection of viable bacteria of gas-producing vinegar contamination microorganisms-lactic acid bacteria Z-1, characterized in that: Specific primers (RA_F: 5'-CGACGCAGAAAACGCCTTAG -3', RA_R: 5'-ACCCTCGATTTCAGCTTGGG-3') were used to perform q-PCR on lactic acid bacteria Z-1. The q-PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 s, annealing at 53°C for 10 s, and extension at 72°C for 5 s, for 40 cycles.

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