CN112176023B - Method for quickly detecting soy sauce gas-producing bacteria without culturing - Google Patents
Method for quickly detecting soy sauce gas-producing bacteria without culturing Download PDFInfo
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Abstract
The invention discloses a method for quickly detecting soy sauce gas-producing bacteria without culturing, which comprises the following steps: s1, collecting a sample of the flatulence soy sauce; s2, performing high-throughput sequencing analysis on a sample of the flatulence soy sauce, and determining the microorganism composition of the flatulence sample; s3, performing PCA analysis on the flatulence sample and the normal soy sauce sample to find out the gas-producing keybacteria of the flatulence sample; s4, designing a specific primer according to the 16S gene sequence of the gas-producing key bacteria; s5, identifying the sample to be detected by adopting a stain-fluorescence quantitative PCR, and determining the Ct value of the sample to be detected; s6, judging that the sample to be detected is qualified if the Ct value exceeds m, and judging that the sample to be detected is unqualified if the Ct value is lower than m. The method for detecting the soy sauce gas-producing bacteria by combining high-throughput sequencing and staining agent-fluorescent quantitative PCR can be used for rapidly detecting the soy sauce sample and determining the soy sauce sample, so that the rapid discrimination of the quality of the soy sauce is realized.
Description
Technical Field
The invention relates to the field of soy quality detection, in particular to a method for rapidly detecting soy gas-producing bacteria without culturing.
Background
The soy sauce is a common seasoning for China families, has delicious taste, rich sauce flavor and red and bright color, and is deeply favored by consumers. The brewing and post-treatment processes of soy sauce are complex, and many pipelines and packaging materials are contacted, so that the possibility of microbial infection is caused, the finished soy sauce product has a bottle expansion during the shelf life, the quality of the soy sauce is influenced, and even the personal safety of consumers is possibly damaged. How to rapidly detect micro-microorganisms in soy finished products before delivery and eliminate possible safety risk factors is a problem to be solved by soy manufacturers.
The soy sauce factory usually adopts a method based on microorganism culture to detect, but because the soy sauce flatulence is usually difficult to culture and the content of the flatulence is too low in factory inspection, the soy sauce can not be effectively screened by the traditional microorganism culture method, and because of different soy sauce production places and different production processes, the gas-producing bond-related bacteria of the soy sauce in different factories are different.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for quickly detecting soy sauce gas-producing bacteria without culturing, which is used for determining soy sauce gas-producing bond bacteria based on high-throughput sequencing targeting, and quickly detecting the gas-producing live bacteria content of a sample to be detected by a method of dye-fluorescence quantitative PCR, so that the safety of the soy sauce in the shelf life is ensured.
The invention is realized by the following technical scheme:
the invention provides a method for quickly detecting soy sauce gas-producing bacteria without culturing, which is characterized by comprising the following steps:
s1, collecting a sample of the flatulence soy sauce;
s2, performing high-throughput sequencing analysis on a sample of the flatulence soy sauce, and determining the microorganism composition of the flatulence sample;
s3, performing PCA analysis on the flatulence sample and the normal soy sauce sample to find out the gas-producing keybacteria of the flatulence sample;
s4, designing a specific primer according to the 16S gene sequence of the gas-producing key bacteria;
s5, identifying the sample to be detected by adopting a stain-fluorescence quantitative PCR, and determining the Ct value of the sample to be detected;
s6, judging that the sample to be detected is qualified if the Ct value exceeds m, and judging that the sample to be detected is unqualified if the Ct value is lower than m.
The method for detecting the soy sauce gas-producing bacteria by combining high-throughput sequencing and staining agent-fluorescent quantitative PCR can be used for rapidly detecting the soy sauce sample and determining the soy sauce sample, so that the rapid discrimination of the quality of the soy sauce is realized. The traditional microorganism culture detection method needs 2-5 days, and is difficult to succeed for strains difficult to culture, but the detection method can detect samples within 2 hours, has higher precision and more accurate results. The invention carries out detection based on the culture-free technology, and effectively avoids the defects of the traditional culture.
The dye added in the dye-fluorescence quantitative PCR can be jointed with dead cell DNA in soy sauce, so that the joint of the dead cell DNA and DNA polymerase is prevented, the expansion of dead cells is avoided, the dye cannot enter living cells, and therefore fragments amplified by the dye-fluorescence quantitative PCR represent the number of the living cells, and the detection accuracy is greatly improved.
In step S6, the m value is calculated as follows: respectively adding different volume parts of gas-filled soy sauce into the sterile soy sauce, carrying out dye-fluorescence quantitative PCR on the inoculated mixed soy sauce to obtain Ct values of all samples, then placing the samples at 30-37 ℃ for culturing for 6-12 months, and marking the Ct value of the sample with the lowest gas-filled soy sauce inoculation amount in the gas-filled samples as m.
The specific primer in the step S4 is a characteristic primer designed according to the 16S sequence of the gas-producing keybacteria obtained by high-throughput sequencing.
In step S5, the DNA dye is EMA, PMA or PMAxx.
In step S5, DNA dye is added to a final concentration of 10-50. Mu.g/mL.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. according to the method for rapidly detecting the soy sauce gas-producing bacteria without culturing, provided by the invention, the soy sauce gas-producing bacteria can be rapidly detected by combining high-throughput sequencing with the stain-fluorescent quantitative PCR, and the soy sauce sample is measured, so that the rapid discrimination of the quality of the soy sauce is realized;
2. the method for rapidly detecting the soy sauce gas-producing bacteria without culturing can detect the sample within 2 hours, has higher precision and more accurate result, and can effectively avoid the defects of the traditional culturing.
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FIG. 1 is a technical roadmap of the invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, and the description thereof is merely illustrative of the present invention and not intended to be limiting.
Example 1
And determining the gas-producing keybacteria in the gas-producing soy sauce and designing a characteristic primer.
(1) Firstly, collecting a plurality of different gas-filled soy samples and normal soy samples, and determining the microorganism composition of the gas-filled soy samples and the normal soy samples through high-throughput sequencing;
(2) Performing PCA analysis (principal component analysis) on the flatulence soy sauce sample and the normal soy sauce sample to find out 1-3 key flatulence strains;
by analyzing the collected sample of the flatulence soy sauce and the sample of the normal soy sauce, lactobacillus is found to be the main microorganism in the flatulence soy sauce, and thus the bacterium is judged as the flatulence-producing bacterium. The 16S sequence of the strain was sequenced, and the sequencing result was as follows:
AGGGGGGCGTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGGGACTGTG
characteristic recognition fragments of the Lactobacillus strain were designed, as indicated by the bolded fragments in the above sequences, and then characteristic test primers were designed. Wherein, the upstream primer 5'-GAACTTGAGTGCAGAAGAG-3' and the downstream primer 5'-CTCAGCGTCAGTTACAGAC-3' are designed. In dye-qPCR, a fluorescent reporter group FAM is added at the 5 'end and a fluorescent reporter group TAMRA is added at the 3' end.
Example 2
The effect of different dyes on the identification of viable bacterial stain-fluorescent quantitative PCR was determined in this example.
The effect of using the dyes ethylenimine azide (EMA) or ethylenimine azide (PMA) or PMAxx (a modified ethylenimine azide dye) on heat-inactivated pure soy sample stain-fluorescent quantitative PCR was mainly determined.
The method mainly comprises the following steps:
(1) Taking 2mL of heat-inactivated soy sauce sample, placing the sample in a centrifuge tube, adding a dye working solution to adjust the dye concentration to 10 mug/mL, fully and uniformly mixing, and incubating in a dark place for 5min to enable the nucleic acid dye to enter damaged cells; after incubation is completed, the centrifugal cover is opened, and the tungsten lamp is illuminated for 5min to complete the combination of the dye and the DNA, and simultaneously, the dye which is not combined with the DNA is passivated;
(2) Extracting total DNA from the treated bacterial liquid according to the national standard, and performing fluorescence PCR analysis according to the characteristic primer in the example 1; the fluorescent PCR reaction conditions were as follows: pre-deforming for 5min at 95 ℃; the conditions in the cycle are: 95 ℃ for 5s;60 ℃ and 60 seconds, and 40 cycles are total; and after the circulation is finished, the temperature is kept at 40 ℃ for 10min.
As can be seen from Table 1, the addition of nucleic acid stain EMA, PMA, PMAxx alone delayed the Ct value, indicating that the nucleic acid stain is able to distinguish between live and dead bacteria. Among them, the PMAxx stain has the highest Ct value and the best effect.
TABLE 1 Effect of different nucleic acid dyes on qPCR identification of heat-inactivated Soy sauce samples
Example 3
In this example, the critical Ct value m of the soy sample for flatulence is determined as follows:
(1) The flatulence soy sauce was inoculated into the sterile soy sauce according to the inoculum size of 0.01%, 0.025%, 0.05%, 0.075%, 0.1%, 0.25%, 0.5%, 0.75%, 1%, respectively, and after the inoculation was completed, the Ct value of the soy sauce was measured as in example 2, and the remaining samples were cultured in a constant temperature sample-keeping chamber at 37℃for 8 months to observe the flatulence, and the results are shown in Table 2.
TABLE 2 Ct values for different flatulence soy inoculations
As shown in Table 2, when the inoculum size was 0.05% or more, the soy sauce was left to stand for 8 months or less, and therefore, it was judged that the Ct value 29.79 at the inoculum size was 0.05% or more, and when the Ct value of the soy sauce to be tested was lower than 29.79, the risk of soy sauce swelling was large, and it was judged as unacceptable.
Example 4
The application of PMAxx-fluorescence quantitative PCR (polymerase chain reaction) in the quality control of soy products is determined by the method, and the method comprises the following steps:
(1) Taking 2mL of soy sauce sample to be detected in a centrifuge tube, adding dye working solution to adjust the dye concentration to 10 mug/mL, fully and uniformly mixing, and incubating in a dark place for 5min to enable the nucleic acid dye to enter damaged cells; after incubation is completed, the centrifugal cover is opened, and the tungsten lamp is illuminated for 5min to complete the combination of the dye and the DNA, and simultaneously, the dye which is not combined with the DNA is passivated;
(2) Extracting total DNA from the treated bacterial liquid according to the national standard, and then carrying out dye-fluorescence quantitative PCR according to the characteristic primer in the example 1; the fluorescent quantitative PCR reaction conditions were as follows: pre-deforming for 5min at 95 ℃; the conditions in the cycle are: 95 ℃ for 5s;60 ℃ and 60 seconds, and 40 cycles are total; and after the circulation is finished, the temperature is kept at 40 ℃ for 10min. After the completion, ct value of the sample to be detected is obtained;
(3) Control experimental group: 2mL of soy sauce sample is poured on a flat plate, then MRS solid culture medium which is dissolved and cooled to about 40 degrees is poured into the flat plate, evenly mixed, cultured for 5 days at 37 ℃ to observe the growth condition of microorganisms, and the colony count is counted, and the result is shown in Table 3.
TABLE 3 application of PMAxx-fluorescent quantitative PCR in quality control of Soy sauce finished products and comparison with traditional microbiological tests
As can be seen from table 3, sample 2 having Ct value lower than the flatulence threshold value m was left to find that the sample was indeed inflated; the Ct value is higher than the sample of the flatulence critical value m, and the sample is reserved to find that the flatulence does not occur within 8 months; in the method for culturing microorganisms, the sample 2 has no colony, is not consistent with the condition of actually generating flatulence, is not accurately detected, and meanwhile, soy sauce samples 1, 3 and 8 with colonies are cultured on an MRS culture medium, and finally, the result shows that the flatulence phenomenon does not occur. Therefore, in conclusion, the experimental result shows that the PMAxx-fluorescence quantitative PCR method is accurate and feasible for detecting soy sauce samples, and compared with the traditional microorganism culture method, the method has the advantages that the sample detection time is saved, the detection accuracy is improved, and the method has important significance for quality control of soy sauce finished products.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (3)
1. A method for quickly detecting soy sauce gas-producing bacteria without culturing is characterized by comprising the following steps:
s1, collecting a sample of the flatulence soy sauce;
s2, performing high-throughput sequencing analysis on a sample of the flatulence soy sauce, and determining the microorganism composition of the flatulence sample;
s3, performing PCA analysis on the flatulence sample and the normal soy sauce sample to find out the gas-producing keybacteria of the flatulence sample;
s4, designing a specific primer according to the 16S gene sequence of the gas-producing key bacteria;
s5, identifying a sample to be detected by adopting a stain-fluorescence quantitative PCR, determining a Ct value of the sample to be detected, and adopting EMA, PMA or PMAxx as DNA dye;
s6, judging that the sample to be detected is qualified if the Ct value exceeds m, and judging that the sample to be detected is unqualified if the Ct value is lower than m;
the m value is calculated as follows: respectively adding different volume parts of gas-filled soy sauce into the sterile soy sauce, performing dye-fluorescence quantitative PCR on the inoculated mixed soy sauce to obtain Ct values of all samples, then placing the samples at 30-37 ℃ for culturing for 6-12 months, and marking the Ct value of the sample with the lowest gas-filled soy sauce inoculation amount in the gas-filled samples as m.
2. The method for rapid detection of soy sauce gas-producing bacteria without culturing according to claim 1, wherein the specific primer in step S4 is a characteristic primer designed according to the 16S sequence of gas-producing bond bacteria obtained by high throughput sequencing.
3. The method for rapidly detecting soy sauce gas-producing bacteria without culturing according to claim 1, wherein in step S5, the final concentration of DNA dye added is 10-50 μg/mL.
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| CN101892299A (en) * | 2010-02-10 | 2010-11-24 | 广州华峰生物科技有限公司 | Clostridium perfringen detection kit and user method thereof |
| CN104630356A (en) * | 2015-01-29 | 2015-05-20 | 华南理工大学 | Method for analyzing unknown microorganisms in soy source fermented grains |
| CN108179171A (en) * | 2017-12-12 | 2018-06-19 | 天津科技大学 | A kind of method of aerogen in Rapid identification swollen bag soy sauce |
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| CN101892299A (en) * | 2010-02-10 | 2010-11-24 | 广州华峰生物科技有限公司 | Clostridium perfringen detection kit and user method thereof |
| CN104630356A (en) * | 2015-01-29 | 2015-05-20 | 华南理工大学 | Method for analyzing unknown microorganisms in soy source fermented grains |
| CN108179171A (en) * | 2017-12-12 | 2018-06-19 | 天津科技大学 | A kind of method of aerogen in Rapid identification swollen bag soy sauce |
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