Rapid acetic bacteria detection primer and method

Abstract

The invention discloses a primer and a method for rapidly detecting acetic bacteria, which adopt a real-time quantitative PCR (Polymerase Chain Reaction) technology to establish a rapid quantitative detection method with high sensitivity, high accuracy and short time aiming at the acetic bacteria, and by comparing 16S rRNA (Ribosomal Ribonucleic Acid) gene sequences of different acetic bacteria and lactic acid bacteria, the acetic bacteria can be rapidly detected. Specific nucleic acid fragments capable of efficiently distinguishing acetic acid bacteria with relatively close genetic relationship are screened, a specific primer sequence is designed, an absolute quantitative detection method for acetic acid bacteria is established, rapid quantitative detection of acetic acid bacteria in the brewing process is realized in a relatively short time, and a foundation is laid for rapid detection of acetic acid bacteria in vinegar enterprises. Theoretical basis and technical support are provided for evaluating the structure and fermentation state of the microbial flora and formulating a microbial regulation strategy.

Description

Technical Field

[0001] The invention relates to a primer and a method for rapid detection of acetic acid bacteria. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Shanxi aged vinegar (SAV) is a famous vinegar produced by traditional solid-state fermentation. It is made by fermentation with the participation of multiple microorganisms and has a history of thousands of years of use in China. Solid-state acetic acid fermentation (AAF) is a natural or semi-controlled fermentation process driven by a repeatable microbial community. Wu Jiajia et al. used traditional and non-traditional methods to study the diversity of LAB populations during the fermentation of Shanxi aged vinegar, and obtained the dynamics of bacteria and changes in metabolites during the AAF process. Among them, the core microbial communities in the brewing process of Shanxi aged vinegar include yeast (Saccharomyces), acetic acid bacteria (Acetobacter, Komagataeibacter) and lactic acid bacteria (Lactobacillus), which are significantly associated with flavor metabolites (sugars and their derivatives, organic acids, amino acids, flavonoids, polyphenols, etc.). As an important microorganism in the vinegar brewing industry, acetic acid bacteria plays an important role in the flavor formation of vinegar.

[0004] At present, the most cutting-edge research methods have been used to study the microorganisms in the relevant traditional vinegar brewing process through culture-free molecular biology, ecology and other technologies, which has a foundational significance for the modernization and standardization of traditional processes. There are many methods for detecting colonies. The traditional method is the plate culture separation method, but the culture time is long and it is difficult to quickly identify the complete microbial community structure. Nowadays, culture-free technologies such as real-time quantitative PCR, DGGE technology, and high-throughput sequencing technology are relatively cutting-edge. They are mainly used for microbial detection, but each has its own advantages and disadvantages. For example, real-time quantitative PCR technology has the advantages of high sensitivity, high accuracy and short detection time, but there are also problems such as: it is difficult to stably preserve the obtained standard, and the current method of quantitative bacterial genome solution concentration at a wavelength of 260nm is affected by many factors, such as buffer and instrument performance; DGGE technology has many operating steps and cannot fully detect low-abundance microorganisms in the sample; and the metagenomic method based on high-throughput technology is expensive, and it generally takes 45 working days from sample collection to sequencing results, which cannot meet the requirements of rapid detection in factories.

[0005] SYBR Green I fluorescence quantitative technology combines the characteristics of fluorescent dyes and PCR, and can quickly and quantitatively detect unknown nucleic acid fragments. Since SYBR Green I dye can be coupled with template double-stranded DNA, when the reactant is amplified, the fluorescent dye can be released to increase the fluorescence value. In the process of configuring the fluorescence quantitative system, the fluorescent dye will gradually bind to the product to be amplified, and then the fluorescence signal is monitored in real time using a fluorescent quantitative PCR instrument to obtain the amplification data of the product. This method has better detection performance than ordinary PCR, and the test results can be directly analyzed by software, which is more objective and accurate, and after obtaining the results, agarose gel electrophoresis is not required, which can save a lot of time. The specificity of the primer can be judged according to the melting curve. If a single peak appears, it means that the primer has strong specificity. At present, the amplification graph of the target specific product can also be quantitatively detected by designing a fluorescent labeled probe that can bind to the amplified product, also called Taqman technology, which is commonly used in clinical testing.

[0006] The existing implementation scheme that is most similar to the present invention: primers, kits, and methods for rapid identification and quantification of Saccharomyces cerevisiae.

[0007] Specific primers were designed for the ADH4 gene of Saccharomyces cerevisiae alcohol dehydrogenase, which can be used to quickly and qualitatively detect Saccharomyces cerevisiae in samples such as Daqu and fermented grains by PCR technology, and the content of Saccharomyces cerevisiae in samples can be determined by fluorescent quantitative PCR; it has the characteristics of high accuracy, good effectiveness and high sensitivity, and is suitable for the identification and quantitative detection of Saccharomyces cerevisiae in fermented foods or fermentation process samples. The following steps are included;

[0008] S1. Prepare suspensions of Saccharomyces cerevisiae with different bacterial concentrations and known concentrations, and extract genomes from the suspensions respectively;

[0009] S2. Use the primers shown to perform fluorescence quantitative PCR on the genome, and draw a standard curve with CT value as the horizontal axis and the number of cells or the Lg value of the number of cells as the vertical axis;

[0010] S3. Extract the genome of the sample to be tested, use the primers shown to perform fluorescence quantitative PCR on the genome of the sample to be tested, substitute the CT value of fluorescence quantitative PCR into the standard curve, and convert to obtain the amount of Saccharomyces cerevisiae in the sample.

[0011] At present, the research progress of traditional vinegar brewing technology is slow due to the inability to effectively detect most microorganisms in the environment using traditional culture methods. Therefore, a rapid quantitative method is needed to quickly and quantitatively detect most microorganisms in the environment. Summary of the invention

[0012] In view of the shortcomings of the prior art, the present invention uses the fermented grains in the brewing process of Shanxi aged vinegar as raw material, adopts SYBRGreen I fluorescent quantitative PCR method to study the rapid detection of acetic acid bacteria in the fermented grains of Shanxi aged vinegar, and aims to establish a simple, rapid and highly sensitive detection method so that the factory can evaluate the fermentation state according to the structure of the microbial flora and formulate microbial regulation strategies in time.

[0013] The technical solution adopted by the present invention is as follows:

[0014] In a first aspect of the present invention, a specific primer pair for rapid detection of Acetobacter pasteurianus is provided, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2 (represented as primer F in the text).

[0015] In a second aspect of the present invention, a kit for rapid detection of Acetobacter pasteurianus is provided, the kit comprising the above-mentioned specific primer pair.

[0016] In a third aspect of the present invention, there is provided use of the above-mentioned specific primer pair and / or the above-mentioned kit in detecting Acetobacter pasteurianus.

[0017] In a fourth aspect of the present invention, a method for rapid detection of Acetobacter pasteurianus is provided, the method comprising the following steps:

[0018] (1) Isolating and purifying bacterial strains from the sample to be tested, and extracting DNA from the strains;

[0019] (2) using DNA as a template, performing PCR gene amplification using the above primer pairs;

[0020] (3) Electrophoresis observation: After the reaction is completed, the PCR product is observed by gel electrophoresis. If the PCR product has a band between 100-250 bp (including the endpoint value) on the electrophoresis map, it means that the sample to be tested contains Acetobacter pasteurianus. If the PCR product is not between 100 and 250 bp or has no band, it means that the sample to be tested does not contain Acetobacter pasteurianus.

[0021] In one or some embodiments of the present invention, the method further comprises the following steps: taking 16S rRNA genomic DNA of Acetobacter pasteurianus as a standard, and using qPCR technology to perform experiments to obtain a qPCR standard curve, thereby establishing an absolute quantitative detection method.

[0022] Furthermore, the reaction system of qPCR technology: DNA template 1 μL, upstream primer (10 μM) 0.4 μL, downstream primer (10 μM) 0.4 μL, 2×Q3 SYBR Green I qPCR Supermix 10 μL and ddH 2O 8.2 μL.

[0023] Furthermore, the operating parameters of the qPCR technology are: denaturation: 95°C, 5 min; 39 cycles: 95°C, 30 s; 60.9°C, 30 s; 72°C, 15 s; final extension: 55°C, 5 s; 95°C, 30 s.

[0024] Furthermore, the regression equation of the standard curve is y=-2.9597x+49.36, where the y-axis is the Ct value, the x-axis is the logarithmic value of the DNA template copy number concentration, and R 2 =0.9543.

[0025] In one or some embodiments of the present invention, in step (1), the sample to be tested is vinegar mash in the process of brewing aged vinegar.

[0026] In one or some embodiments of the present invention, in step (2), the PCR gene amplification system is: 2×TransFast Taq PCR SuperMix (+dye) 12 μL, Nuclease-free Water 10 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL and DNA template 1 μL.

[0027] The PCR operating parameters were as follows: denaturation: 94°C, 4 min; 72°C, 10 min; 40 cycles: 94°C, 45 s; 54.6°C, 30 s; 72°C, 1 min; final extension: 72°C, 10 min.

[0028] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects:

[0029] The present invention adopts real-time quantitative PCR technology to establish a high-precision, low-time, and high-sensitivity detection method for acetic acid bacteria, and its sensitivity is finally detected within the range of 3.13×10 3 ~3.13×10 4 A rapid quantitative detection method of 100 copies / μL was developed. By comparing the 16S rRNA gene sequences of different acetic acid bacteria and lactic acid bacteria, specific nucleic acid fragments that can effectively distinguish closely related acetic acid bacteria were screened, specific primer sequences were designed, and an absolute quantitative detection method for acetic acid bacteria was established. This method can achieve rapid quantitative detection of acetic acid bacteria in the brewing process in a relatively short period of time, providing a theoretical basis and technical support for vinegar companies to quickly detect acetic acid bacteria, evaluate the structure and fermentation status of microbial flora, and formulate microbial regulation strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 It is a specific nucleic acid fragment 1 obtained after alignment of 16S rRNA gene sequences of six strains of Lactobacillus reuteri, Acetobacter pasteurianus, Lactobacillus helveticus, Enterobacter hallii, Bacillus cereus and Bacillus amyloliquefaciens.

[0032] Figure 2 It is a specific nucleic acid fragment 2 obtained after alignment of 16S rRNA gene sequences of six strains of Lactobacillus reuteri, Acetobacter pasteurianus, Lactobacillus helveticus, Enterobacter hallii, Bacillus cereus, and Bacillus amyloliquefaciens.

[0033] Figure 3 Agarose gel electrophoresis diagram for primer-specific PCR verification. Note: M: Marker, E: Primer E, F: Primer F, G: Primer G, H: Primer H, 1: Acetobacter pasteurianus, 2: Lactobacillus reuteri, 3: Lactobacillus helveticus, 4: Bacillus cereus, 5: Bacillus amyloliquefaciens, 6: Enterobacter hallii.

[0034] Figure 4 Agarose gel electrophoresis diagram for optimization of primer PCR program. Note: M: Marker, F: Primer F, 1: Acetobacter pasteurianus, 2: Lactobacillus reuteri, 3: Lactobacillus helveticus, 4: Bacillus cereus, 5: Bacillus amyloliquefaciens, 6: Enterobacter hallii.

[0035] Figure 5 is the standard curve of Acetobacter pasteurianus 16S rRNA genomic DNA. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0037] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0039] Example 1 Screening of specific sequences of acetic acid bacteria

[0040] Usually when designing specific primers, we need to find the region with large differences between different sequences. The primers designed in this region have higher specificity. The 16SrRNA sequences of the six strains were compared by MAGE software to find the difference intervals. After comparison by MEGA software (asterisks indicate the same gene sequences, and no asterisks indicate different gene sequences), there are two main intervals with large differences in the difference sequences. The first interval is between 1200 and 1500 bp ( Figure 1 ), the second segment is located between 1100 and 1400 bp ( Figure 2 ), so it can be used as a specific nucleic acid fragment to design primers. After designing with primer5 special primer software, four pairs of specific primers E, F, G, and H were obtained. Figure 3 After verification by agarose gel electrophoresis, primer F had the best effect. After parameter optimization and adjustment, the primer pair F with the best specific effect was obtained.

[0041] By comparing the 16S rRNA gene sequences of different acetic acid bacteria and lactic acid bacteria, it was initially Figure 3 Among the 4 primer pairs, the specific fragments were screened. The primer F pair had a better effect. Later, the annealing temperature of primer F was appropriately reduced by 2-5°C. After optimization, agarose gel electrophoresis was performed to verify the results, and a clearer effect was obtained. Figure 4 The optimized primer pair F had a significant effect in distinguishing specific nucleic acid fragments of closely related acetic acid bacteria.

[0042] S1. First, acetic acid bacteria strains were selected from the fermented mash of Shanxi aged vinegar as the research object. 10 g of fermented mash sample was weighed into a sterilized 250 mL triangular flask containing 90 mL of sterile water and 3 g of glass beads, and then placed in a shaker at 30 ° C and 200 r / min for 30 min, and then allowed to stand for 5 min to obtain a bacterial suspension. The bacterial suspension was diluted and 10 -3 , 10 -4 and 10 -5 The three dilutions of the sample bacterial suspension were spread on acetic acid bacteria growth medium plates and cultured in a 30°C incubator for 48-72 hours. Single colonies on the plates were picked and numbered as acetic acid bacteria-like strains.

[0043] S2. Then, a slant medium for the growth of acetic acid bacteria was prepared, and the screened acetic acid bacteria-like strains were separated and purified, and then transferred to a test tube slant, cultured at 30°C, and stored at 4°C after the culture was completed.

[0044] S3. Finally, the genome was extracted and PCR molecular identification was performed using the universal forward primer F-AGAGTTTGATCCTGGCTCAG and the reverse primer R-ACGGCTACCTTGTTACGACTT. The PCR reaction system and operating parameters are listed in Tables 1 and 2 below, respectively.

[0045] Table 1 PCR reaction system

[0046]

[0047] Table 2 PCR operation parameters

[0048]

[0049]

[0050] S4. Compare the sequence obtained by PCR amplification with the amplicon sequence of the model strain in the NCBI website system to obtain the specific information of the target strain.

[0051] S5. Then, the extracted genome of acetic acid bacteria was used as a template, and PCR amplification was performed with the four pairs of primers shown in Table 3, respectively. The operating parameters were as shown in Table 4, and the products were identified by 1.5% agarose gel electrophoresis. Figure 3 As shown in Figure 4, the amplification effects of other primers were significantly inferior to primer pair F.

[0052] Table 316S rRNA primer sequences

[0053]

[0054] Table 4 PCR operation parameters

[0055]

[0056] Example 2 Verification of acetic acid bacteria specific primer pairs

[0057] S1. Using the genome of the extracted acetic acid bacteria as a template, the above-mentioned specific primer pair F (F-GCCGTCTCAGTTCGGATTGC and R-TGCGTCCTTGCGGTTCG) as primers, PCR was performed, and the negative controls were Lactobacillus reuteri, Lactobacillus helveticus, Bacillus cereus, Bacillus amyloliquefaciens, and Enterobacter hallii. The PCR system and PCR conditions were the same as those in Example 1.

[0058] S2. After PCR, the product was subjected to gel electrophoresis and the bands on the gel were observed by gel imaging to confirm the specificity of the primers. Acetobacter pasteurelli had obvious target bands around 100-250 bp, while the other negative controls had no bands (such as Figure 4As shown in the figure, it indicates that the designed specific primer pair has specificity for Acetobacter pasteurelli.

[0059] Example 3

[0060] S1. Dilute the genomic DNA solution of Acetobacter pasteurianus with a measured concentration by 10 times. Refer to Table 5 for the reaction system and Table 6 for the operating parameters. Perform fluorescence quantitative PCR detection. Repeat 3 times for each concentration. 2 The fluorescence quantitative PCR system with O as the template was used as the blank control, and the target sequence of Acetobacter pasteurianus and the blank control were detected using a Bio-Rad fluorescence quantitative instrument.

[0061] Table 5 Fluorescence quantitative PCR reaction system

[0062]

[0063] Table 616S rRNA fluorescence quantitative PCR operating parameters

[0064]

[0065] S2, 10-fold dilution placed in -20℃ refrigerator for backup Acetobacter pasteurian genome, set 3.13×10 4 copies / μL、3.13×10 5 copies / μL、3.13×10 6 copies / μL、3.13×10 7 copies / μL、3.13×10 8 copies / μL、3.13×10 9 The six gradients of 1000 copies / μL were then tested using a fluorescent quantitative PCR instrument to detect the genomic template of Acetobacter pasteurianus with a multiple-ratio gradient dilution. The standard curve was Figure 5 , regression equation: y = -2.9597x + 49.36, where the y-axis is the Ct value, and the x-axis is the logarithmic value of the DNA template copy number concentration, R 2 =0.9543. The above fluorescence quantitative PCR reaction system was used for testing and found that its sensitivity and final detection limit were 3.13×10 4 copies / μL.

[0066] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A specific primer pair for rapid detection of Acetobacter pasteurianus, characterized in that: The nucleotide sequences of the specific primer pair are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. A kit for rapid detection of Acetobacter pasteurianus, characterized in that: The kit comprises the specific primer pair described in claim 1.

3. Use of the specific primer pair according to claim 1 and / or the kit according to claim 2 in detecting Acetobacter pasteurianus.

4. A rapid detection method for Acetobacter pasteurianus, characterized in that: The method comprises the following steps: (1) Isolating and purifying bacterial strains from the sample to be tested, and extracting DNA from the strains; (2) using DNA as a template and performing PCR gene amplification using the specific primer pair described in claim 1; (3) Electrophoresis observation: After the reaction is completed, the PCR product is observed by gel electrophoresis. If the PCR product has a band between 100-250 bp (including the endpoint value) on the electrophoresis map, it means that the sample to be tested contains Acetobacter pasteurianus. If the PCR product is not between 100 and 250 bp or has no band, it means that the sample to be tested does not contain Acetobacter pasteurianus.

5. The rapid detection method for Acetobacter pasteurianus according to claim 4, characterized in that: The method further comprises the following steps: taking 16S rRNA genomic DNA of Acetobacter pasteurianus as a standard substance, performing experiments using qPCR technology to obtain a qPCR standard curve, thereby establishing an absolute quantitative detection method.

6. The rapid detection method for Acetobacter pasteurianus according to claim 5, characterized in that: The reaction system of qPCR technology: DNA template 1μL, upstream primer (10μM) 0.4μL, downstream primer (10μM) 0.4μL, 2×Q3 SYBR Green I qPCR Supermix 10μL and ddH2O 8.2μL.

7. The rapid detection method for Acetobacter pasteurianus according to claim 5, characterized in that: The operating parameters of qPCR technology were as follows: denaturation: 95°C, 5 min; 39 cycles: 95°C, 30 s; 60°C, 30 s; 72°C, 15 s; final extension: 55°C, 5 s; 95°C, 30 s.

8. The rapid detection method for Acetobacter pasteurianus according to claim 5, characterized in that: The regression equation of the standard curve is y=-2.9597x+49.36, where the y-axis is the Ct value, the x-axis is the logarithmic value of the DNA template copy number concentration, and R 2 =0.9543.

9. The rapid detection method for Acetobacter pasteurianus according to claim 4, characterized in that: In step (1), the sample to be tested is the fermented grains in the process of brewing aged vinegar.

10. The rapid detection method for Acetobacter pasteurianus according to claim 4, characterized in that: In step (2), the PCR gene amplification system is: 2×TransFast Taq PCR SuperMix (+dye) 12 μL, Nuclease-free Water 10 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL and DNA template 1 μL; The PCR operating parameters were as follows: denaturation: 94°C, 4 min; 72°C, 10 min; 40 cycles: 94°C, 45 s; 54.6°C, 30 s; 72°C, 1 min; final extension: 72°C, 10 min.

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