A degenerate primer for identifying ethyl acetate-producing strains and its application

Abstract

The present invention discloses degenerate primers for identifying ethyl acetate-producing strains and their applications, belonging to the field of molecular biology. The present invention respectively designed 6 pairs of degenerate primer pairs based on the alcohol acyltransferases encoded by the functional genes ATF1 and EAT1 for ethyl acetate synthesis. By comparing the detection effectiveness, accuracy, and sensitivity of each primer pair, 2 pairs of degenerate primer pairs with significantly excellent effects were screened. Through multiplex PCR technology, it can quickly detect whether yeast and test samples have the potential to produce ethyl acetate, and can determine the gene categories responsible for ethyl acetate synthesis in the test samples.

Description

Technical Field

[0001] The present invention relates to a degenerate primer for identifying ethyl acetate-producing strains and its application, belonging to the field of biotechnology. Background Art

[0002] As one of the "four esters" in Luzhou-flavor liquor, ethyl acetate plays an important role in the typical flavor presentation of Luzhou-flavor liquor in terms of the ratio of its amount to that of ethyl hexanoate. In recent years, due to the phenomenon of too high ethyl acetate content during the brewing process, even exceeding the content of ethyl hexanoate, the coordination and typicality of the liquor body are reduced, restricting the improvement of the quality of Luzhou-flavor liquor. There are multiple yeasts jointly involved in the fermentation of Luzhou-flavor liquor, such as Pichia, Candida, Saccharomyces, etc. These yeasts have strong abilities to produce esters and other flavor compounds, such as ethyl acetate, ethyl isovalerate, isoamyl alcohol, phenylethyl alcohol, etc., which have important impacts on the flavor of liquor. Therefore, quickly identifying the ethyl acetate-producing strains in the traditional brewing process and realizing the source control of the ethyl acetate content are problems that need to be urgently solved.

[0003] Yeasts mainly generate ethyl acetate by transferring the acyl group of acetyl coenzyme A to ethanol through the transfer action of alcohol acyltransferase. In yeasts, the genes encoding alcohol acyltransferase are mainly ATF1 and EAT1. The alcohol acyltransferases encoded by genes ATF1 and EAT1 are respectively in the cytoplasmic matrix and mitochondria of yeast cells, and their abilities to synthesize ethyl acetate vary greatly. Due to the diversification of the coding genes in different yeasts and the large sequence differences between the ATF1 and EAT1 genes in the same yeast, there has not been a primer that can simultaneously detect the ATF1 or EAT1 genes of different yeasts so far. Summary of the Invention

[0004] In order to solve the above problems, through the comparison of the amplification performances of 6 pairs of degenerate primers, the present invention provides a primer for rapid detection of ethyl acetate-producing yeasts and its application.

[0005] The first object of the present invention is to provide a degenerate primer pair for identifying or assisting in identifying ethyl acetate-producing strains, and the degenerate primer pair includes a degenerate primer pair A for gene ATF1 and / or a degenerate primer pair B for gene EAT1; the degenerate primer pair A is composed of nucleotide fragments with nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the degenerate primer pair B is composed of nucleotide fragments with nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4.

[0006] The second object of the present invention is to provide a kit for identifying or assisting in identifying ethyl acetate-producing strains, and the kit contains the degenerate primer pair.

[0007] In one embodiment of the present invention, the kit further contains a PCR reaction buffer.

[0008] The third object of the present invention is to provide a method for screening or detecting ethyl acetate-producing strains using the degenerate primer pair or the kit, and the specific steps of the method are as follows:

[0009] (1) Extract the DNA of the sample to be tested;

[0010] (2) Using the DNA of the sample to be tested in step (1) as a template, perform PCR amplification using the degenerate primer pair;

[0011] (3) Determine whether the sample to be tested contains ethyl acetate-producing strains according to the size of the PCR amplification product.

[0012] In one embodiment of the present invention, in step (2), the annealing condition for PCR amplification is annealing at 48-52 °C for 55-65 s.

[0013] In one embodiment of the present invention, in step (2), the PCR amplification conditions for degenerate primer pair A are 95 °C for 3 min; 95 °C for 30 s; 52 °C for 1 min; 72 °C for 1 min; 30 cycles, and the PCR amplification conditions for degenerate primer pair B are 95 °C for 3 min; 95 °C for 30 s; 50 °C for 1 min; 72 °C for 1 min; 30 cycles; the multiplex PCR amplification conditions for degenerate primer pairs A and B are 95 °C for 3 min; 95 °C for 30 s; 48 °C for 1 min; 72 °C for 1 min; 30 cycles.

[0014] In one embodiment of the present invention, in step (2), the reaction system for the PCR amplification is 1 μl of the DNA of the sample to be tested, 12.5 μl of 2×Taq PCR MasterMix (with loading dye), 1 μl of the forward primer, 1 μl of the reverse primer, and deionized water is added to make up 25 μl.

[0015] In one embodiment of the present invention, in step (3), when using degenerate primer pair A, the size of the PCR amplification product is 420-440 bp; when using degenerate primer pair B, the size of the PCR amplification product is 220-270 bp; when using degenerate primers A and B, the sizes of the PCR amplification products are 300-360 bp and 220-270 bp.

[0016] The present invention also provides the application of the degenerate primer pair or the kit in screening or detecting ethyl acetate-producing strains.

[0017] The present invention also provides the application of the degenerate primer pair in the preparation of products for screening or detecting ethyl acetate-producing strains.

[0018] The present invention also provides the application of the degenerate primer pair in metagenomic detection and analysis.

[0019] Beneficial effects achieved by the present invention:

[0020] (1) The method of the present invention avoids the disadvantages of the traditional identification method, such as the cumbersome, time-consuming, blind and easy-to-miss screening processes in strain screening, fermentation and fermentation product identification, etc. It has stronger purposefulness and greatly improves the work efficiency.

[0021] (2) The degenerate primer pair provided by the present invention has good effectiveness, high accuracy and high sensitivity when used for detection.

[0022] (3) The degenerate primer pair of the present invention can achieve rapid detection of ethyl acetate-producing yeasts in the liquor fermentation system through PCR technology.

[0023] (4) The present invention can identify the gene categories responsible for ethyl acetate synthesis in the detected samples. Description of the Drawings

[0024] Figure 1 It is a result graph of the amino acid sequence alignment of alcohol acyltransferases encoded by the ATF1 genes of 8 different yeasts in the embodiment of the present invention.

[0025] Figure 2 It is a result graph of the amino acid sequence alignment of alcohol acyltransferases encoded by the EAT1 genes of 8 different yeasts in the embodiment of the present invention.

[0026] Figure 3This is the electrophoresis result diagram of the PCR amplification products for the detection effectiveness experiment of degenerate primers in the embodiments of the present invention. In A, M is the DNA Marker; 1-5: The PCR amplification results with ATF1-F / R-1 as primers and the genomes of Saccharomyces cerevisiae, Wickerhamomyces anomalus, Wickerhamomyces ciferrii, Pichia kudriavzevii, and Candida orthopsilosis as templates respectively; 6-10: The PCR amplification results with EAT1-F / R-1 as primers and the genomes of Saccharomyces cerevisiae, Wickerhamomyces anomalus, Wickerhamomyces ciferrii, Pichia kudriavzevii, and Candida orthopsilosis as templates respectively. In B, 1-5: With ATF1-F / R-2 as primers; 6-10: With ATF1-F / R-3 as primers; 11-15: With EAT1-F / R-2 as primers; 16-20: With EAT1-F / R-3 as primers; the templates used are all the genomes of Saccharomyces cerevisiae, Wickerhamomyces anomalus, Wickerhamomyces ciferrii, Pichia kudriavzevii, and Candida orthopsilosis.

[0027] Figure 4 This is the electrophoresis result diagram of the PCR amplification products for the detection accuracy experiment of degenerate primers in the embodiments of the present invention. Among them, the M lane is the DNA Marker; 1-3: With ATF1-F / R-1 as primers; 4-6: With ATF1-F / R-2 as primers; 7-9: With EAT1-F / R-1 as primers; 10-12: With EAT1-F / R-3 as primers; the templates used are all the genomes of Issatchenkia orientalis, Aspergillus amstelodami, and Lactobacillus fermentum.

[0028] Figure 5This is the electrophoresis result diagram of the PCR amplification products for the detection sensitivity experiment of degenerate primers in the embodiments of the present invention. In A, M is the DNA Marker; 1-4: using ATF1-F / R-1 as primers; 5-8: using ATF1-F / R-2 as primers; in B, M is the DNA Marker; 1-4: using EAT1-F / R-1 as primers; 5-8: using EAT1-F / R-3 as primers; the template concentrations used are respectively 100 ng / μl, 10 ng / μl, 1 ng / μl, and 0.1 ng / μl.

[0029] Figure 6 This is the electrophoresis result diagram of the PCR amplification products of the microbial genomes of the fermented grains of Baijiu at different fermentation days in the embodiments of the present invention. Lanes 1-4 are respectively for fermentation days 5, 10, 15, and 30. Detailed implementation manners

[0030] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments.

[0031] In the following embodiments, the materials, reagents, instruments, and methods used, unless otherwise specified, are all conventional materials, reagents, instruments, and methods in the art and can be obtained through commercial channels.

[0032] The primers were synthesized by Genewiz Biotechnology Co., Ltd. 2×Taq PCR MasterMix (with loading dye) was purchased from Novoprotein Scientific Inc.

[0033] Example 1 Design of degenerate primer pairs

[0034] Currently, the microorganisms reported at home and abroad with the ability to synthesize ethyl acetate mainly include the genera Saccharomyces, Wickerhamomyces, Candida, and Kluyveromyces. Since the alcohol acyltransferases encoded by the genes ATF1 and EAT1 are located in the cytoplasm and mitochondria of yeast cells respectively, and their abilities to synthesize ethyl acetate vary greatly, we designed their degenerate primers separately.

[0035] (1) Collect the amino acid sequences of the alcohol acyltransferases encoded by the gene ATF1 in yeasts of different genera from UniProt. Since the only yeast retrieved was Saccharomyces cerevisiae S288C (Uniprot accession number: P40353). Therefore, the amino acid sequence of this yeast was blasted in NCBI, and it was found that the aligned amino acid sequences all belonged to the genus Saccharomyces. Eight amino acid sequences of different species in the genus Saccharomyces with the highest similarity were selected for multiple sequence alignment in the MEGA-X software (the results are as Figure 1As shown in the figure, different conserved regions were selected for the design of degenerate primers:

[0036] 1) Design the upstream primer from the conserved region at positions 247 - 252 and the downstream primer from the conserved region at positions 384 - 392. The sequence of the resulting degenerate primer ATF1 - F / R - 1 is as follows:

[0037] Upstream primer: 5’ - GCWGTGGATWCTCAAT - 3’ (SEQ ID NO.1);

[0038] Downstream primer: 5’ - AATATTYAASCCACCAKTAT - 3’ (SEQ ID NO.2);

[0039] 2) Obtain the degenerate primer ATF1 - F / R - 2 from the conserved regions at positions 308 - 314 (upstream) and 471 - 476 (downstream). Its sequence is as follows:

[0040] Upstream primer: 5’ - ATCTTGGATGATTWYACATT - 3’;

[0041] Downstream primer: 5’ - GACGAGWATCCACWGC - 3’;

[0042] 3) Obtain the degenerate primer ATF1 - F / R - 3 from the conserved regions at positions 343 - 350 (upstream) and 506 - 511 (downstream). Its sequence is as follows:

[0043] Upstream primer: 5’ - TGATWTMRTTMTACCATATG - 3’;

[0044] Downstream primer: 5’ - CATTMAWATGTTGWGYAAA - 3’;

[0045] (2) Collect the amino acid sequences of the alcohol acyltransferase encoded by the gene EAT1 in yeasts of different species from UniProt. The yeasts retrieved are Saccharomyces cerevisiae S288C (Uniprot accession number: P53208), Wickerhamomyces anomalus NRRL Y - 366 - 8 (A0A1E3P8S6), Cyberlindnera jadinii NRRL Y - 1542 (A0A1E4S2P1), Kluyveromyces marxianus NBRC 104275 (W0T4A7). The amino acid sequences were subjected to multiple sequence alignment in the MEGA - X software (the results are as Figure 2 shown), and different conserved regions were selected for the design of degenerate primers:

[0046] 1) Design the upstream primer from the conserved region 65 - 72 and the downstream primer from 139 - 145. The sequence of the degenerate primer EAT1 - F / R - 1 obtained is as follows:

[0047] Upstream primer: 5’ - GCAAATGTYACACATACWCAAT - 3’ (SEQ ID NO.3);

[0048] Downstream primer: 5’ - GAGGTTGTKSAATTGTAT - 3’ (SEQ ID NO.4);

[0049] 2) Obtain the degenerate primer EAT1 - F / R - 2 from the conserved region 44 - 51 (upstream) and 139 - 145 (downstream). Its sequence is as follows:

[0050] Upstream primer: 5’ - TBGGWTWYTCWWTRGGTG - 3’;

[0051] Downstream primer: 5’ - AACWKKTTCRGYWKGC - 3’;

[0052] 3) Obtain the degenerate primer EAT1 - F / R - 3 from the conserved region 45 - 51 (upstream) and 344 - 349 (downstream). Its sequence is as follows:

[0053] Upstream primer: 5’ - TTGATKTAAGAAAYCATGG - 3’;

[0054] Downstream primer: 5’ - AACWKKTTCRGYWKGC - 3’.

[0055] Example 2 Performance Verification of Degenerate Primers

[0056] In Example 1, a total of 6 pairs of degenerate primers were provided. To screen the optimal degenerate primers, in this example, the effectiveness, accuracy, and sensitivity of the 6 pairs of degenerate primers were evaluated respectively.

[0057] (1) Comparison of Detection Effectiveness of Different Primer Pairs

[0058] Take 5 strains with the ability to produce ethyl acetate screened from the fermented grains of Baijiu: Saccharomyces cerevisiae, Wickerhamomyces anomalus, Wickerhamomyces ciferrii, Pichia kudriavzevii, Candida orthopsilosis, and extract their respective genomes.

[0059] The method for extracting microbial DNA is as follows: Add 200 μl of PBS buffer to a centrifuge tube containing microbial cells, mix well to resuspend the cells, and then add 700 μl of phenol:chloroform:isoamyl alcohol (volume ratio 25:24:1), and shake well. Shake on a nucleic acid extractor for 1 min and centrifuge at 10,000 rpm for 10 min. Aspirate the supernatant and add an equal volume of chloroform:isoamyl alcohol (volume ratio 24:1), shake well, and centrifuge at 10,000 rpm for 10 min. Aspirate the supernatant, add 2 volumes of absolute ethanol, and precipitate at -20°C for 2 h. Centrifuge at 12,000 rpm for 10 min, pour out the supernatant, dry for 1 h, add 50 μl of sterile water, and store at -20°C.

[0060] Use the six pairs of degenerate primers in Example 1 to perform PCR on each genome, and then detect by electrophoresis. Set up a 25 μl system as follows: 1 μl of template DNA, 12.5 μl of 2× Taq PCR MasterMix (with loading dye), 1 μl of forward primer, 1 μl of reverse primer, and make up to 25 μl with deionized water.

[0061] The amplification conditions for primer pairs ATF1-F / R-1, ATF1-F / R-2, and ATF1-F / R-3 are: 95°C for 3 min; 95°C for 30 s; 52°C for 1 min; 72°C for 1 min; 30 cycles.

[0062] The amplification conditions for primer pairs EAT1-F / R-1, EAT1-F / R-2, and EAT1-F / R-3 are: 95°C for 3 min; 95°C for 30 s; 50°C for 1 min; 72°C for 1 min; 30 cycles.

[0063] The result shows that: as Figure 3 shown in A, using the genomes of the 5 tested strains as templates, the PCR products of primer pairs ATF1-F / R-1 and EAT1-F / R-1 both have single bands at 431 bp and 249 bp respectively, indicating that these primers can indeed amplify the functional genes ATF1 and EAT1 for ethyl acetate synthesis from ethyl acetate-producing strains, and there are no obvious other bands, showing good effectiveness.

[0064] As Figure 3 shown in lanes 1-5 of B, the PCR product of primer pair ATF1-F / R-2 has a single band at 490 bp (except for lane 5), indicating that this primer has a certain effectiveness, can amplify the functional gene ATF1 for ethyl acetate synthesis in most (about 80%) of the ethyl acetate-producing strains, and there are no obvious other bands, showing a certain effectiveness.

[0065] As Figure 3As shown in lanes 6-10 of B, the amplification effect of primer pair ATF1-F / R-3 was significantly inferior to that of primer pairs ATF1-F / R-1 and ATF2-F / R-1. Multiple amplified bands appeared, indicating obvious non-specific amplification and poor effectiveness.

[0066] As Figure 3 As shown in lanes 11-15 of B, no bands were electrophoresed for the PCR products of primer pair EAT1-F / R-2.

[0067] As Figure 3 As shown in lanes 16-20 of B, the PCR products of primer pair EAT1-F / R-3 had a single bright band at 1050 bp (lanes 19 and 20), indicating that this primer had a certain effectiveness. It could amplify the functional gene EAT1 for ethyl acetate synthesis in a small part (about 40%) of ethyl acetate-producing strains, and there were no obvious other bands, showing a certain effectiveness.

[0068] (2) Comparison of detection accuracy of different primer pairs

[0069] Three strains that had been confirmed not to carry the alcohol acyltransferase genes ATF1 and EAT1 (Issatchenkia orientalis, Aspergillus amstelodami, Lactobacillus fermentum, all from the Baijiu fermentation system) were taken, and their genomes were extracted. Then, PCR was performed using the primer pairs ATF1-F / R-1, EAT1-F / R-1, ATF1-F / R-2, and EAT1-F / R-3 with a certain effectiveness respectively. The genome extraction method and PCR amplification conditions were the same as in step (1).

[0070] As Figure 4 As shown, no bands were obtained in the amplification results of all primer pairs. This indicates that the primer pairs have good specificity and detection accuracy and will not lead to false positive judgments for strains that do not carry the alcohol acyltransferase genes ATF1 and EAT1.

[0071] (3) Comparison of detection sensitivity of different primer pairs

[0072] The genome of Saccharomyces cerevisiae was extracted and diluted to 1000 ng / μl as the initial template concentration, and then serially diluted 10-fold to 100 ng / μl, 10 ng / μl, 1 ng / μl, and 0.1 ng / μl.

[0073] Four pairs of degenerate primers, ATF1-F / R-1, EAT1-F / R-1, ATF1-F / R-2, and EAT1-F / R-3, were used to perform PCR at different template concentrations. The PCR reaction system and PCR reaction conditions were the same as those in Example 2. 5 μl of the PCR amplification reaction product was taken for gel electrophoresis, and the sensitivity of each primer pair was judged based on the presence or absence of bands.

[0074] As Figure 5 shown, in Figure A, lanes 1, 2, 3, and 4 are the amplification results using the degenerate primer ATF1-F / R-1 with initial template concentrations of 100 ng / μl, 10 ng / μl, 1 ng / μl, and 0.1 ng / μl, respectively. The detection sensitivity of the degenerate primer ATF1-F / R-1 is approximately 1 ng / μl.

[0075] In Figure A, lanes 5, 6, 7, and 8 are the amplification results using the degenerate primer ATF1-F / R-2 with initial template concentrations of 100 ng / μl, 10 ng / μl, 1 ng / μl, and 0.1 ng / μl, respectively. The detection sensitivity of the degenerate primer ATF1-F / R-2 is approximately 100 ng / μl.

[0076] In Figure B, lanes 1, 2, 3, and 4 are the amplification results using the degenerate primer EAT1-F / R-1 with initial template concentrations of 100 ng / μl, 10 ng / μl, 1 ng / μl, and 0.1 ng / μl, respectively. The detection sensitivity of the degenerate primer EAT1-F / R-1 is approximately 10 ng / μl.

[0077] In Figure B, lanes 5, 6, 7, and 8 are the amplification results using the degenerate primer EAT1-F / R-3 with initial template concentrations of 100 ng / μl, 10 ng / μl, 1 ng / μl, and 0.1 ng / μl, respectively. The detection sensitivity of the degenerate primer EAT1-F / R-3 is approximately 100 ng / μl.

[0078] Based on the comprehensive comparative analysis results above, the degenerate primers ATF1-F / R-1 and ATF1-F / R-1 provided by the present invention for identifying the alcohol acyltransferase genes ATF1 and EAT1 in ethyl acetate-producing strains showed significantly excellent effects in terms of detection effectiveness, accuracy, and sensitivity.

[0079] Example 3 Identification of the Potential of Baijiu Fermentation Grains to Produce Ethyl Acetate

[0080] Take the fermented grains after 5, 10, and 15 days of fermentation, respectively extract the total microbial DNA, and the extraction method is as follows: (1) Take 30 g of fermented grains and mix them thoroughly with 100 ml of PBS buffer, shake well for 10 min, filter with 4 layers of sterilized gauze, and collect the filtrate. (2) Take 30 ml of the filtrate and put it into a 50 ml centrifuge tube, centrifuge at 12000 rpm for 10 min, and collect the bacterial cells. (3) Grind the collected bacterial cells in liquid nitrogen. (4) Take 200 mg of the ground powder and put it into a centrifuge tube that has been pre-added with 600 μl of sodium laurate buffer and 600 μl of phenol:chloroform:isoamyl alcohol (volume ratio 25:24:1), and shake well. (5) Centrifuge at 12000 rpm at 4 °C for 10 min, aspirate the supernatant and add an equal volume of chloroform:isoamyl alcohol (volume ratio 24:1), and mix well. (6) Centrifuge at 12000 rpm at 4 °C for 10 min, aspirate the supernatant and add an equal volume of isoamyl alcohol, and precipitate at 4 °C for 15 min. (7) Remove the supernatant, add 500 μl of ethanol to wash the precipitate, centrifuge at 12000 rpm at 4 °C for 10 min, remove the supernatant, and dry thoroughly at room temperature. (8) Add 50 μl of sterile water and store at -20 °C.

[0081] Use the two pairs of degenerate primers designed in Example 1 to perform PCR on the microbial genomes of the fermented grains after 5, 10, and 15 days of fermentation, and then detect by electrophoresis. The PCR reaction system is the same as in Example 2. Perform multiplex PCR, and simultaneously perform PCR amplification reactions on the primer pairs ATF1-F / R-1 and EAT1-F / R-1 in one reaction system. The PCR reaction system is as follows: 1 μl of template DNA, 12.5 μl of 2× Taq PCR MasterMix (with loading dye), 1 μl of each forward primer, 1 μl of each reverse primer, and make up to 25 μl with deionized water. The amplification reaction conditions for the primer pairs are: 94 °C for 10 min; 94 °C for 1 min; 48 °C for 30 s; 72 °C for 1 min; 30 cycles.

[0082] The results showed that: two bands were detected in the fermented grains after 5, 10, and 15 days of fermentation, with single bands at 431 bp and 249 bp respectively (as Figure 6 shown), indicating that there are microorganisms with ATF1 and EAT1 functional genes in these fermented grains at the same time. However, no bands were amplified from the fermented grains after 30 days of fermentation, indicating that there are no microorganisms with the potential to produce ethyl acetate in the fermented grains at this time.

[0083] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A degenerate primer pair for identifying or assisting in identifying ethyl acetate-producing strains in yeast, characterized in that, The degenerate primer pairs include degenerate primer pair A targeting gene ATF1 and degenerate primer pair B targeting gene EAT1; the degenerate primer pair A consists of nucleotide fragments with nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the degenerate primer pair B consists of nucleotide fragments with nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.

4.

2. Kit for identifying or assisting in identifying ethyl acetate-producing strains in yeast, characterized in that, The kit contains the degenerate primer pair described in claim 1.

3. The kit according to claim 2, characterized in that, The kit also contains a PCR reaction buffer.

4. The degenerate primer pair according to claim 1 or the kit according to claim 2 or 3, in a method for screening or detecting ethyl acetate-producing strains, characterized in that, The specific steps of the method are as follows: (1) Extract the DNA of the sample to be tested; (2) Using the DNA of the sample to be tested in step (1) as a template, perform PCR amplification using the degenerate primer pair; (3) Determine whether the sample to be tested contains ethyl acetate-producing strains according to the size of the PCR amplification product.

5. The method according to claim 4, wherein In step (2), the annealing condition for PCR amplification is annealing at 48 - 52°C for 55 - 65 s.

6. The method according to claim 4 or 5, characterized in that In step (2), the PCR amplification conditions for degenerate primer pair A are 95°C for 3 min; 95°C for 30 s; 52°C for 1 min; 72°C for 1 min; 30 cycles, the PCR amplification conditions for degenerate primer pair B are 95°C for 3 min; 95°C for 30 s; 50°C for 1 min; 72°C for 1 min; 30 cycles; the multiplex PCR amplification conditions for degenerate primer pairs A and B are 95°C for 3 min; 95°C for 30 s; 48°C for 1 min; 72°C for 1 min; 30 cycles.

7. The method according to claim 4, wherein In step (3), when using degenerate primer pair A, the size of the PCR amplification product is 420 - 440 bp; when using degenerate primer pair B, the size of the PCR amplification product is 220 - 270 bp; when using degenerate primers A and B, the sizes of the PCR amplification products are 300 - 360 bp and 220 - 270 bp.

8. Use of the degenerate primer pair described in claim 1 or the kit described in claim 2 or 3 in screening or detecting ethyl acetate-producing strains in yeast.

9. Use of the degenerate primer pair described in claim 1 in the preparation of a product for screening or detecting ethyl acetate-producing strains in yeast.

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