A PSR primer, kit and detection method for identifying pediococcus acidilactici based on specific gene target

Abstract

This invention discloses a PSR primer, kit, and detection method for identifying Pediococcus lactis based on a specific gene target. Based on the conserved sequence of the target gene, this invention designs and optimizes PSR-specific primers, establishes a PSR amplification system, and further provides a kit containing the primers and its application method. The PSR detection method established by this invention features high sensitivity, strong specificity, rapid reaction, simple operation, and low cost. It is suitable for the rapid and accurate identification of Pediococcus lactis and has promising application prospects in food fermentation, probiotic detection, and microbial quality control.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a PSR primer, kit, and detection method for identifying Pediococcus lactis based on specific gene targets. Background Technology

[0002] Beer itself is considered an unfavorable culture medium for microbial growth due to its inherent and extrinsic antimicrobial barriers. Intrinsic barriers include low pH, ethanol, extremely low fermentable sugar content, low oxygen levels, antimicrobial hop compounds, and high concentrations of carbon dioxide. Extrinsic antimicrobial processing steps include malting (bio-acidification), saccharification, wort boiling, pasteurization or aseptic filtration, and low-temperature storage. However, when one or more of these antimicrobial barriers are absent or reduced, certain microorganisms can still grow in malt, wort, and beer. Among these, a few beer-specific lactic acid bacteria have evolved or adapted to the hop environment and can cause beer spoilage. Pediococcus lactis is a prime example (reference source). Therefore, rapid and accurate identification and monitoring of potential spoilage bacteria or functional bacteria, including Pediococcus lactis, are crucial for ensuring the quality and safety of food and beverages (especially beer), for production process control, and for the quality control of probiotic products.

[0003] Currently, the detection of Pediococcus lactis mainly relies on traditional microbial culture methods. While these methods do not require sophisticated equipment, they suffer from long detection cycles (typically 3-5 days), cumbersome procedures, and the inability to effectively culture certain damaged or dormant cells, leading to false negatives. Identification methods based on physiological and biochemical characteristics are also time-consuming and prone to misclassification due to phenotypic similarity. Molecular biology methods, such as conventional PCR, while improving speed and specificity, still rely on sophisticated thermal cycling equipment, hindering rapid on-site detection. Furthermore, some reported target genes may exhibit cross-reactivity between closely related species, requiring further improvement in specificity.

[0004] Polymerase spiral reaction (PSR), as a novel isothermal amplification technique, has shown excellent application prospects in the rapid detection of pathogenic microorganisms in recent years. Compared with traditional PCR technology, PSR does not require complex thermal cycling processes, can achieve gene amplification at isothermal conditions, is simple to operate, and can significantly improve the sensitivity and specificity of detection. Therefore, PSR detection methods based on specific gene targets can provide a new solution for the rapid and efficient detection of Pediococcus lactis.

[0005] While some molecular detection methods have been reported for other putrefactive lactic acid bacteria (such as Pediococcus damnosus), specific and highly sensitive isothermal detection methods for Pediococcus lactis, especially those based on PSR technology, remain lacking. Existing primers or targets may not effectively distinguish Pediococcus lactis from its closely related species, or may lack sufficient sensitivity in complex sample matrices. Therefore, developing a PSR detection method based on highly specific gene targets for the accurate and rapid identification of Pediococcus lactis has urgent industrial demand and significant application value. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a PSR primer for identifying Pediococcus lactis based on a specific gene target.

[0007] Another objective of this invention is to provide a kit for identifying Pediococcus lactis based on specific gene targets.

[0008] Another objective of this invention is to provide a detection method for identifying Pediococcus lactis based on specific gene targets.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A set of PSR primers for identifying Pediococcus lactis based on specific gene targets, including primers PaciT2-Ft1 and PaciT2-Bt1, with the nucleotide sequences shown below:

[0011] PaciT2-Ft1:

[0012] 5'-CTAGCAACATGTAAGGGCAGCAGACGTTCGGAAGTG-3';

[0013] PaciT2-Bt1:

[0014] 5'-CGGGAATGTACAACGATCTTTGACCGCATTGCTTCT-3'.

[0015] A kit for identifying Pediococcus lactis based on specific gene targets, comprising the aforementioned PSR primers for identifying Pediococcus lactis based on specific gene targets.

[0016] In the reagents described, the concentrations of both PaciT2-Ft1 and PaciT2-Bt1 are 0.8 μM.

[0017] The reagents also include 2×PSR reaction buffer and Bst enzyme.

[0018] The formulation of the 2×PSR reaction buffer is as follows: 40.0 mM Tris-HCl, 20.0 mM ammonium sulfate, 20.0 mM potassium chloride, 16.0 mM magnesium sulfate, 0.2% (v / v) Tween 20, 1.4 M betaine, and 10.0 mM dNTPs.

[0019] A detection reagent for identifying Pediococcus lactis based on specific gene targets, comprising the aforementioned PSR primers for identifying Pediococcus lactis based on specific gene targets.

[0020] The above-mentioned primers or reagents are used in the identification of Pediococcus lactis.

[0021] An experimental research method for identifying Pediococcus lactis for non-disease diagnosis using the above-mentioned reagents includes the following steps:

[0022] (1) Extract bacterial DNA from the sample to be tested as a template, and ensure that the OD of the template DNA aqueous solution is within acceptable limits. 260 / OD 280 The value is in the range of 1.8 to 2.0;

[0023] (2) Incubate in a 63℃ water bath for 60 minutes to perform polymerase helical amplification reaction; wherein, the polymerase helical amplification reaction system is a 25 μL reaction system: 12.5 μL of 2×PSR reaction buffer, 0.8 μM each of primers PaciT2-Ft1 and PaciT2-Bt1, 2 μL of template DNA, 1 μL of Bst enzyme, and add water to make up to 25 μL;

[0024] (3) Take 5 μL of PSR reaction product and perform electrophoresis on a 2% agarose gel. If a band appears, it is judged to be positive for Pediococcus lactis.

[0025] Application of the target PaciT2 in the identification of Pediococcus lactis.

[0026] The nucleotide sequence of the target PaciT2 is shown in SEQ ID NO.1.

[0027] The present invention has the following advantages and effects compared with the prior art:

[0028] This invention discloses a specific detection target for *Pediococcus lactis*, along with corresponding primers and a detection method for PSR detection. The specific detection target, obtained through comparative genomics and bioinformatics mining, is selected from the PaciT2 gene and exhibits both high accuracy and strong specificity. This invention targets the newly discovered specific detection target for *Pediococcus lactis*, PaciT2. Gene-designed specific primers were used to establish a PSR detection method for Pediococcus lactis. The detection method of this invention has the characteristics of high sensitivity, good specificity, fast reaction speed, low cost and simple operation.

[0029] This invention provides a PSR detection method for identifying Pediococcus lactis based on specific gene targets. Compared with other detection methods, it does not require expensive equipment and professional personnel, shortens the detection cycle, expands the detection limit, and is more suitable for on-site testing needs. It has important application value in the identification of Pediococcus lactis, food safety risk assessment, and pathogen tracing of foodborne diseases. Attached Figure Description

[0030] Figure 1 The results are PCR electrophoresis detection results of three specific target sites in Example 2.

[0031] Figure 2 The results of PCR electrophoresis detection of clones after molecular cloning of the three specific targets in Example 2 are shown.

[0032] Figure 3 The electrophoretic detection results are those of three sets of PSR primers for Pediococcus lactis based on the specific gene target PaciT2 in Example 3.

[0033] Figure 4 The electrophoretic detection results are for the specificity evaluation of the PSR detection method for Pediococcus lactis based on the specific gene target PaciT2 in Example 4.

[0034] Figure 5 The electrophoretic detection results are for sensitivity evaluation of the PSR detection method for Pediococcus lactis based on the specific gene target PaciT2 in Example 5. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0036] Example 1: Screening for highly specific targets in Pediococcus lactis

[0037] The reference genome of *Pediococcus lactis* (code: ASM991365v1) was obtained from the NCBI Genome Database. This genome contains 1932 coding sequences (CDS). Additionally, 243 genomes from within the *Pediococcus lactis* species and 177 genomes from other species within the *Pediococcus* genus (i.e., extraspecific and intragenus-specific) were collected for subsequent specificity analysis.

[0038] Based on the aforementioned genomic data, 1932 CDS sequences in the reference genome underwent multi-level specificity screening. Blast+ was used to perform intraspecific, intragenus-extraspecific, and extragenus-extraspecific (NT database) sequence alignments, with the following screening thresholds: the average coverage × identity product (CVP) of intraspecific alignments was greater than 98%; the average CVP × identity of both intragenus-extraspecific and extragenus-extraspecific NT database alignments was less than 5%. After rigorous screening, 13 highly specific candidate targets were obtained. The three most specific targets were further selected for subsequent experimental validation. For the identified highly specific targets, MEGA software and the MEME website were used for multiple sequence alignment to obtain conserved sequences. The following three specific targets were obtained: PaciT1, PaciT2, and PaciT3.

[0039] Example 2: Preparation of positive control plasmid for Pediococcus lactis-specific gene target

[0040] To construct plasmid standards for each target, PCR amplification was performed using the corresponding target-specific primer pairs. The resulting amplicons were purified and then cloned into the pUCm-T vector. They were then propagated in competent *E. coli* DH5α cells according to the manufacturer's instructions. Positive clones confirmed by agarose gel electrophoresis were sent for Sanger sequencing (Guangzhou Aiji Biotechnology Co., Ltd.) to ensure sequence fidelity. Finally, plasmid DNA was extracted from the validated clones using a commercial plasmid extraction kit (TIANGEN BIOTECH) and quantified using a micro spectrophotometer. These purified plasmids were used as positive controls in subsequent isothermal amplification experiments.

[0041] Table 1. PCR primers for three specific target sites of Pediococcus lactis

[0042]

[0043] PCR results as follows Figure 1 As shown, all three identified specific targets could be successfully amplified by PCR. However, multiple attempts to prepare plasmids for PaciT1 and PaciT3 failed, while only PaciT2 successfully prepared a plasmid. Figure 2 Therefore, an experiment was conducted to design a subsequent PSR detection method for PaciT2.

[0044] Example 3: Establishment of a PSR detection method for Pediococcus lactis (i.e., the PSR detection method for Pediococcus lactis based on the specific gene target PaciT2).

[0045] Three sets of primers, including a forward primer (Ft) and a reverse primer (Bt), were designed using Primer Premier 5 for the conserved sequence of PaciT2. The designed primers were compared with the NCBI NT database using the online tool Primer-BLAST to verify the accuracy of the detection primers of this invention.

[0046] Table 2. PSR primers for detecting WP_036685256.1

[0047]

[0048] A PSR detection method for Pediococcus lactis was established using the above primer pairs.

[0049] The total volume of the PSR reaction system is 25 μL, including 12.5 μL of 2×PSR reaction buffer, 0.8 μM each of primers PaciT2-Ft and PaciT2-Bt, 2 μL of template DNA, 1 μL of Bst enzyme, and water to make up to 25 μL.

[0050] The three sets of primers were reacted at 63℃ for 60 min, and the results were verified by agarose gel electrophoresis.

[0051] Interpretation of test results: Take 5 μL of PSR reaction product and perform electrophoresis on a 2% agarose gel. If diffuse bands appear, it is judged as positive for Pediococcus lactis. If no bands appear, it is judged as negative for Pediococcus lactis.

[0052] Electrophoresis image as shown Figure 3 As shown, lanes 1-3, 4-6, and 7-9 are the PSR results of the amplification of internal standard, DNA, and ultrapure water using the first, second, and third sets of primers for PaciT2, respectively. The gel electrophoresis results of the first set of primers show relatively clear band reactions.

[0053] Finally, the primer sequences used for PSR detection of PaciT2 were determined to be:

[0054] PaciT2-Ft1: 5'-CCGAAACCGTGATGGGACCATTAAACGGCGTGGAGA-3';

[0055] PaciT2-Bt1: 5'-CAGGGTAGTGCCAAAGCCTACAGTGTTGCGGTCCTG-3'.

[0056] Example 4: Specificity evaluation of the PSR detection method for Pediococcus lactis of the present invention

[0057] The specificity evaluation of the PSR detection method for *Pediococcus lactis* of the present invention was performed using *Pediococcus lactis* and 24 non-*Pediococcus lactis* strains, namely *Lactobacillus gasseri*, *Lactobacillus brevis*, *Lactobacillus plantarum*, *Enterococcus faecalis*, *Lactobacillus brevis*, *Lactobacillus iners*, *Lactobacillus jensenii*, *Corynebacterium glutamicum*, *Bacillus subtilis*, *Staphylococcus haemolyticus*, *Vibrio parahaemolyticus*, *Listeria monocytogenes*, *Pseudomonas aeruginosa*, and *Staphylococcus aureus*. Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Salmonella enteritidis, Bacillus cereus, and Shigella flexneri.

[0058] One strain of *Pediococcus lactis* and 24 strains of non-*Pediococcus non-lactate* were inoculated into 10 mL of TSB liquid medium. After enrichment at 37°C for 12 h, 1 mL of the bacterial suspension was taken and genomic DNA was extracted using the bacterial DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0059] One μL of the extracted genomic DNA was used for a PSR reaction, under the conditions described in Example 3. The PSR product was subjected to electrophoresis, and the detection results were determined using the method for identifying *Pediococcus lactis* as described in Example 3. The electrophoresis results of the PSR products are shown in Table 3. Figure 4 As shown. Figure 4 In the table, M represents the DNA Marker of DL 1000, lane 1 is for Pediococcus lactis, and lanes 2-25 are for non-Pediococcus lactis. Specific species and strain names are shown in Table 3.

[0060] Experimental results show that the PSR detection method for Pediococcus lactis based on specific gene targets provided in Example 3 correctly detects Pediococcus lactis positive and has high sensitivity; for 23 non-Pediococcus lactis strains, this method detects them all negatively. Therefore, this method has strong specificity.

[0061] Table 3. Specificity of the PSR detection method for Pediococcus lactis

[0062]

[0063] Note: + indicates a positive result; - indicates a negative result. *Lactobacillus gasseri* Lgas230001, *Lactobacillus brevis* Lbre200001, *Enterococcus faecalis* Efae29212, *Lactobacillus curvatureii* Lcri230001, *Lactobacillus indolentii* Limne230001, *Lactobacillus jenny* Ljen230001, *Corynebacterium glutamicum* Cglu230001, *Staphylococcus hemolyticus* Shae230001, *Staphylococcus epidermidis* S.epi 6508, and *Shigella flexneri* Sfle230001 were all purchased from Guangzhou Peptide Valley Technology Co., Ltd.

[0064] Example 5: Determination of the detection limit of the PSR detection method for Pediococcus lactis

[0065] The plasmid concentration of *Pediococcus lactis* was determined using a micro-volume UV spectrophotometer, with an initial concentration of 2.778 ng / μL. Genomic DNA was serially diluted with nucleic acid-free water to obtain plasmids with concentrations ranging from 2.778 ng / μL to 0.028 fg / μL. Amplification was performed using the established and optimized PSR reaction system, and the amplification products were detected by agarose gel electrophoresis to determine successful amplification. 1 μL of plasmid from each dilution was used as template and added to the 25 μL PSR reaction system. The amplification results are attached. Figure 5 As shown; where, Figure 5The first column shows the DL 1000 DNA marker. Lanes 1-10 correspond to plasmid concentrations of the positive control, 2.778 ng / μL, 277.8 pg / μL, 27.78 pg / μL, 2.778 pg / μL, 277.8 fg / μL, 27.78 fg / μL, 2.778 fg / μL, 0.28 fg / μL, and 0.028 fg / μL, respectively. Lanes 1-6 all show clearly visible bands. Since the concentration for lane 6 should be 277.8 fg / μL, the detection limit for *Pediococcus lactis* DNA using the primer scheme WP_036685256.1 should be 277.8 fg / μL. Therefore, the PSR detection method for Pediococcus lactis based on specific gene targets provided by this invention has high sensitivity for the detection of Pediococcus lactis genomic DNA.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A set of PSR primers for identifying Pediococcus lactis based on specific gene targets, characterized in that: The PSR primers for identifying Pediococcus lactis based on specific gene targets include primers PaciT2-Ft1 and PaciT2-Bt1, whose nucleotide sequences are shown below: PaciT2-Ft1: 5'-CTAGCAACATGTAAGGGCAGCAGACGTTCGGAAGTG-3'; PaciT2-Bt1: 5'-CGGGAATGTACAACGATCTTTGACCGCATTGCTTCT-3'.

2. A kit for identifying Pediococcus lactis based on specific gene targets, characterized in that... Includes the PSR primers for identifying Pediococcus lactis based on specific gene targets as described in claim 1.

3. A detection reagent for identifying Pediococcus lactis based on specific gene targets, characterized in that... Includes the PSR primers for identifying Pediococcus lactis based on specific gene targets as described in claim 1.

4. The kit according to claim 2 or the detection reagent according to claim 3, characterized in that: In the PSR primers, the concentrations of PaciT2-Ft1 and PaciT2-Bt1 are both 0.8 μM; It also includes 2×PSR reaction buffer and Bst enzyme; The formulation of the 2×PSR reaction buffer is as follows: 40.0 mM Tris-HCl, 20.0 mM ammonium sulfate, 20.0 mM potassium chloride, 16.0 mM magnesium sulfate, 0.2% (v / v) Tween 20, 1.4 M betaine, and 10.0 mM dNTPs.

5. The application of the PSR primers for identifying Pediococcus lactis based on specific gene targets as described in claim 1, the kit for identifying Pediococcus lactis based on specific gene targets as described in claim 2, and / or the detection reagent for identifying Pediococcus lactis based on specific gene targets as described in claim 3 in the identification of Pediococcus lactis.

6. An experimental research method for identifying Pediococcus lactis for non-disease diagnosis, comprising the following steps: (1) Extract bacterial DNA from the sample to be tested as a template, and ensure that the OD of the template DNA aqueous solution is within acceptable limits. 260 / OD 280 The value is in the range of 1.8 to 2.0; (2) Polymerase helical amplification reaction was carried out by incubating in a water bath at 63℃ for 60 minutes; wherein, The polymerase helical amplification reaction system was a 25 μL reaction system: 12.5 μL of 2×PSR reaction buffer, 0.8 μM of each PSR primer, 2 μL of template DNA, 1 μL of Bst enzyme, and water to make up to 25 μL. (3) Take 5 μL of PSR reaction product and perform electrophoresis on a 2% agarose gel. If a band appears, it is judged to be positive for Pediococcus lactis. The PSR primers described are the PSR primers for identifying Pediococcus lactis based on specific gene targets as described in claim 1.

7. The application of the target PaciT2 in the identification of Pediococcus lactis, characterized in that: The nucleotide sequence of the target PaciT2 is shown in SEQ ID NO.1.

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