A method for detecting a highly corrosive pseudomonas rathonii

Abstract

The present application relates to a kind of detection methods of strong putrefaction Pseudomonas lundensis, comprising extracting nucleic acid in the sample to be measured;Then with nucleic acid as template, using specific RAA primer pair is recombined enzyme-mediated amplification, and obtains amplification product;RAA primer pair targets the aprX gene of Pseudomonas lundensis comprising single nucleotide polymorphism site associated with strong putrefaction phenotype;Finally, the amplification product is added to CRISPR / Cas detection system, incubated at constant temperature, then real-time detects its fluorescence signal;Detection system includes Cas protein, specific crRNA and fluorescent reporter probe, and crRNA can recognize single nucleotide polymorphism site associated with strong putrefaction phenotype in Pseudomonas lundensis aprX gene.The present application improves the detection accuracy, and detection process is simple to operate, and reaction system is simple, not only improves detection efficiency, also reduces detection cost.

Description

Technical Field

[0001] This invention relates to a method for detecting highly saprophytic Pseudomonas reninae, applicable to the field of biotechnology. Background Technology

[0002] *Pseudomonas lundensis* is a typical psychrophilic and highly spoilage-causing bacterium, a key species leading to dairy product spoilage. Its secreted extracellular degradative enzymes remain active even after heat processing, continuously decomposing proteins and fats in dairy products, causing quality degradation during storage, such as whey separation, fat floating, and off-flavors. Current research on *Pseudomonas lundensis* has found that its secreted thermostable protease (alkaline protease X, aprX) is a key factor in dairy product deterioration. Sequence differences in the aprX gene encoding this thermostable protease are an important intrinsic factor in the differentiation of spoilage capacity among strains. The aprX gene of highly spoilage-causing *Pseudomonas lundensis* contains specific single nucleotide polymorphism (SNP) sites, directly related to its highly spoilage phenotype. Therefore, detection of these SNP sites can effectively achieve precise screening for highly spoilage-causing *Pseudomonas lundensis*.

[0003] However, among existing conventional detection methods, the ordinary polymerase chain reaction (PCR) method can only detect the presence or absence of the aprX gene and cannot distinguish the specific SNP sites mentioned above. Since the aprX gene is ubiquitous in Pseudomonas, detection results based on the aprX gene are usually positive, limiting its application value. While real-time quantitative polymerase chain reaction (qPCR) can quantify, distinguishing single-base differences requires the design of TaqMan-MGB probes, which is not only costly but also involves stringent primer and probe design, making it difficult to apply in-situ detection. Sequencing methods offer high accuracy, but they are time-consuming and complex, making them unsuitable for rapid screening of large batches of samples. Existing isothermal amplification techniques such as recombinase-mediated isothermal amplification (RAA) or loop-mediated isothermal amplification (LAMP) are mostly species-level and cannot distinguish single-base differences. Therefore, there is a lack of existing technologies for a rapid, accurate, and low-cost detection method to screen for highly saprophytic Pseudomonas reninae. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention proposes a method for detecting highly saprophytic Pseudomonas reninsteine.

[0005] The technical solution adopted in this invention is: a method for detecting highly saprophytic Pseudomonas reninsteine, comprising: S1. Extract nucleic acid from the sample to be tested; S2. Using nucleic acid as a template, recombinase-mediated amplification was performed using specific RAA primer pairs capable of amplifying all Pseudomonas repens DNA containing the aprX gene to obtain the amplification product; the RAA primer pairs targeted the aprX gene sequence fragment of Pseudomonas repens containing a single nucleotide polymorphism site associated with a highly putrefactive phenotype. S3. Add the amplification product to the CRISPR / Cas detection system, incubate at a constant temperature, and then detect its fluorescence signal in real time. The detection system contains Cas protein, crRNA for specific detection of highly saprophytic Pseudomonas repens and a fluorescent reporter probe, and the crRNA can recognize the single nucleotide polymorphism site in the aprX gene of Pseudomonas repens that is associated with the highly saprophytic phenotype.

[0006] Furthermore, in step S2, the sequences of the RAA primer pair are shown in SEQ ID No. 1 and SEQ ID No. 2.

[0007] Further, in step S2, the amount of template added is 1~3 μL, the concentration of RAA primer pair is 10 μM, and the amount of RAA primer pair added is 1.2~2 μL.

[0008] Furthermore, in step S2, the amplification temperature for recombinase-mediated amplification is 37℃~42℃.

[0009] Further, in step S3, the sequence of crRNA is shown in SEQ ID No. 3.

[0010] Furthermore, in step S3, the Cas protein is Cas12a.

[0011] Furthermore, in step S3, the fluorescent reporter probe is a short nucleotide sequence with a fluorescent group and a quencher group labeled at both ends, respectively.

[0012] Further, in step S3, the sequence of the fluorescent reporter probe is shown in SEQ ID No. 4.

[0013] Furthermore, in step S3, the constant temperature incubation temperature is 37℃~42℃.

[0014] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: The detection method for highly saprophytic Pseudomonas reninae of this invention combines the rapid amplification capability of RAA with the single-base recognition accuracy of the CRISPR / Cas12a system, and specifically designs RAA primer pairs and crRNA. This enables rapid amplification and recognition of SNP sites in Pseudomonas reninae associated with the highly saprophytic phenotype, overcoming the problem that traditional detection methods cannot distinguish high-risk strains. Simultaneously, it allows for quantification of strains, improving detection accuracy. Furthermore, the detection process is simple to operate, with a straightforward reaction system, thus improving detection efficiency and reducing detection costs. Attached Figure Description

[0015] The following sections will describe some specific embodiments of the invention in a detailed manner, by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar components or parts. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings: Figure 1 This is an electrophoresis diagram of the RAA orthogonal experimental results in Embodiment 1 of the present invention; Figure 2 yes Figure 1 Fluorescence curves of the RAA orthogonal experiment results in the illustrated embodiment (the numbers at the end of the curves are the experiment numbers); Figure 3 yes Figure 1 The fluorescence curve of the amplification product detected by CRISPR / Cas at 37°C in the example shown (the letters at the end of the curve are the strain sample numbers). Figure 4 yes Figure 1 The fluorescence curve of the amplification product detected by CRISPR / Cas at 39°C in the example shown (the letters at the end of the curve are the strain sample numbers). Figure 5 yes Figure 1 The fluorescence curve of the amplification product detected by CRISPR / Cas at 42°C in the example shown (the letters at the end of the curve are the strain sample numbers). Figure 6 This is a bar chart of fluorescence values ​​at various dilutions in Embodiment 2 of the present invention; Figure 7 yes Figure 6 The dilution standard curves shown in the examples are as follows; Figure 8 This is a bar chart of fluorescence values ​​at various dilutions in Embodiment 3 of the present invention; Figure 9 yes Figure 8 The dilution standard curves shown in the examples are as follows; Figure 10 yes Figure 1The phylogenetic tree diagram of four representative strains of Pseudomonas reninae carrying the aprX gene in the illustrated example. Detailed Implementation

[0016] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0018] Example 1 This embodiment provides a method for detecting highly saprophytic Pseudomonas reninskii, including: S1. Extract nucleic acid from the sample to be tested; S2. Using nucleic acid as a template, recombinase-mediated amplification was performed using specific RAA primer pairs capable of amplifying all Pseudomonas repens DNA containing the aprX gene to obtain the amplified product. The amount of template added was 2 μL, the concentration of the RAA primer pair was 10 μM, and the amount of RAA primer pair added was 1.2 μL. The amplification temperature was 39℃. The RAA primer pair targeted the aprX gene of Pseudomonas repens containing a single nucleotide polymorphism site associated with a highly putrefactive phenotype. The sequences of the RAA primer pair are shown in SEQ ID No. 1 and SEQ ID No. 2.

[0019] Specifically, the RAA primer pair design and amplification condition optimization method in step S2 is as follows: Multiple RAA primer pairs are designed targeting the conserved region of the *Pseudomonas reninae* aprX gene that contains the SNP site associated with the highly detrimental phenotype. The primer design region is selected from highly conserved sequences flanking the SNP site associated with the highly detrimental phenotype, ensuring that the amplification product contains the SNP site associated with the highly detrimental phenotype, but the amplification is not dependent on the SNP site. This ensures that RAA amplification can cover all *Pseudomonas reninae* strains containing the aprX gene. A candidate primer pair is initially screened for orthogonal optimization. Then, a three-factor, three-level orthogonal design is used to optimize the RAA reaction conditions. The specific three factors and their levels are as follows: Factor A: Amount of nucleic acid template added; Levels: 1 μL, 2 μL, 3 μL; Factor B: Primer addition amount (concentration 10 μM); levels: 1.2 μL, 1.6 μL, 2.0 μL; Factor C: Amplification temperature; Levels: 37℃, 39℃, 42℃.

[0020] L9 (3) 3Nine experiments were conducted using an orthogonal array, with genomic DNA from *Pseudomonas reninae* carrying SNPs associated with a highly putrefactive phenotype as templates, and three replicates per group. A commercially available basic RAA nucleic acid amplification kit was used. The total volume of the basic reaction system was 50 μL, including 25 μL buffer, 5 μL 280 mM magnesium acetate, primer and nucleic acid template amounts adjusted according to the orthogonal design, and the remainder ddH2O. The reaction time was 30 min. The specific orthogonal experimental design is shown in the table below. From each of the above 9 experimental groups, 5 μL of amplified product was subjected to 3% agarose gel electrophoresis to observe the clarity and brightness of the target bands and to perform semi-quantitative scoring. Then, 2 μL of the amplified product was detected using the CRISPR / Cas detection system in step S3, and the endpoint fluorescence value was recorded, with the average of three replicates taken. (Reference) Figure 1 , Figure 2 The experimental results showed that under the conditions of the fourth group of experiments, the electrophoretic bands were clear and bright, and the fluorescence value detected by Cas was the highest. This indicates that the optimal RAA reaction conditions are: nucleic acid template addition of 2 μL, primer addition of 1.2 μL, and amplification temperature of 39℃. The sequences of the final RAA primer pairs are shown in SEQ ID No. 1 and SEQ ID No. 2.

[0021] S3. The amplification product was added to the CRISPR / Cas detection system and incubated at an incubator, and then its fluorescence signal was detected in real time. The detection system contained Cas12a protein, crRNA for specific detection of highly putrefactive Pseudomonas reninae, and a fluorescent reporter probe. The crRNA could recognize the single nucleotide polymorphism site in the aprX gene of Pseudomonas reninae that was associated with the highly putrefactive phenotype. The sequence of the crRNA is shown in SEQ ID No. 3. The fluorescent reporter probe was a short nucleotide sequence with fluorescent and quenching groups labeled at both ends, and the sequence of the fluorescent reporter probe is shown in SEQ ID No. 4. The incubation temperature was 42℃.

[0022] Specifically, the establishment and optimization method of the CRISPR / Cas detection system in step S3 is as follows: using the universal crRNA backbone sequence: 5'-UAAUUUCUACUAAGUGUAGAU-3', followed by a guide sequence complementary to the target sequence. SNP site discovery involved whole-genome sequencing of broadly screened *Pseudomonas repens* isolates, identifying four classes of *Pseudomonas repens* carrying the aprX gene. Representative strains from these four classes were selected, including one highly saprophytic strain (numbered TA0410) and three moderately saprophytic strains (numbered LY0405, JN0201, and LY0708). Sequence alignment of the aprX gene in each of the four representative strains was performed, and phylogenetic trees were constructed. Figure 10As shown in the table below, the aprX sequences of strains TA0410, JN0201, and LY0405 have extremely high similarity. The crRNA was designed based on the differences in SNP sites among these three bacteria. Referring to the PAM site requirements for Cas12a protein recognition, SNP site 336 among the three bacteria was selected as the distinguishing target, resulting in the crRNA sequence shown in SEQ ID No. 3. The total volume of the CRISPR / Cas detection system was 20 μL, including 100 nM Cas12a protein, 100 nM crRNA, 1 mM fluorescent reporter probe, 2 μL 10× reaction buffer, 2 μL RAA amplification product, and the remainder being ddH2O.

[0023] Genomic DNA from the four strains was collected as templates to verify the method's ability to distinguish between highly pathogenic Pseudomonas spp. The four templates were amplified using the RAA primer pair and optimal reaction conditions in step S2. The four amplification products were then mixed with the CRISPR / Cas detection system described above. Each mixture was incubated at 37℃, 39℃, and 42℃ for 30 minutes, respectively. Finally, fluorescence signals were collected every 30 seconds using a real-time PCR instrument.

[0024] Reference Appendix Figure 3-5 The experimental results showed that at 37℃, strains TA0410, LY0405, and LY0708 all produced strong fluorescence signals with little difference in intensity. However, strain TA0410 had the highest fluorescence value, reaching 250,000 RFU at the endpoint. At 39℃, strain TA0410 maintained a high level of fluorescence signal, with an endpoint fluorescence value exceeding 170,000 RFU, while the fluorescence signals of the other strains decreased significantly, resulting in a significant increase in distinguishability. At 42℃, strain TA0410 produced a strong fluorescence signal, with an endpoint fluorescence value exceeding 500,000 RFU, while the other strains showed no obvious fluorescence signal, with endpoint fluorescence values ​​below 200 RFU. The results showed that the optimal reaction temperature of the CRISPR / Cas detection system was 42℃. Under this condition, the method could accurately distinguish whether Pseudomonas reninae carried SNP sites associated with the highly putrefactive phenotype. This also demonstrated the effectiveness of the method in detecting Pseudomonas reninae carrying SNP sites associated with the highly putrefactive phenotype. The method can accurately distinguish between highly similar sequences by targeting the differentially expressed SNP sites of highly putrefactive bacteria.

[0025] Example 2 In this embodiment, a pair of conventional PCR primers were designed targeting the aprX gene region containing the SNP site associated with the highly detrimental phenotype. Their nucleotide sequences are shown in SEQ ID No. 5 and SEQ ID No. 6. Genomic DNA of a highly detrimental *Pseudomonas repens* strain carrying the SNP site was used as a template for PCR amplification. The total PCR reaction volume was 50 μL, including: 25 μL 2×Taq Master Mix, 2 μL each of forward and reverse PCR primers (10 μM), 2 μL template DNA, and the remainder ddH2O. The reaction conditions were: pre-denaturation at 95℃ for 5 minutes, followed by a cycle of denaturation at 95℃ for 30 seconds, annealing at 58℃ for 30 seconds, and extension at 72℃ for 30 seconds, for a total of 35 cycles. The final cycle included a 5-minute extension at 72℃. After electrophoresis on a 1.5% agarose gel, the target band was excised and purified using a gel extraction kit. By amplifying and purifying the strain DNA using conventional PCR, the instability and inaccurate concentration issues associated with directly using strain DNA are avoided, thus providing a stable template containing well-defined, pure, and sequenced SNP sites related to the highly corrosive phenotype.

[0026] The purified PCR product was ligated to the T vector. The ligation system consisted of 5 μL Solution I, 1 μL T vector, and 4 μL purified PCR product, and ligation was performed at 16°C for 30 minutes. The ligated product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing ampicillin, incubated overnight at 37°C. Single colonies were picked for colony PCR identification. Positive clones were inoculated into LB liquid medium for expansion culture. Recombinant plasmids were extracted using a plasmid extraction kit, and the constructed recombinant plasmids were confirmed by sequencing to ensure the presence of SNPs associated with a strong putrefactive phenotype. The stability, precise concentration, and accurate copy number conversion of the plasmid facilitate subsequent sensitivity assays and ensure the reliability of sensitivity and standard curve results.

[0027] The recombinant plasmid was serially diluted 10-fold to obtain 10 1 Copy / μL to 10 8 Eight sets of standards were prepared, with a copy number / μL. The formula for converting recombinant plasmid concentration to copy number is: Copy number (copies / μL) = [plasmid concentration (ng / μL) × 6.02 × 10⁻⁶] 23 The plasmid length (bp) × 660 was calculated, and then, using each dilution plasmid as a template, RAA amplification and CRISPR / Cas detection were performed using the detection method of Example 1. Each dilution was repeated in triplicate, and a template-free control was also included. The fluorescence curves of each reaction were recorded, and a standard curve was plotted using the fluorescence value after 20 minutes of CRISPR rapid detection and the logarithm of the corresponding plasmid copy number.

[0028] refer to Figure 6 , Figure 7 Experimental results showed that when the template concentration exceeded 10... 7 After copy / μL, the fluorescence values ​​of the RAA process products reached their peak values ​​during the CRISPR detection stage, making quantification impossible. The limit of detection was 10. 3 copies / μL, quantitative range of 10 3 ~10 7 The fluorescence value showed a good linear relationship with the logarithm of the copy number (copy / μL), R 2 >0.99, the standard curve equation is fluorescence value = 19788 × lg (copy number) + 28007. This example clarifies the theoretical sensitivity and quantitative range of this method using recombinant plasmid standards, demonstrating that this method has good amplification efficiency and quantitative performance, and can rapidly and accurately screen for highly pathogenic Pseudomonas humicis.

[0029] Example 3 In this embodiment, highly pathogenic *Pseudomonas ginseng* was artificially spiked onto sterile UHT-sterilized milk to obtain 10... 2 CFU / mL to 10 8 Seven groups of highly pathogenic *Pseudomonas hunzans*-spikeped milk were prepared. 100 mL of spiked milk was centrifuged at 12000 g for 15 min at 4 °C, the supernatant was discarded, and the precipitate was retained. The precipitate was washed four times with 30 mL of TE buffer, and bacterial DNA was extracted using a kit suitable for extracting bacterial DNA from complex matrices. The extracted milk bacterial DNA was used as a template. Each template group was then detected according to the detection method in Example 1, with three replicates per group and a template-free control. Fluorescence curves for each reaction were recorded, and a standard curve was plotted using the fluorescence value after 20 minutes of CRISPR rapid detection and the logarithm of the corresponding bacterial concentration.

[0030] refer to Figure 8 , Figure 9 Experimental results show that, using actual milk spiked for detection, the limit of detection for this method is 10. 2 CFU / mL, quantification range in 10 2 ~10 8 Within the CFU / mL range, the fluorescence value showed a good linear relationship with the logarithm of the bacterial concentration, R 2 >0.99, the standard curve equation is fluorescence value = 19788 × lg (copy number) + 28007. In actual raw milk, the concentration of Pseudomonas is generally around 10. 2 ~10 3Raw milk with a higher concentration of CFU / mL generally exhibits obvious spoilage characteristics, which can be detected without testing. This embodiment further verifies the applicability and actual detection limit of this method in complex samples through a milk matrix spiked experiment, proving that this method meets the early detection requirements of highly putrefactive Pseudomonas renensis in dairy products and has practical application value.

[0031] The nucleotide sequences of the specific primers and probes are shown in the table below: Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: The detection method for highly saprophytic Pseudomonas reninae of this invention combines the rapid amplification capability of RAA with the single-base recognition accuracy of the CRISPR / Cas12a system, and specifically designs RAA primer pairs and crRNA. This enables rapid amplification and recognition of SNP sites in Pseudomonas reninae associated with the highly saprophytic phenotype, overcoming the problem that traditional detection methods cannot distinguish high-risk strains. Simultaneously, it allows for quantification of strains, improving detection accuracy. Furthermore, the detection process is simple to operate, with a straightforward reaction system, thus improving detection efficiency and reducing detection costs.

[0032] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting highly saprophytic Pseudomonas reninsteine, characterized in that, include: S1. Extract nucleic acid from the sample to be tested; S2. Using the nucleic acid as a template, recombinase-mediated amplification is performed using a specific RAA primer pair capable of amplifying all Pseudomonas repens DNA containing the aprX gene to obtain the amplification product; the RAA primer pair targets the aprX gene sequence fragment of Pseudomonas repens containing a single nucleotide polymorphism site associated with a strong putrefactive phenotype. S3. Add the amplification product to the CRISPR / Cas detection system, incubate at a constant temperature, and then detect its fluorescence signal in real time. The detection system includes a Cas protein, a crRNA for specific detection of highly saprophytic Pseudomonas reninae, and a fluorescent reporter probe. The crRNA can recognize single nucleotide polymorphism sites in the aprX gene of Pseudomonas reninae that are associated with the highly saprophytic phenotype.

2. The method for detecting highly saprophytic Pseudomonas reninskii according to claim 1, characterized in that: In step S2, the sequences of the RAA primer pair are shown in SEQ ID No. 1 and SEQ ID No.

2.

3. The method for detecting highly saprophytic Pseudomonas reninskii according to claim 1, characterized in that: In step S2, the amount of template added is 1~3 μL, the concentration of the RAA primer pair is 10 μM, and the amount of RAA primer pair added is 1.2~2 μL.

4. The method for detecting highly saprophytic Pseudomonas reninsteine ​​according to claim 1, characterized in that: In step S2, the amplification temperature for the recombinase-mediated amplification is 37℃~42℃.

5. The method for detecting highly saprophytic Pseudomonas reninskii according to claim 1, characterized in that: In step S3, the sequence of the crRNA is shown in SEQ ID No.

3.

6. The method for detecting highly saprophytic Pseudomonas reninskii according to claim 1, characterized in that: In step S3, the Cas protein is Cas12a.

7. The method for detecting highly saprophytic Pseudomonas reninskii according to claim 1, characterized in that: In step S3, the fluorescent reporter probe is a short nucleotide sequence with fluorescent and quenching groups labeled at both ends.

8. The method for detecting highly saprophytic Pseudomonas reninskii according to claim 1, characterized in that: In step S3, the sequence of the fluorescent reporter probe is shown in SEQ ID No.

4.

9. The method for detecting highly saprophytic Pseudomonas reninskii according to claim 1, characterized in that: In step S3, the temperature for constant temperature incubation is 37℃~42℃.

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