Klebsiella pneumoniae based on CRISPR / cas12a and detection of ethanol dehydrogenase gene of Klebsiella pneumoniae
By combining PCR amplification and fluorescence detection with a CRISPR/Cas12a kit, the problems of long detection time and high cost of Klebsiella pneumoniae were solved, enabling rapid and accurate detection of Klebsiella pneumoniae and its alcohol dehydrogenase gene.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CAPITAL INST OF PEDIATRICS
- Filing Date
- 2026-01-27
- Publication Date
- 2026-05-12
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to a method for detecting Klebsiella pneumoniae and its alcohol dehydrogenase gene based on CRISPR / cas12a. Background Technology
[0002] Klebsiella pneumoniae is an opportunistically pathogenic Gram-negative Enterobacteriaceae bacterium. High-alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) in the intestinal flora is a significant contributing factor to non-alcoholic fatty liver disease (NAFLD). NAFLD is a precursor to cirrhosis and hepatocellular carcinoma, the most common chronic liver disease worldwide. Alcohol dehydrogenase (ADH) is a crucial enzyme in Klebsiella pneumoniae that controls ethanol synthesis via the 2,3-butanediol pathway. Identifying and eliminating the source of Klebsiella pneumoniae infection can effectively prevent bacterial infection, reduce the incidence of NAFLD, and lower the prevalence of hepatitis and cirrhosis.
[0003] Identifying Klebsiella pneumoniae and its alcohol dehydrogenase gene at the species level is crucial for patients with non-alcoholic fatty liver disease. Currently, commonly used detection methods for Klebsiella pneumoniae include culture-based matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDITOF MS) and 16S rDNA sequencing. Traditional culture methods are time-consuming, and detection typically relies on expensive, sophisticated instruments. Therefore, the need for developing a rapid and accurate detection platform for Klebsiella pneumoniae and its alcohol dehydrogenase gene is growing.
[0004] The acquired immune system of bacteria and archaea consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), enabling highly sensitive and specific defense against the invasion of exogenous nucleic acids. Cas proteins include cas12a, cas12b, cas13a, cas13b, and cas14. The CRISPR-Cas system comprises CRISPR RNA (crRNA) that recognizes and degrades exogenous nucleic acids and Cas proteins that recognize and cis-cleave target DNA or RNA. Cas proteins can be indiscriminately activated to trans-cleave non-target single-stranded DNA (ssDNA). Non-target ssDNA is labeled with fluorophores and catalytic agents, respectively, to produce corresponding fluorescent signals after side branch cleavage. The intensity of the fluorescence signal is then measured using a fluorometer for nucleic acid detection. Summary of the Invention
[0005] The technical problem solved by this invention is to provide a detection method for Klebsiella pneumoniae and its alcohol dehydrogenase gene based on CRISPR / cas12a.
[0006] To address the aforementioned technical problems, the first aspect of this invention provides a kit for simultaneously detecting Klebsiella pneumoniae and its alcohol dehydrogenase gene adh, comprising the following A1) or A2): A1) The crRNA that binds to gene rcsA, the crRNA that binds to gene adh, and Cas12a protein; A2) The CRISPR-Cas12a / crRNA complex that binds to the gene rcsA and the CRISPR-Cas12a / crRNA complex that binds to the gene adh. The nucleotide sequence of the crRNA that binds to gene rcsA is sequence 3; The nucleotide sequence of the crRNA that binds to the adh gene is sequence 6. The CRISPR-Cas12a / crRNA complex that binds the rcsA gene includes the crRNA that binds the rcsA gene and the Cas12a protein. The CRISPR-Cas12a / crRNA complex that binds the adh gene comprises the crRNA that binds the adh gene and the Cas12a protein.
[0007] The kit described above also includes primer sets for amplifying the rcsA gene and primer sets for amplifying the adh gene.
[0008] In the kit described above, the primer set for amplifying the gene rcsA consists of single-stranded DNA molecules as shown in Sequence 1 and single-stranded DNA molecules as shown in Sequence 2; The primer set for amplifying the adh gene consists of single-stranded DNA molecules as shown in sequence 4 and single-stranded DNA molecules as shown in sequence 5.
[0009] The kit described above also includes an ssDNA probe.
[0010] In the kit described above, the nucleotide sequence of the ssDNA probe is sequence 7.
[0011] In the kit described above, the ssDNA probe is labeled with a fluorescent group and a quencher group at both ends.
[0012] In the kit described above, the Cas12a protein is EnGen® Lba Cas12a.
[0013] In a second aspect, the present invention provides the application of the reagent kit described in the first aspect in the preparation of products having any of the following functions: B1) Detect whether the test strain is Klebsiella pneumoniae and also contains the adh gene; B2) Detect whether the test strain is an ethanol-producing Klebsiella pneumoniae.
[0014] The applications include the following: C1) Extract genomic DNA from the test strain and perform PCR amplification using the primer set for amplifying gene rcsA and the primer set for amplifying gene adh as described in the first aspect, respectively, to obtain the PCR product of gene rcsA and the PCR product of gene adh. C2) Construct a CRISPR-Cas12a system using the PCR product of the gene rcsA or the PCR product of the gene adh, respectively, and react to obtain the reaction product of gene rcsA and the reaction product of gene adh. The CRISPR-Cas12a system includes the CRISPR-Cas12a / crRNA complex that binds to the rcsA gene in the first aspect, the PCR product of the rcsA gene, and the ssDNA probe. Alternatively, the CRISPR-Cas12a system may include the CRISPR-Cas12a / crRNA complex that binds to the gene adh in the first aspect, the PCR product of the gene adh, and the ssDNA probe. The fluorescence intensity of the rcsA reaction product and the adh reaction product of the gene is detected to determine whether the test strain is Klebsiella pneumoniae and contains the adh gene.
[0015] In the above text, if the fluorescence intensity of the rcsA reaction product of the gene is greater than or equal to that of the negative control, and the fluorescence intensity of the adh reaction product of the gene is greater than or equal to that of the negative control, then the test strain is or is a candidate for Klebsiella pneumoniae and simultaneously contains the adh gene. If the fluorescence intensity of the rcsA reaction product is less than that of the negative control, and the fluorescence intensity of the adh reaction product is less than that of the negative control, then the test strain is not or is not a candidate Klebsiella pneumoniae and simultaneously contains the adh gene.
[0016] In the above text, the negative control is water.
[0017] In the above text, the CRISPR-Cas12a / crRNA complex is the product obtained by incubating the Cas12a protein and the crRNA in a buffer solution.
[0018] In the above text, the annealing temperature for PCR amplification of the primer set for amplifying the adh gene is 59℃; In the above text, the annealing temperature for PCR amplification of the primer set for the amplified gene rcsA was 57℃.
[0019] In the above text, the concentration of the crRNA that binds to the rcsA gene in its corresponding CRISPR-Cas 12a system is 100 nM; In the above text, the concentration of the crRNA that binds to the adh gene in its corresponding CRISPR-Cas 12a system is 140 nM.
[0020] In the above text, gene rcsA: Gene ID: 11848468, updated on October 6, 2023; gene adh: Gene ID: 11847576, updated on 4-Feb-2024.
[0021] This invention develops a method for combining PCR amplification and CRISPR. A detection technology called "CRISPR-PCR," combining Cas12a fluorescence detection, was developed for the simultaneous detection of Klebsiella pneumoniae and ethanol-producing genes. Fluorescent signals are generated using the principle of cleaving non-target ssDNA for nucleic acid detection. The specific and conserved capsular polysaccharide synthesis regulator rcsA was selected as the target sequence for identifying Klebsiella pneumoniae, and the alcohol dehydrogenase gene adh was selected as the target sequence for identifying ethanol production. The platform's detection conditions were optimized to determine its sensitivity and specificity. A CRISPR / Cas12a-based dual-target detection kit was developed, achieving highly sensitive, highly specific, and rapid identification of Klebsiella pneumoniae and its ethanol-producing ability by simultaneously targeting the rcsA and adh genes. Attached Figure Description
[0022] Figure 1 This is a schematic diagram illustrating the workflow and principle of the CRISPR-PCR fluorescence detection system.
[0023] Figure 2 Screening for the optimal annealing temperature for PCR amplification.
[0024] Figure 3 CRISPR for Klebsiella pneumoniae Sensitivity of PCR fluorescence detection.
[0025] Figure 4 This study aimed to improve the specificity of CRISPR-PCR fluorescence detection of the Klebsiella pneumoniae gene rcsA.
[0026] Figure 5 This study aimed to improve the specificity of CRISPR-PCR fluorescence detection of the Klebsiella pneumoniae gene adh. Detailed Implementation
[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0029] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.
[0030] Table 1 shows the primers and crRNA sequences for Klebsiella pneumoniae.
[0031] Table 2 lists the bacterial strains used in the experiment.
[0032] Example 1: CRISPR-PCR Detection Method I. Materials used in the test and the testing principle 1. Materials High-ethanol-producing Klebsiella pneumoniae (HiAlc) Kpn W14 (described in the following literature: Yuan, J.; Chen, C.; Cui, J.; Lu, J.; Yan, C.; Wei, X., Zhao X.; Li N.; Li S.; Xue G.; et al. Fatty liver disease caused by high-alcohol-producing Klebsiella pneumoniae. Cell. Metab. 2019, 30, 675-688.) contains the gene. oh The strain (named W14 in the literature) was the experimental strain used in this study. This strain was initially stored at -80°C in a cryopreservation solution containing 20% glycerol. It was streaked in MacConkey containers and incubated overnight at 37°C. Single colonies were resuspended in LB broth. It was then incubated overnight at 37°C with shaking at 200 rpm. Next, it was resuspended in fresh LB broth at a 1:100 volume ratio and incubated at 37°C with shaking at 200 rpm until OD600 = 1.0 (bacterial concentration approximately 1 × 10⁻⁶). 9 (CFU / mL) to obtain bacterial suspension.
[0033] Take 1 mL of bacterial culture and isolate genomic DNA from it using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). Add 200 μL of DNA preservation solution and incubate overnight at 4°C. Then, quantify the purified DNA using a Nano Drop spectrophotometer. Store the DNA sample at -20°C.
[0034] 2. Principle Two-step method to combine PCR amplification and CRISPR Combining Cas12a fluorescence detection with a novel approach, named "CRISPR-PCR," the primers and probes are based on the capsular polysaccharide synthesis regulators of Klebsiella pneumoniae. rcsA and alcohol dehydrogenase gene oh Designed (Table 1). Gene rcsA: Gene ID: 11848468, updated on October 6, 2023; Gene adh: Gene ID: 11847576, updated on 4-Feb-2024.
[0035] Figure 1 This is a schematic diagram illustrating the workflow and principle of the CRISPR-PCR fluorescence detection system. The CRISPR-PCR fluorescence detection process includes three steps: genome extraction, PCR amplification of the target fragment, and CRISPR fluorescence detection. The principle of CRISPR-PCR fluorescence detection is as follows: First, the target DNA fragment is amplified by PCR; then, the Cas12a protein binds to crRNA to form a CRISPR-Cas12a / crRNA complex. Guided by crRNA, the Cas12a protein binds to the target double-stranded DNA, activating its trans-cleavage activity and indiscriminately cleaving the single-stranded DNA probe. The lysed probe emits fluorescence, which can be detected using a real-time quantitative PCR instrument.
[0036] II. Establishment of CRISPR-PCR Detection Method 1. PCR amplification To each rcsA and genealogy Perform amplification: Using the genomic DNA of the sample to be tested as a template, as shown in Table 1 rcsA Forward primer and rcsA PCR amplification was performed using reverse primers to obtain... rcsA Amplification products; Using the genomic DNA of the sample to be tested as a template, as shown in Table 1 oh Forward primer and oh PCR amplification was performed using reverse primers to obtain... oh Amplification products.
[0037] The samples to be tested were Klebsiella pneumoniae or its cell suspension.
[0038] The total volume of each of the above PCR amplification reactions is 25 µL, containing 12.5 µL of prex Ex Taq (Probe qPCR, TaKaRa (Dalian, China) PrimeSTAR). ® The following materials were prepared: HS (Premix) Code No. R040A, 10 μL deionized water, 0.5 μL forward primer (20 nM), 0.5 μL reverse primer (20 nM), and 1.5 μL template DNA. PCR detection was performed using a Bio-Rad CFX96 real-time PCR system.
[0039] The thermal cycling conditions were as follows: initial incubation at 95°C for 3 min, followed by alternating incubation at 95°C, 57°C / 59°C for 30 s, 30 s, and 30 s for 35 cycles. A further incubation at 72°C for 7 min was performed to complete the extension step.
[0040] The annealing temperature for amplifying the above gene rcsA is 57℃; The above genes oh The amplification annealing reaction temperature was 59℃.
[0041] 2. CRISPR Cas12a reaction Fluorescent signals are generated by cleaving non-target ssDNA for nucleic acid detection.
[0042] CRISPR-Cas12a / crRNA complex preparation method: As shown in Table 1... rcsA crRNA sequence or oh The crRNA sequence and Cas12a (Cas12a protein EnGen® Lba Cas12a) were dissolved in NEBuffer 2.1 (newengland biolabs product catalog number: NEBuffer™ r2.1 B6002SVIAL) to obtain a mixture, with each crRNA at a concentration of 100 nM and the Cas12a at a concentration of 75 nM; the mixture was incubated at 37°C for 10 min to obtain the reaction product, denoted as CRISPR-Cas12a / rcsA crRNA complex (including) rcsA crRNA and Cas12a) and CRISPR-Cas12a / oh crRNA complex (including) oh crRNA and Cas12a).
[0043] 100 μL detection rcsA The CRISPR-Cas12a reaction system includes: 50 μL 2 × NEBuffer 2.1, 18 μL CRISPR-Cas12a / rcsA crRNA complex, 2 μL rcsA Amplification product, 27.5 μL of distilled water, and 2.0 μL of the ssDNA probe (200 nM) shown in Table 1.
[0044] 100 μL detection oh The CRISPR-Cas12a reaction system includes: 50 μL 2 × NEBuffer 2.1, 18 μL CRISPR-Cas12a / oh crRNA complex, 2 μL oh Amplification product, 27.5 μL of distilled water, and 2.5 μL of the ssDNA probe (250 nM) shown in Table 1.
[0045] The above CRISPR-Cas 12a reaction system was incubated at 37°C for 60 minutes to obtain the CRISPR-Cas 12a reaction product.
[0046] Genes were detected through calculation. rcsA The concentration of crRNA in the corresponding CRISPR-Cas 12a reaction system was 100 nM.
[0047] Genes were detected through calculation. oh The concentration of crRNA in the corresponding CRISPR-Cas 12a reaction system was 140 nM.
[0048] Fluorescence was detected for 60 minutes during incubation using a QuantStudio 7 Flex (Applied Biosystems) instrument. The temperature was maintained at 37°C for 60 cycles, with fluorescence reads taken once per cycle. Fluorescence was measured every minute, and the final determination of whether a sample was the target sample was based on its fluorescence intensity.
[0049] If detection rcsA The fluorescence intensity of the CRISPR-Cas 12a reaction product was greater than or equal to that of its corresponding negative control, and the detection... oh If the CRISPR-Cas 12a reaction product is greater than or equal to its corresponding negative control, then the test sample is or is a candidate for Klebsiella pneumoniae and also contains the gene adh. If detection rcsA The fluorescence intensity of the CRISPR-Cas 12a reaction product was lower than that of its corresponding negative control, and the detection... ohIf the CRISPR-Cas 12a reaction product of the amplification product is smaller than its corresponding negative control, then the test sample is not or is not a candidate for Klebsiella pneumoniae and also contains the adh gene.
[0050] The corresponding negative control is achieved by replacing the amplification product in the corresponding system with DW (deionized water).
[0051] Example 2: Optimization of CRISPR-PCR Detection Method for Klebsiella pneumoniae I. Optimization of PCR reaction temperature 1. PCR amplification High-ethanol-producing Klebsiella pneumoniae (HiAlc) Kpn Using W14 genomic DNA as a template, PCR amplification was performed according to the method in Example 1, except that the 57℃ / 59℃ thermal cycling conditions were replaced with any one of the different temperature gradients of 55-60℃, with the temperature of each gradient increasing by 1℃.
[0052] 2. CRISPR Cas12a reaction The amplification products obtained at different annealing temperatures were subjected to CRISPR according to the method in Example 1, Part 2. Cas12a reaction.
[0053] Results of reaction product detection are as follows Figure 2 As shown, it can be seen that, targeting genes oh The optimal annealing temperature for PCR amplification is 59℃, and the optimal annealing temperature for gene rcsA amplification is 57℃. The reaction time is fastest and the fluorescence value is relatively high under the optimal reaction temperature conditions.
[0054] II. Optimization of ssDNA probe concentration 1. PCR amplification High-ethanol-producing Klebsiella pneumoniae (HiAlc) Kpn Using W14 genomic DNA as a template, PCR amplification was performed according to the method in Example 1, Section 2, to obtain the gene. oh PCR amplification products of the gene and PCR amplification products of the rcsA gene. 2. CRISPR Cas12a reaction Genes were processed according to the method in Example 1, Part 2. oh The PCR amplification products of the PCR amplification products of the rcsA gene were subjected to CRISPR. The only difference in the CRISPR-Cas 12a reaction was the volume of the ssDNA probe in the CRISPR-Cas 12a reaction system for detecting different genes: 0.5 μL, 0.8 μL, 1 μL, 1.5 μL, 2 μL, 2.5 μL, and 3 μL, corresponding to final concentrations of 50 nM, 80 nM, 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM, respectively.
[0055] Klebsiella pneumoniae W14 was used as a positive template, and DW (deionized water) was used as a negative control.
[0056] The results of the detection reaction products are as follows: 2.0 μL (final concentration 200 nM) of ssDNA fluorescent probe showed the best detection effect on gene rcsA, and 2.5 μL (final concentration 250 nM) of ssDNA fluorescent probe showed the best detection effect on gene adh.
[0057] III. Optimization of crRNA Concentration 1. PCR amplification High-ethanol-producing Klebsiella pneumoniae (HiAlc) Kpn Using W14 genomic DNA as a template, PCR amplification was performed according to the method in Example 1, Part 2, to obtain the gene. oh PCR amplification products of the gene and PCR amplification products of the rcsA gene. 2. CRISPR Cas12a reaction Genes were processed according to the method in Example 1, Part 2. oh The PCR amplification products of the PCR amplification products of the rcsA gene were subjected to CRISPR. The only difference in the CRISPR-Cas12a reaction was the volume of crRNA used in the preparation of the CRISPR-Cas12a / crRNA complex: 0.5 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, and 9 μL. The calculations yielded the crRNA volume in the CRISPR reaction. The final concentrations of crRNA in the Cas12a reaction system were 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM, 120 nM, 140 nM, 160 nM, and 180 nM. The crRNA concentration corresponding to the optimal fluorescence intensity was then determined.
[0058] Klebsiella pneumoniae W14 was used as a positive template, and DW was used as a negative control.
[0059] The results of the reaction product detection are as follows: The optimal crRNA concentrations for gene adh and gene rcsA were determined to be 7 μL (final concentration 140 nM) and 5 μL (final concentration 100 nM), respectively.
[0060] Example 3: Sensitivity and specificity of the CRISPR-PCR detection method for Klebsiella pneumoniae I. Sensitivity Testing To evaluate the sensitivity of CRISPR-PCR detection of Klebsiella pneumoniae, high-ethanol-producing Klebsiella pneumoniae (HiAlc) was used in a 10-fold serial dilution. Kpn Genomic DNA from W14 bacterial culture was used as a template for PCR amplification.
[0061] The initial concentration of extracted DNA was determined by controlling the optical density of high-ethanol-producing Klebsiella pneumoniae cultured in LB medium at OD. 600 =1.0 [approximately 1 × 10] 9 The bacterial concentration corresponding to [(CFU) / mL].
[0062] The bacterial concentration was determined to be 1 × 10⁻⁶ according to the method in Example 1, Section 2. 9 CFU / mL - 1 CFU / mL for each bacterium.
[0063] The results are as follows Figure 3 As shown, it can be seen that the gene adh has no detection limit, while the gene rcsA In a fluorescently detectable DNA template, the LOD is 10. 4 The CFU / mL result was consistent with the agarose gel electrophoresis results.
[0064] II. Specific Detection To verify the specificity of CRISPR-PCR detection of Klebsiella pneumoniae, genomic DNA was extracted from 60 clinical isolates of Klebsiella pneumoniae and 34 non-Klebsiella pneumoniae isolates (Table 2) using the Wizard Genomic DNA Purification Kit. Klebsiella pneumoniae w14 was used as the positive control (PC), and DW was used as the negative control (NC).
[0065] The expression levels of genes rcsA and adh in the genomic DNA of the above-mentioned strains were detected according to the method in Example 1, Part 2.
[0066] The results of rcsA gene detection of 60 strains of Klebsiella pneumoniae are as follows: Figure 4 The figure above shows 60 clinical strains of Klebsiella pneumoniae, with 1-60 representing the total number of strains. The vertical axis represents the change in fluorescence value after 60 minutes of reaction at 37°C in a real-time quantitative PCR instrument. NC is the negative control (top). The clinical strains of Klebsiella pneumoniae were positive (n=60).
[0067] The rcsA gene detection results of 60 clinical isolates of Klebsiella pneumoniae and 34 non-Klebsiella pneumoniae were analyzed. Figure 4As shown in the left figure below, the template results for 34 clinical isolates of non-Klebsiella pneumoniae were negative (Negative, n=34), while the template results for 60 clinical isolates of Klebsiella pneumoniae were positive (Positeve, n=60). An unpaired t-test was used to analyze the differences with the NC (Negative, Positeve, n=60). p<0.0001 (bottom left). This indicates that the detected gene rcsA was positive in all clinical isolates of Klebsiella pneumoniae and negative in non-Klebsiella pneumoniae strains, and the difference between positive and negative fluorescence values was statistically significant.
[0068] Box plot of rcsA gene detection results from 34 clinical isolates of non-Klebsiella pneumoniae is shown below. Figure 4 The right image below shows the negative fluorescence of 34 clinical isolates of non-Klebsiella pneumoniae under blue light.
[0069] The results of gene ADH detection for 60 strains of Klebsiella pneumoniae are as follows: Figure 5 As shown in the figure above, 1-60 represent 60 clinical strains of Klebsiella pneumoniae, and the vertical axis represents the change in fluorescence value after 60 minutes of reaction at 37°C in a real-time quantitative PCR instrument; NC is the negative control (top).
[0070] Box plot of gene adh detection results for 34 clinical isolates of non-Klebsiella pneumoniae is shown below. Figure 5 As shown in the figure below, the adh gene test was also positive in non-Klebsiella pneumoniae strains, indicating that it cannot specifically distinguish between clinical isolates of Klebsiella pneumoniae and non-Klebsiella pneumoniae strains.
[0071] The above results indicate that the fluorescence intensity of the rcsA and adh genes in the 60 Klebsiella pneumoniae strains was higher than that in the negative control. Therefore, the strains were identified as Klebsiella pneumoniae and also contained the adh gene, suggesting that they might be ethanol-producing Klebsiella pneumoniae.
[0072] Commercial ethanol detection kits (BioVision, Milpitas, CA, USA) using headspace gas chromatography yielded results consistent with the method of this invention.
[0073] Example 4: Exploration of primers and crRNA for CRISPR-PCR detection of Klebsiella pneumoniae I. Primer Sequence Determination The primer pairs designed for the rcsA gene are shown in Table 3 below: Table 3 lists the primers for amplifying gene rcsA.
[0074] The primer pairs designed for the adh gene are shown in Table 4 below: Table 4 lists the primers for amplifying the adh gene.
[0075] High-ethanol-producing Klebsiella pneumoniae (HiAlc) Kpn Using W14 genomic DNA as a template, PCR amplification was performed according to the method in Example 1, Part 2, using the three primer pairs for gene rcsA shown in Table 3 and the three primer pairs for gene adh shown in Table 4, respectively, to obtain different primer pairs. rcsA Amplification products and different primer pairs oh Amplification products.
[0076] The results were compared with other primer pairs. rcsA The amplification product from the forward / reverse primer pair showed a single, clearly defined band, indicating that these were the optimal primers for amplification. Compared to other primer pairs, oh The amplification products from forward / reverse primers have single, bright bands, indicating they are the optimal primers for amplification.
[0077] II. crRNA sequence exploration 1. PCR amplification High-ethanol-producing Klebsiella pneumoniae (HiAlc) Kpn Using W14 genomic DNA as a template, PCR amplification was performed according to the method in Example 1, Part 2, to obtain... rcsA amplification products and oh Amplification products.
[0078] 2. CRISPR Cas12a reaction The crRNAs designed for the rcsA gene are shown in Table 5 below.
[0079] Table 5 shows the crRNA sequences.
[0080] The crRNAs designed for the adh gene are shown in Table 6 below.
[0081] Table 6 shows the crRNA sequences.
[0082] The above rcsA amplification products and oh The amplified products were subjected to CRISPR according to the method in Example 1, Part 2. The Cas12a reaction, in which rcsA CRISPR of amplified products The crRNAs used in the Cas12a reaction were the two types of crRNAs shown in Table 5; among them oh CRISPR of amplified products The crRNAs used in the Cas12a reaction were the two types shown in Table 5. Reaction products were obtained for different crRNAs.
[0083] Detection rcsA The reaction products of different crRNAs amplification products are as follows: rcsA The fluorescence intensity of the reaction product of crRNA sequence 2 was not significantly enhanced compared to the negative control group; sequence 3 showed... rcsA The fluorescence intensity of the crRNA reaction product was significantly greater than that of the negative control group.
[0084] Detection oh The reaction products of different crRNAs amplification products are as follows: oh The fluorescence intensity of the reaction product of crRNA sequence 2 was not significantly enhanced compared to the negative control group; sequence 6 showed... oh The fluorescence intensity of the crRNA reaction product was significantly greater than that of the negative control group.
[0085] III. ssDNA Probe Exploration 1. PCR amplification High-ethanol-producing Klebsiella pneumoniae (HiAlc) Kpn Using W14 genomic DNA as a template, PCR amplification was performed according to the method in Example 1, Part 2, to obtain... rcsA amplification products and oh Amplification products.
[0086] 2. CRISPR Cas12a reaction The ssDNA probes are designed as shown in Table 7 below.
[0087] Table 7 lists ssDNA probes.
[0088] The above rcsA amplification products and oh The amplified products were subjected to CRISPR according to the method in Example 1, Part 2. The Cas12a reaction was performed, with the only difference being the selection of different ssDNA probes as shown in Table 7. Reaction products were obtained for each ssDNA probe.
[0089] Detection rcsAThe results of the amplification products of different ssDNA probes are as follows: The fluorescence intensity of the reaction product of ssDNA probe 2 was not significantly enhanced compared with the negative control group; the fluorescence intensity of the reaction product of the ssDNA probe shown in sequence 7 was significantly greater than that of the negative control group.
[0090] Detection oh The results of the amplification products of different ssDNA probes are as follows: The fluorescence intensity of the reaction product of ssDNA probe 2 was not significantly enhanced compared with the negative control group; the fluorescence intensity of the reaction product of the ssDNA probe shown in sequence 7 was significantly greater than that of the negative control group.
[0091] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. A kit for simultaneous detection of Klebsiella pneumoniae and its alcohol dehydrogenase gene adh, comprising the following A1) or A2): A1) The crRNA that binds to gene rcsA, the crRNA that binds to gene adh, and Cas12a protein; A2) The CRISPR-Cas12a / crRNA complex that binds to the gene rcsA and the CRISPR-Cas12a / crRNA complex that binds to the gene adh. The nucleotide sequence of the crRNA that binds to gene rcsA is sequence 3; The nucleotide sequence of the crRNA that binds to the adh gene is sequence 6. The CRISPR-Cas12a / crRNA complex that binds the rcsA gene includes the crRNA that binds the rcsA gene and the Cas12a protein. The CRISPR-Cas12a / crRNA complex that binds the adh gene comprises the crRNA that binds the adh gene and the Cas12a protein.
2. The reagent kit according to claim 1, characterized in that: The kit also includes primer sets for amplifying the rcsA gene and primer sets for amplifying the adh gene.
3. The reagent kit according to claim 2, characterized in that: The primer set for amplifying the gene rcsA consists of single-stranded DNA molecules as shown in sequence 1 and single-stranded DNA molecules as shown in sequence 2; The primer set for amplifying the adh gene consists of single-stranded DNA molecules as shown in sequence 4 and single-stranded DNA molecules as shown in sequence 5.
4. The kit according to any one of claims 1-3, characterized in that: The kit also includes an ssDNA probe.
5. The reagent kit according to claim 4, characterized in that: The nucleotide sequence of the ssDNA probe is sequence 7.
6. The kit according to claim 4 or 5, characterized in that: The ssDNA probe is labeled with a fluorescent group and a quencher group at both ends, respectively.
7. The use of the kit according to any one of claims 1-6 in the preparation of a product having any of the following functions: B1) Detect whether the test strain is Klebsiella pneumoniae and also contains the adh gene; B2) Detect whether the test strain is an ethanol-producing Klebsiella pneumoniae.