Primer and probe for detecting citrobacter vinegaticus, fluorescent quantitative PCR (Polymerase Chain Reaction) kit and method and application

Abstract

The invention discloses a primer and a probe for detecting citrobacter vinegaticus, a fluorescent quantitative PCR (Polymerase Chain Reaction) kit and a method as well as application. The primer and the probe comprise a forward primer, a reverse primer, a forward primer, a reverse primer and a reverse primer, wherein the forward primer is 5 '-GTACTACTGGCGCATTC-3'; the reverse primer is 5 '-GGGTAGCAGGATTTAATAAC-3', and the reverse primer is 5 '- And a probe, namely 5 '-6-FAM-TTGGCCCCTTCGTTGCCG-MGB-3', and a probe, namely 5 '-6- By using the primer and the probe or the kit disclosed by the invention, the detection of the citrobacter vinegaticus can be quickly and accurately realized, a good linear relation is realized in a bacterial quantity range of 1.4 * 10 < 1 >-1.4 * 10 < 9 > CFU / mL, the specificity is good, and the citrobacter vinegaticus is not interfered by citrobacter freurauensis. The method is high in specificity and sensitivity, and a rapid and accurate detection means is provided for laboratory diagnosis of citrobacter vinegaticus.

Description

Technical Field

[0001] The present invention relates to the field of microbial detection, and in particular to primers and probes, a fluorescent quantitative PCR kit, a method and applications for detecting Citrobacter rodentium. Background Art

[0002] Citrobacter Portugueseum ( Citrobacter portucalensis ) is a Gram-negative facultative anaerobic bacterium and a member of the genus Citrobacter of the family Enterobacteriaceae. It was first isolated and identified in 2017. So far, there have been relatively few studies on this bacterium. Despite this, it has been reported that the bacterium is an opportunistic pathogen, and its potential pathogenicity to humans and animals cannot be ignored. In addition, Citrobacter Portuguese can exist in the host for a long time and accumulate drug resistance, and then develop into a multidrug-resistant strain. In recent years, extremely resistant strains and pan-resistant strains have also been isolated and reported many times. In view of the pathogenicity and drug resistance characteristics it exhibits, in the era of precision medicine, rapid and accurate identification and continuous monitoring of Citrobacter Portuguese clinically is very necessary. However, because Citrobacter Portuguese is different from another common clinical pathogenic species of the genus Citrobacter, Citrobacter freudii ( Citrobacter freundii ) have similar genetic backgrounds and metabolites, and the resolution of clinical microbial mass spectrometry technology is limited, making it difficult to achieve accurate distinction, which has also led to the neglect of Portuguese Citrobacter in clinical monitoring. The development of a fluorescent PCR probe detection method for Portuguese Citrobacter that cannot be effectively distinguished by microbial mass spectrometers will greatly improve the accuracy and specificity of clinical identification of this bacterium. The primers and probes can bind to the nucleic acid sequence of the species-specific core gene of Portuguese Citrobacter, and achieve high-precision identification of the target pathogen through the presence or absence of fluorescent signals or intensity differences. The application of this technology can not only make up for the shortcomings of the Portuguese Citrobacter microbial mass spectrometry identification method, but also provide faster and more accurate diagnostic methods for its related infectious diseases, thereby guiding precision treatment, reducing the abuse of antibiotics, and improving clinical treatment effects, which has far-reaching significance for public health and safety. Summary of the invention

[0003] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a reagent for detecting a target nucleic acid for use in detecting Portuguese Citrobacter rodentium or preparing a Portuguese Citrobacter rodentium detection product.

[0004] Another object of the present invention is to provide a primer pair and a probe for detecting Portuguese Citrobacter rodentium, a method for detecting Portuguese Citrobacter rodentium using the primer pair and the probe, and an application thereof.

[0005] The third object of the present invention is a kit containing the above primer pair and probe and its application.

[0006] The object of the present invention is achieved by the following technical solutions: Application of a reagent for detecting a target nucleic acid in detecting Citrobacter portugalensis or preparing a detection product for Citrobacter portugalensis, wherein the sequence of the target nucleic acid is (also shown as SEQ ID NO: 1): ATGAGCACGAGAATTCCTGTTGGTATCCGCACCTGTCTGACCACAGAAGCGAGTGAGGCACCTCAGTTCGACGAAAATACATTAATACGTGTTTACGCTTTGCATGAATTGCACCGCCTGCGTCAGCGAAAATTAACGCGGGGCGCATTAATTGACTATCATACCCGCTACAAACTGGTACTACTGGCGCATTCCCAGCCAGAGTATCGGGAGCTTGGCCCCTTCGTTGCCGCAATCCATGAATGGCAAGATCTCGATGCTTTTTTTACCGAGTATCGCCTACGATTGATCCATCTGTTATTAAATCCTGCTACCCGGCGTAACCATACCAATGTATTGATGCACGTACAGGGCTATTTCCGTAATCAGCTCGATCTGCGTCAACGTCAGGAATTGACCTCGATAATCGATAACTATCGACGCGAGGCGCAGCCGCTACTTGCACCCTTAATGCTCCTCAAACACTATATGGCTGAGCACCCGAACACCTGGTTGTCCGGGCAACGTTATTTTGAACTCTGGCCTGCGATTTGGCGTTTACAGGGCAATGATTAA; Furthermore, the amino acid sequence of the protein encoded by the target nucleic acid sequence is (also shown as SEQ ID NO: 2): MSTRIPVGIRTCLTTEASEAPQFDENTLIRVYALHELHRLRQRKLTRGALIDYHTRYKLVLLAHSQPEYRELGPFVAAIHEWQDLDAFFTEYRLRLIHLLLNPATRRNHTNVLMHVQGYFRNQLDLRQRQELTSIIDNYRREAQPLLAPLMLLKHYMAEHPNTWLSGQRYFELWPAIWRLQGND.

[0007] Furthermore, the reagent for detecting the target nucleic acid includes: Forward primer: 5'-GTACTACTGGCGCATTC-3'; Reverse primer: 5'-GGGTAGCAGGATTTAATAAC-3'; and Probe: 5'-6-FAM-TTGGCCCCTTCGTTGCCG-MGB-3'.

[0008] Furthermore, the reagent for detecting the target nucleic acid further includes: reference dye, Premix Type reagent and water; Even further, the reference dye is Rox Reference Dye Ⅱ; the Premix Type reagent is Premix Ex Taq (Probe qPCR); the water is DEPC water.

[0009] A primer pair and a probe for detecting Citrobacter portucalensis, including: Forward primer: 5'-GTACTACTGGCGCATTC-3'; Reverse primer: 5'-GGGTAGCAGGATTTAATAAC-3'; and Probe: 5'-6-FAM-TTGGCCCCTTCGTTGCCG-MGB-3'.

[0010] A kit for detecting Citrobacter portucalensis, including the above primer pair and probe.

[0011] Further, the kit further includes: reference dye, Premix Type reagent and water; Even further, the reference dye is Rox Reference Dye Ⅱ; the Premix Type reagent is Premix Ex Taq (Probe qPCR); the water is DEPC water.

[0012] Application of the above primer pair and probe in detecting Citrobacter portucalensis or preparing a Citrobacter portucalensis detection product.

[0013] Application of the above kit in detecting Citrobacter portucalensis or preparing a Citrobacter portucalensis detection product.

[0014] A method for detecting Citrobacter portucalensis using the above primer pair and probe, including the following steps: Extract DNA from the sample, add the obtained DNA to a PCR fluorescence quantitative detection system containing the above primer pair and probe, and carry out the reaction.

[0015] Further, the content of Citrobacter portucalensis in the sample is greater than or equal to 1.4×10 1 CFU / mL, and if fluorescence is generated during the reaction, it is positive.

[0016] Further, the DNA in the extracted sample is extracted by a bacterial genomic DNA extraction kit.

[0017] Further, the PCR fluorescence quantitative detection system containing the above primer pair and probe also contains a reference dye, Premix Type reagent and water; Furthermore, the reference dye is Rox Reference Dye Ⅱ; the Premix Type reagent is Premix Ex Taq (Probe qPCR); the water is DEPC water; Even further, after adding the obtained DNA to the PCR fluorescence quantitative detection system containing the above primer pair and probe, the concentrations of each component in the system are: 0.2 µM probe, 0.2 µM forward primer, 0.2 µM reverse primer, 0.5×Rox Reference Dye Ⅱ and 1×Premix Ex Taq (Probe qPCR).

[0018] Further, the reaction procedure is: 95°C for 30 s; 95°C for 5 s, 60°C for 34 s, for 40 cycles.

[0019] The present invention has the following advantages and effects compared with the prior art: Using the kit of the present invention can quickly and accurately detect Citrobacter portucalensis; the kit of the present invention has a good linear relationship within the bacterial amount range of 1.4×10 1 ~1.4×10 9 CFU / ml; it has good specificity and is not interfered by Citrobacter freundii. In summary, this method has strong specificity and high sensitivity, and provides a rapid and accurate detection means for the laboratory diagnosis of Citrobacter portucalensis. Description of the Drawings

[0020] Figure 1 It is the result graph of the amplification curve for optimizing the primer and probe concentrations.

[0021] Figure 2It is the amplification curve result graph of the specificity test; among them, the 4 positive amplification curves P1 - P4 are Citrobacter portucalensis PY0607, GYCP1, GYCP2, and GYCP3 respectively. Among the 41 negative amplification curves, F1 - F30 are different clinical isolates of Citrobacter freundii, and 1 - 10 are the standard strains of Citrobacter freundii (ATCC 43864), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC29213), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 700603), Acinetobacter baumannii (ATCC 19606), Proteus mirabilis (HI4320), Streptococcus group B (ATCC 12386), Enterococcus faecalis (ATCC 29212), and Candida albicans (ATCC14053) respectively, and NC is the negative control.

[0022] Figure 3 It is the gel electrophoresis result graph of the specificity test; among them, M: D2000 DNA Marker, F1 - F30 are different clinical isolates of Citrobacter freundii, the P1 - P4 lanes are Citrobacter portucalensis PY0607, GYCP1, GYCP2, and GYCP3 respectively, and the 1 - 10 lanes are the standard strains: Citrobacter freundii (ATCC 43864), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoniae (ATCC 700603), Acinetobacter baumannii (ATCC 19606), Proteus mirabilis (HI4320), Streptococcus group B (ATCC12386), Enterococcus faecalis (ATCC 29212), and Candida albicans (ATCC 14053).

[0023] Figure 4 It is the amplification curve result graph of the sensitivity test; the amplification curves from left to right, 1 - 9 are the amplification curves of 1.4×10 9 、1.4×10 8 、1.4×10 7 、1.4×10 6 、1.4×10 5 、1.4×10 4 、1.4×10 3 、1.4×10 2 、1.4×10 1 CFU / mL respectively, 10 is the amplification curve of 1.4×10 0 CFU / mL and the amplification curve of the negative control (NC) coincide. Detailed implementation manners

[0024] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto. For the test methods without specific experimental conditions noted in the following embodiments, they are generally carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, can be obtained commercially.

[0025] The instruments and reagents used in the embodiments are as follows: The ABI 7500 fluorescence quantitative PCR instrument was purchased from ABI Company, the bacterial genomic DNA extraction kit was purchased from GENFINE Company, Premix Ex Taq (Probe qPCR) was purchased from TaKaRa Company, DEPC water was purchased from Biosharp Company, the nucleic acid electrophoresis instrument Sub-Cell Model 192 and the gel imaging system ChemiDoc XRS+ were purchased from Bio-Rad Company, and the DNAmarker D2000 was purchased from TIANGEN Company.

[0026] The Citrobacter portucalensis PY0607, GYCP1, GYCP2, and GYCP3 used in the experiment were all Citrobacter portucalensis strains isolated clinically, which have been sequenced and identified as Citrobacter portucalensis, and their genomic sequences have been uploaded to NCBI; the NCBI genomic query number of Citrobacter portucalensis PY0607 is SAMN45949205, the NCBI genomic query number of Citrobacter portucalensis GYCP1 is SAMN45949638, the NCBI genomic query number of Citrobacter portucalensis GYCP2 is SAMN45949639, and the NCBI genomic query number of Citrobacter portucalensis GYCP3 is SAMN45949640.

[0027] Example 1 Design of Probes and Primer Pairs In the present invention, potential Citrobacter portucalensis species-specific core gene families were screened out, and the gene sequences of the screening results were retrieved online in NCBI BLASTN to verify the NR library, and the gene families with homology in Citrobacter freundii were excluded. Finally, the Citrobacter portucalensis species-specific core gene was obtained. The nucleic acid sequence of this gene is: ATGAGCACGAGAATTCCTGTTGGTATCCGCACCTGTCTGACCACAGAAGCGAGTGAGGCACCTCAGTTCGACGAAAATACATTAATACGTGTTTACGCTTTGCATGAATTGCACCGCCTGCGTCAGCGAAAATTAACGCGGGGCGCATTAATTGACTATCATACCCGCTACAAACTGGTACTACTGGCGCATTCCCAGCCAGAGTATCGGGAGCTTGGCCCCTTCGTTGCCGCAATCCATGAATGGCAAGATCTCGATGCTTTTTTTACCGAGTATCGCCTACGATTGATCCATCTGTTATTAAATCCTGCTACCCGGCGTAACCATACCAATGTATTGATGCACGTACAGGGCTATTTCCGTAATCAGCTCGATCTGCGTCAACGTCAGGAATTGACCTCGATAATCGATAACTATCGACGCGAGGCGCAGCCGCTACTTGCACCCTTAATGCTCCTCAAACACTATATGGCTGAGCACCCGAACACCTGGTTGTCCGGGCAACGTTATTTTGAACTCTGGCCTGCGATTTGGCGTTTACAGGGCAATGATTAA; The protein sequence encoded by this gene is as follows: MSTRIPVGIRTCLTTEASEAPQFDENTLIRVYALHELHRLRQRKLTRGALIDYHTRYKLVLLAHSQPEYRELGPFVAAIHEWQDLDAFFTEYRLRLIHLLLNPATRRNHTNVLMHVQGYFRNQLDLRQRQELTSIIDNYRREAQPLLAPLMLLKHYMAEHPNTWLSGQRYFELWPAIWRLQGND。

[0028] Based on the above-mentioned specific core gene of Citrobacter portucalensis, a primer and a probe for specific detection of Citrobacter portucalensis were designed, wherein: the primer sequences are as follows: Forward primer: 5'-GTACTACTGGCGCATTC-3'; Reverse primer: 5'-GGGTAGCAGGATTTAATAAC-3'; The probe sequence is as follows: 5'-6-FAM-TTGGCCCCTTCGTTGCCG-MGB-3'. The fluorescent reporter group labeled at the 5' end of the probe is FAM, and the fluorescent quenching group labeled at the 3' end is MGB.

[0029] Example 2 Optimal PCR Reaction System and Conditions Absorb 1 mL of Citrobacter portucalensis PY0607 bacterial solution with a concentration of 1×10 9 CFU / mL, and extract DNA using a bacterial genomic DNA extraction kit. The obtained product can be directly used for fluorescence quantitative PCR or stored at -20 °C for later use.

[0030] The fluorescence quantitative PCR detection system for detecting Citrobacter portucalensis includes: probe, forward primer, reverse primer, reference dye, Premix Type reagent and water. Among them, the sequence of the probe is: 5'-6-FAM-TTGGCCCCTTCGTTGCCG-MGB-3'; the sequence of the forward primer is: 5'-GTACTACTGGCGCATTC-3'; the sequence of the reverse primer is: 5'-GGGTAGCAGGATTTAATAAC-3'; the reference dye is Rox Reference Dye Ⅱ; the PremixType reagent is Premix Ex Taq (Probe qPCR); the water is DEPC water.

[0031] Add the extracted DNA to the above fluorescence quantitative PCR detection system to make the total volume of the reaction system 20 µL, and the amounts of each component are: 0.4 µL probe (10 µM), 0.4 µL forward primer (10 µM), 0.4 µL reverse primer (10 µM), 0.2 µL Rox Reference Dye Ⅱ (50×), 10 µL 2×Premix Ex Taq (Probe qPCR), 6.6 µL DEPC water and 2 µL DNA.

[0032] Perform PCR amplification reaction; the reaction conditions are 95 °C for 30 s; 95 °C for 5 s, 60 °C for 34 s, for 40 cycles.

[0033] Example 3 Influence of PCR Reaction System and Conditions (1) Influence of primer concentration and probe concentration The experiment refers to Example 2, the difference is that after adding DNA, the final concentrations of each group of probe, forward primer and reverse primer in the reaction system are shown in Table 1, and the results of the threshold cycle number (Cycle threshold, Ct value) of PCR amplification at different concentrations are shown in Table 1, and its amplification curve is as Figure 1 shown.

[0034] Table 1 Ct values for optimizing primer and probe concentrations 。

[0035] Example 4 Specificity test Absorb 1 mL of bacterial suspension with a concentration of 1×10 9 CFU / mL, and extract DNA using a bacterial genomic DNA extraction kit. The obtained product is directly used for fluorescence quantitative PCR. The bacterial suspensions are: suspensions of 4 strains of Citrobacter portucalensis (PY0607, GYCP1, GYCP2, GYCP3), suspensions of 31 strains of Citrobacter freundii (30 different clinical isolates of Citrobacter freundii, 1 standard strain of Citrobacter freundii (ATCC 43864)), suspensions of Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoniae (ATCC 700603), Acinetobacter baumannii (ATCC 19606), Proteus mirabilis (HI4320), Streptococcus agalactiae (ATCC 12386), Enterococcus faecalis (ATCC29212) and Candida albicans (ATCC 14053). Additionally, a negative control group (NC) is set by taking 1 mL of the same medium used when culturing the bacterial suspension.

[0036] Add the extracted DNA to the fluorescence quantitative PCR detection system to make the total volume of the reaction system 20 µL. The amounts of each component are as follows: 0.4 µL of probe (10 µM), 0.4 µL of forward primer (10 µM), 0.4 µL of reverse primer (10 µM), 0.2 µL of Rox Reference Dye Ⅱ (50×), 10 µL of 2×Premix Ex Taq (Probe qPCR), 6.6 µL of DEPC water, and 2 µL of DNA.

[0037] Perform PCR amplification reaction; the reaction conditions are 95℃ for 30 s; 95℃ for 5 s, 60℃ for 34 s, for 40 cycles.

[0038] After PCR amplification, perform agarose gel electrophoresis analysis on the PCR products.

[0039] The results of the PCR amplification curve are as Figure 2 shown, and the results of the gel electrophoresis are as Figure 3As shown, P1 to P4 are Citrobacter portucalensis PY0607, GYCP1, GYCP2, and GYCP3 respectively, F1 to F30 are different clinical isolates of Citrobacter freundii, and 1 to 10 are standard strains of Citrobacter freundii (ATCC 43864), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC27853), Klebsiella pneumoniae (ATCC700603), Acinetobacter baumannii (ATCC 19606), Proteus mirabilis (HI4320), Streptococcus group B (ATCC 12386), Enterococcus faecalis (ATCC 29212), and Candida albicans (ATCC 14053). NC is the negative control. The results show that this system can specifically detect Citrobacter portucalensis.

[0040] Example 5 Sensitivity Test Prepare a bacterial suspension with an OD of 1.0 using freshly cultured Citrobacter portucalensis PY0607, and dilute it 10-fold serially to 10 600nm , take 50 µL of the bacterial solution for plate coating, place it in a 37°C, 5% CO -9 incubator and culture overnight, perform colony counting, calculate the bacterial solution concentration, and the concentrations are 1.4×10 2 CFU / mL, 1.4×10 0 CFU / mL, 1.4×10 1 CFU / mL, 1.4×10 2 CFU / mL, 1.4×10 3 CFU / mL, 1.4×10 4 CFU / mL, 1.4×10 5 CFU / mL, 1.4×10 6 CFU / mL, 1.4×10 7 CFU / mL, 1.4×10 8 CFU / mL, and 1.4×10 9 CFU / mL. Take 1 mL of the bacterial solution from each dilution to extract DNA and perform real-time fluorescence quantitative PCR to determine the lower limit of bacterial amount detection. Additionally, set up a negative control group (NC) by taking 1 mL of the same medium used when culturing the bacterial solution.

[0041] Real-time fluorescence quantitative PCR is as follows: The extracted DNA is added to the fluorescence quantitative PCR detection system to make the total volume of the reaction system 20 µL. The amounts of each component are as follows: 0.4 µL of probe (10 µM), 0.4 µL of forward primer (10 µM), 0.4 µL of reverse primer (10 µM), 0.2 µL of Rox Reference Dye Ⅱ (50×), 10 µL of 2×Premix ExTaq (Probe qPCR), 6.6 µL of DEPC water, and 2 µL of DNA. PCR amplification reaction is carried out; the reaction conditions are 95°C for 30 s; 95°C for 5 s, 60°C for 34 s, for 40 cycles.

[0042] The results of the PCR amplification curve are as Figure 4 shown, indicating that under this system and reaction conditions, the lowest detection limit of the viable bacteria count of Citrobacter portugalensis is 1.4×10 1 CFU / mL.

[0043] 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of a reagent for detecting a target nucleic acid in detecting Citrobacter rodentium or preparing a product for detecting Citrobacter rodentium, characterized in that: The sequence of the target nucleic acid is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the target nucleic acid sequence is shown in SEQ ID NO:

2.

3. A primer pair and probe for detecting Portuguese Citrobacter rodentium, characterized in that: include: Forward primer: 5′-GTACTACTGGCGCATTC-3′; Reverse primer: 5'-GGGTAGCAGGATTTAATAAC-3'; and Probe: 5'-6-FAM-TTGGCCCCTTCGTTGCCG-MGB-3'.

4. A kit for detecting Portuguese Citrobacter, characterized in that: Comprising the primer pair and probe described in claim 3.

5. The kit according to claim 4, characterized in that The kit also includes a reference dye, a PremixType reagent, and water.

6. Use of the primer pair and probe according to claim 3, the kit according to claim 4 or the kit according to claim 5 in detecting Portuguese Citrobacter rodentium or preparing a Portuguese Citrobacter rodentium detection product.

7. A method for detecting Citrobacter rodentium using the primer pair of claim 3, characterized in that: The following steps are involved: The DNA in the sample is extracted, and the obtained DNA is added to a PCR fluorescence quantitative detection system containing the primer pair and probe according to claim 3 to carry out a reaction.

8. The method according to claim 7, characterized in that The PCR fluorescence quantitative detection system containing the primer pair and probe also contains reference dye, Premix Type reagent and water.

9. The method according to claim 8, characterized in that The reference dye is Rox Reference Dye Ⅱ; the Premix Type reagent is Premix ExTaq (Probe qPCR); the water is DEPC water; After the obtained DNA is added to the PCR fluorescence quantitative detection system containing the above primer pair and probe, the concentrations of the components in the system are: 0.2 µM probe, 0.2 µM forward primer, 0.2 µM reverse primer, 0.5×Rox Reference DyeⅡ and 1×Premix Ex Taq (Probe qPCR).

10. The method according to claim 7, characterized in that The reaction procedure is: 95°C for 30s; 95°C for 5s, 60°C for 34s, 40 cycles.

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