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

Abstract

The invention discloses a primer and a probe for detecting proteus mirabilis, a fluorescent quantitative PCR (Polymerase Chain Reaction) kit, a method and application. The primer and the probe comprise an upstream primer, a downstream primer, an upstream primer, a downstream primer and a downstream primer, wherein the upstream primer is 5 '-GCCCTTTCCCTATGACACAAC-3'; the downstream primer is 5 '-ATCGTTCTACCACAGCTAATGG-3', and the downstream primer is 5 '-ATCGCTCTACCACAGCTAATGG-3'; and the probe is 5 '-6-FAM-CCTTCTGTAAAATCG-MGB-3', and the probe is 5 '-6- By using the primer and the probe or the kit disclosed by the invention, the proteus mirabilis can be rapidly and accurately detected; the kit disclosed by the invention has a good linear relationship in a bacterium amount range of 3.43 * 10 < 2 >-3.43 * 10 < 9 > CFU / mL; the specificity is good, and the method is not interfered by various proteusbacillus vulgaris and various common pathogenic bacteria.

Description

[0001] Primers and probes for detecting Proteus mirabilis, fluorescent quantitative PCR kit and method and application thereof Technical Field

[0002] 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 Proteus mirabilis. Background Art

[0003] Urinary tract infection is one of the most common diseases in the community and hospitals. Its high incidence and high recurrence rate lead to long-term treatment for patients. The long duration and high cost burden have led to serious public health problems and caused a huge economic burden worldwide. Proteus mirabilis is one of the main pathogens causing urinary tract infections, and the illness it causes is serious, long-lasting and difficult to treat. In addition, the problem of drug resistance of Proteus mirabilis is becoming increasingly prominent, which has also led to the recurrence and chronic development of urinary tract infections caused by it, posing a great challenge to clinical anti-infection treatment. Proteus mirabilis ( Proteus mirabilis ) is a Gram-negative bacillus belonging to the genus Proteus ( Proteus ), due to similar genetic background and metabolites, it is similar to other species in the genus Proteus, such as Proteus vulgaris ( Proteus vulgaris ), Proteus houchenii ( Proteus hauseri ), Myxoproteus ( Proteus myxofaciens ) are difficult to distinguish. Therefore, developing a highly specific and sensitive fluorescent PCR probe assay can enable rapid and accurate identification of Proteus mirabilis, providing a powerful tool for early clinical diagnosis and enabling timely targeted treatment. This is crucial for controlling the spread of infection and reducing complications. It can also be used to monitor the drug resistance patterns of clinical Proteus mirabilis strains, providing a basis for the rational use of antibiotics and helping to curb the spread of multidrug-resistant bacteria. Summary of the Invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a reagent for detecting target nucleic acid for use in detecting Proteus mirabilis or preparing a Proteus mirabilis detection product.

[0005] Another object of the present invention is to provide a primer pair and a probe for detecting Proteus mirabilis, a method for detecting Proteus mirabilis using the primer pair and the probe, and an application thereof.

[0006] The third object of the present invention is a kit containing the above primer pair and probe and its application.

[0007] The purpose of the present invention is achieved through the following technical solutions: The application of the reagent for detecting target nucleic acid in detecting Proteus mirabilis or preparing Proteus mirabilis detection product, the amino acid sequence of the protein coded by the target nucleic acid sequence is (also shown in SEQ ID NO: 2): mkktkhlfktkgcnipftsvmmvsfvlmgcndqstqptkpaipevgvytvkeeilpltvdlpartasyrvsqvrpqvdgilekrlftegsdvkkdqplyqidkkpyetqvakaqaaydnvkklaarykslratnaisqqeyddvvsrldqakadlnqakinlgytqvlspisgkisrsfvsegalvssgqsqplatitqldpiyvditqpitnqlklksliqqgvvtdsedkmdvtlfledgsqypytgtlsfsevqvepstgsvtlrakfpnpqhqllpgmytkarimqgtqekaivvpqqaitfdargksityivdqqqvlqardveilkpqgngwiikqglqagdrimteghhrakpgvkvnavpannislkttle.

[0008] Further, the sequence of the target nucleic acid is (also shown in SEQ ID NO: 1): Further, the reagent for detecting the target nucleic acid comprises: an upstream primer: 5'-GCCCTTTTCCCTTATGACACAAC-3'; a downstream primer: 5'-ATCGTTCTTACCACAGAGCTAATGG-3'; and a probe: 5'-6-FAM-CCTTCTGTAAAATCG-MGB-3'.

[0009] Further, the reagent for detecting the target nucleic acid further comprises: a reference dye, a Premix Type reagent and water; further, the reference dye is Rox Reference Dye II; the Premix Type reagent is Premix Ex Taq (Probe qPCR); and the water is DEPC water.

[0010] A primer pair and a probe for detecting Proteus mirabilis, comprising: an upstream primer: 5'-GCCCTTTTCCCTTATGACACAAC-3'; a downstream primer: 5'-ATCGTTCTTACCACAGAGCTAATGG-3'; and a probe: 5'-6-FAM-CCTTCTGTAAAATCG-MGB-3'.

[0011] A kit for detecting Proteus mirabilis, comprising the primer pair and the probe.

[0012] Further, the kit further comprises: a reference dye, a Premix Type reagent and water; Further, the reference dye is Rox Reference Dye II; the Premix Type reagent is Premix Ex Taq (Probe qPCR); and the water is DEPC water.

[0013] The primer pair and the probe are used for detecting Proteus mirabilis or preparing a Proteus mirabilis detection product.

[0014] The kit is used for detecting Proteus mirabilis or preparing a Proteus mirabilis detection product.

[0015] A method for detecting Proteus mirabilis using the primer pair and the probe, comprising the following steps: DNA in a sample is extracted, and the obtained DNA is added to a PCR fluorescent quantitative detection system containing the primer pair and the probe, and then a reaction is performed.

[0016] Further, the content of the Proteus mirabilis in the sample is greater than or equal to 3.43*10 2 CFU / mL, and fluorescence is generated in the reaction, which is positive.

[0017] Further, the DNA in the extracted sample is extracted by a bacterial genomic DNA extraction kit.

[0018] Further, the PCR fluorescent quantitative detection system containing the primer pair and the probe further contains reference dye, Premix Type reagent and water. Further, the reference dye is Rox Reference Dye II; the Premix Type reagent is Premix Ex Taq (Probe qPCR); and the water is DEPC water. Further, after the obtained DNA is added to the PCR fluorescent quantitative detection system containing the primer pair and the probe, the concentration of each component in the system is: 0.6 µM probe, 0.2 µM upstream primer, 0.2 µM downstream primer, 0.5* Rox Reference Dye II and 1* Premix Ex Taq (Probe qPCR).

[0019] Further, the program of the reaction is: 95℃ 30s; 95℃ 5s, 60℃ 34s, 35 cycles.

[0020] The present application has the following advantages and effects relative to the prior art: The primer and the probe or the kit of the present application can quickly and accurately realize detection of Proteus mirabilis; the kit has a good linear relationship in the range of 3.43*10 2 ~ 3.43*10 9 CFU / mL; the specificity is good, and is not interfered by ordinary Proteus vulgaris, Proteus hauseri, Proteus myxofaciens, Morganella morganii, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Enterococcus faecalis and Candida albicans. In summary, the method has strong specificity and high sensitivity, and provides a rapid and accurate detection means for laboratory diagnosis of Proteus mirabilis. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a Proteus genus pan-genome analysis and whole genome phylogenetic analysis schematic diagram in embodiment 1 of the present application.

[0022] Figure 2 is an amplification curve schematic diagram of primer and probe concentration optimization.

[0023] Figure 3 The figure is a gel electrophoresis result diagram of the specificity test; wherein, M: DL50bp DNA Marker, lanes 1-5 are Proteus mirabilis HI4320, Proteus mirabilis DP2019, Proteus howe, Proteus mucosus, and Proteus vulgaris, and lanes 6-16 are Proteus mirabilis HI4320, Proteus mirabilis DP2019, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Morganella morganii, Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, and Candida albicans. Figure 4 It is the amplification curve result diagram of the specificity test; among them, the two positive amplification curves M1~M2 are Proteus mirabilis DP2019 and HI4320, and the 12 negative amplification curves N1~N12 are Proteus houseii, Proteus mucosus, Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Morganella morganii, Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, and Candida albicans.

[0024] Figure 5 This is the amplification curve result of the sensitivity test; the amplification curves from left to right 1 to 8 are 3.43×10 9 ~3.43×10 2 CFU / mL, 9 is 3.43×10 1 CFU / mL, 10 is 3.43×10 0 The CFU / mL amplification curves 9 and 10 overlap with the amplification curve of the negative control (NC). DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to the examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. Experimental methods in the following examples, where specific experimental conditions are not specified, were generally performed under conventional experimental conditions or those recommended by the manufacturer. Materials and reagents used, unless otherwise specified, were commercially available.

[0026] The instruments and reagents used in the examples are as follows: QuantStudio 3 fluorescence quantitative PCR instrument purchased from ThermoFisher Scientific, bacterial genomic DNA extraction kit purchased from GENFINE, Premix Ex Taq (Probe qPCR) purchased from TaKaRa, DEPC water purchased from Biosharp, nucleic acid electrophoresis instrument Sub-Cell Model 192 and gel imaging system ChemiDoc XRS+ purchased from Bio-Rad, and DNA marker DL50 purchased from TIANGEN.

[0027] The Proteus mirabilis HI4310 and DP2019 used in the experiment are both clinically isolated Proteus mirabilis strains, which have been sequenced, identified as Proteus mirabilis, and uploaded to NCBI; the GenBank query number of Proteus mirabilis HI4310 is AM942759.1, and the GenBank query number of Proteus mirabilis DP2019 is CP110673.1.

[0028] The Proteus hauseri (ATCC 13315), Proteus myxofaciens (ATCC 19692), Proteus vulgaris (ACCC 11002), Morganella morganii (ACCC 60117), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Acinetobacter baumannii (ATCC 19606), Candida albicans (ATCC 14053), Pseudomonas aeruginosa (ATCC 27853), Streptococcus agalactiae (ATCC 12386), Enterococcus faecalis (ATCC 29212) and Klebsiella pneumoniae (ATCC 700603) used in the experiment are standard strains that can be purchased.

[0029] Example 1 Design of probe and primer pair The present application screens potential Proteus mirabilis species-specific core gene families, puts the screening result gene sequence into NCBI BLASTN online retrieval, verifies the NR library, eliminates homologous gene families existing in other species, and finally obtains the Proteus mirabilis species-specific core gene family through Proteus mirabilis genus pan-genome analysis and whole genome phylogenetic analysis as shown in Figure 1 The nucleic acid sequence of the gene is also shown in SEQ ID NO: 1: The protein sequence encoded by the gene is (also shown in SEQ ID NO: 2): mkktkhlfktkgcnipftsvmmvsfvlmgcndqstqptkpaipevgvytvkeeilpltvdlpartasyrvsqvrpqvdgilekrlftegsdvkkdqplyqidkkpyetqvakaqaaydnvkklaarykslratnaisqqeyddvvsrldqakadlnqakinlgytqvlspisgkisrsfvsegalvssgqsqplatitqldpiyvditqpitnqlklksliqqgvvtdsedkmdvtlfledgsqypytgtlsfsevqvepstgsvtlrakfpnpqhqllpgmytkarimqgtqekaivvpqqaitfdargksityivdqqqvlqardveilkpqgngwiikqglqagdrimteghhrakpgvkvnavpannislkttle.

[0030] A primer and a probe for specifically detecting P. mirabilis are designed according to the specific core gene sequence of P. mirabilis, wherein the primer sequence is as follows: The upstream primer is 5'-GCCCTTTTCCCTTATGACACAAC-3'; The downstream primer is 5'-ATCGTTCTTACCACAGAGCTAATGG-3'; The probe sequence is as follows: 5'-6-FAM CCTTCTGTAAAATCG-MGB-3'. The probe sequence is as follows: The fluorescent reporter group labeled at the 5' end of the probe is FAM, and the fluorescent quencher group labeled at the 3' end is MGB.

[0031] Example 2 Optimal PCR reaction system and conditions Single colonies are selected into 1.5 ml LB medium for overnight activation, 1 ml of activated bacterial solution is taken, DNA is extracted using a bacterial genome DNA extraction kit, and the obtained product is used for fluorescent quantitative PCR or stored at -20°C for standby use.

[0032] The fluorescent quantitative PCR detection system for detecting P. mirabilis includes a probe, an upstream primer, a downstream primer, a reference dye, a Premix Type reagent, and water. Among them, The upstream primer is 5'-GCCCTTTTCCCTTATGACACAAC-3'; The downstream primer is: 5'-ATCGTTCTTACCACAGAGCTAATGG-3'; The sequence of the probe is: 5'-6-FAM CCTTCTGTAAAATCG-MGB-3'.

[0033] The reference dye is Rox Reference Dye II; the premix type reagent is Premix Ex Taq (Probe qPCR); and the water is DEPC water.

[0034] The extracted DNA is added to the above-mentioned fluorescence quantitative PCR detection system, so that the total amount of the reaction system is 20 μL, and the amount of each component is: 1.2 μL of probe (10 μM), 0.4 μL of upstream primer (10 μM), 0.4 μL of downstream primer (10 μM), 0.2 μL of Rox Reference Dye II (50x), 10 μL of 2x Premix Ex Taq (Probe qPCR), 5.8 μL of DEPC water, and 2 μL of DNA.

[0035] PCR amplification reaction is carried out; the reaction conditions are 95℃ for 30s; 95℃ for 5s, 60℃ for 34s, 35 cycles.

[0036] Example 3 Influence of PCR reaction system and conditions (1) Influence of primer concentration and probe concentration According to Experimental Reference Example 2, the difference is that after the DNA is added, the final concentration of each probe, upstream primer and downstream primer in the reaction system is shown in Table 1, and the threshold cycle number (Ct value) of PCR amplification under different concentrations is shown in Table 1, and the amplification curve is shown in Figure 2 .

[0037] Table 1 Ct value of primer and probe concentration optimization

[0038] (2) PCR amplification reaction is carried out at different annealing temperatures, and the best reaction condition is determined as 95℃ for 30s; 95℃ for 5s, 60℃ for 34s, 35 cycles.

[0039] Example 4 Specificity test Single colony was selected to activate overnight in 1.5 ml LB medium, 1 ml activated bacterial solution was taken, DNA was extracted using bacterial genome DNA extraction kit, and the obtained product was used for fluorescent quantitative PCR. The bacterial solution was: Proteus mirabilis strain (HI4320, DP2019), Proteus hauseri (ATCC 13315), Proteus myxofaciens (ATCC 19692), Proteus vulgaris (ACCC 11002), Morganella morganii (ACCC 60117), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213), Acinetobacter baumannii (ATCC 19606), Candida albicans (ATCC 14053), Pseudomonas aeruginosa (ATCC 27853), Streptococcus agalactiae (ATCC 12386), Enterococcus faecalis (ATCC 29212) and Klebsiella pneumoniae (ATCC 700603). The bacterial solution was obtained by culturing the strain in Luria Bertani (LB) liquid medium on a shaker at 220 r / min overnight.

[0040] The extracted DNA was added to the fluorescent quantitative PCR detection system, so that the total amount of the reaction system was 20 μL, and the amount of each component was: 1.2 μL probe (10 μM), 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer (10 μM), 0.2 μL Rox Reference Dye II (50x), 10 μL 2x Premix Ex Taq (Probe qPCR), 5.8 μL DEPC water and 2 μL DNA.

[0041] PCR amplification reaction was carried out; the reaction conditions were 95℃ for 30s; 95℃ for 5s, 60℃ for 34s, 35 cycles.

[0042] After PCR amplification, the PCR product was analyzed by agarose gel electrophoresis.

[0043] The results of gel electrophoresis are shown in Figure 3 , and the results of PCR amplification curve are shown in Figure 4 . Figure 3 , lanes 1-5 are Proteus mirabilis HI4320, Proteus mirabilis DP2019, Proteus hauseri, Proteus myxofaciens, Proteus vulgaris, lanes 6-16 are Proteus mirabilis HI4320, Proteus mirabilis DP2019, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Morganella morganii, Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae and Candida albicans. Figure 4Amplification curves M1 and M2 are for Proteus mirabilis DP2019 and HI4320, respectively, showing positive results. Amplification curves N1 to N12 are for Proteus houseii, Proteus mucosus, Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Morganella morganii, Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, and Candida albicans. These results demonstrate that the system can specifically detect Proteus mirabilis.

[0044] Example 5 Sensitivity Test Prepare OD values ​​using freshly cultured Proteus mirabilis HI4320 600nm The bacterial suspension was 1.0, and the colony count after cultivation showed that the original bacterial solution concentration was 3.43×10 9 CFU / mL, 10-fold serial dilution to 10 -9 , that is, the bacterial solution concentration is 3.43×10 0 CFU / mL. (3.43±0.99)×10 0 、(3.43±0.99)×10 1 、(3.43±0.99)×10 2 、(3.43±0.99)×10 3 、(3.43±0.99)×10 4 、(3.43±0.99)×10 5 、(3.43±0.99)×10 6 、(3.43±0.99)×10 7 、(3.43±0.99)×10 8 、(3.43±0.99)×10 9 A series of gradient bacterial concentrations was prepared. 1 mL of each dilution was used to extract DNA for real-time quantitative PCR to determine the detection limit of bacterial counts. A negative control group (NC) was also established (except that the DNA was replaced with DEPC water; all other reaction systems and conditions were identical to those in the other groups).

[0045] Real-time fluorescence quantitative PCR was performed as follows: the extracted DNA was added to a 20 µL fluorescence quantitative PCR detection system containing the following components: 1.2 µL probe (10 µM), 0.4 µL upstream primer (10 µM), 0.4 µL downstream primer (10 µM), 0.2 µL Rox Reference Dye II (50×), 10 µL 2× Premix ExTaq (Probe qPCR), 5.8 µL DEPC water, and 2 µL DNA. PCR amplification was performed under the following conditions: 95°C for 30 s, followed by 35 cycles of 95°C for 5 s and 60°C for 34 s.

[0046] PCR amplification curve results are as follows Figure 5 As shown in Table 2, the minimum detection limit of viable bacteria of Proteus mirabilis under this system and reaction conditions is 3.43×10 2 CFU / mL.

[0047] Table 2

[0048] Note: - indicates that no signal value is detected The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting a target nucleic acid in detecting Proteus mirabilis or preparing a Proteus mirabilis detection product, characterized in that: The amino acid sequence of the protein encoded by the target nucleic acid sequence is shown in SEQ ID NO:

2.

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

1.

3. A primer pair and probe for detecting Proteus mirabilis, characterized in that: include: Upstream primer: 5′-GCCCTTTTCCCTTATGACACAAC-3′; Downstream primer: 5′-ATCGTTCTTACCACAGAGCTAATGG-3′; and Probe: 5'-6-FAM-CCTTCTGTAAAATCG-MGB-3'.

4. A kit for detecting Proteus mirabilis, characterized in that: The method comprises the primer pair and probe according to claim 3.

5. The kit according to claim 4, characterized in that The kit also includes a reference dye, 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 Proteus mirabilis or preparing a product for detecting Proteus mirabilis.

7. A method for detecting Proteus mirabilis using the primer pair according to claim 3, characterized in that: The following steps are involved: DNA is extracted from the sample, 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 according to claim 3 further contains a reference dye, a 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); and the water is DEPC water. After the obtained DNA is added to the PCR fluorescence quantitative detection system containing the above-mentioned primer pair and probe, the concentrations of the components in the system are: 0.6 μM probe, 0.2 μM upstream primer, 0.2 μM downstream 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 was as follows: 95°C for 30 s; 95°C for 5 s, 60°C for 34 s, for 35 cycles.

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