Kit for detecting urinary tract infection pathogenic bacteria and preparation method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a kit for detecting urinary tract infection pathogenic bacteria and a preparation method of the kit. The multiplex fluorescent quantitative PCR detection method is constructed aiming at common urinary tract infection pathogenic bacteria for the first time, and the method takes conserved regions of escherichia coli, staphylococcus aureus, enterococcus faecium, proteusbacillus vulgaris, klebsiella pneumoniae and pseudomonas aeruginosa as detection targets; according to the present invention, the multiplex fluorescence quantitative PCR primer probe is obtained by using the on-line primer design software so as to achieve the detection analysis of the common urinary tract infection pathogenic bacteria, and the method has advantages of high efficiency, rapidness, high sensitivity, high specificity and the like, can rapidly achieve the identification of the urinary tract infection pathogenic bacteria, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a kit for detecting urinary tract infection pathogenic bacteria and a preparation method thereof. Background Art

[0002] Urinary tract pathogen infection, also known as urinary tract infection (UTI), is a common infectious disease, usually caused by bacteria. Urinary tract infections are generally divided into upper urinary tract infections and lower urinary tract infections. According to the route of bacterial invasion, it can be further divided into: hematogenous infection, ascending infection, and descending infection. Most women have ascending infection because the female urethra is straight and short, and bacteria can easily ascend to the bladder to cause infection. In addition, the external urethral orifice of women is very close to the vaginal orifice and anus, and is easily contaminated by bacteria excreted in feces and vagina. Coupled with the reduced ability of elderly women to completely empty the bladder, bacteria can easily multiply in the residual urine. Clinically, the common lower urinary tract infection, namely cystitis, is a common and frequently-occurring disease in elderly women, and its incidence rate is 8-10 times higher than that in men.

[0003] With the development and maturity of nucleic acid detection technology, more and more nucleic acid detection technologies for urinary tract infection pathogens have emerged. Relying on nucleic acid detection technology, the diagnostic results of pathogens and drug resistance genes can be obtained within a few hours, which can make up for the deficiencies of traditional culture methods. However, the existing detection technologies cannot cover common urinary tract pathogenic bacteria, and it is also impossible to detect the above-mentioned pathogenic bacteria in one system. In summary, establishing a set of detection methods for urinary tract infection pathogenic bacteria that are fast, accurate, sensitive, and specific is an urgent technical problem to be solved in this field. Summary of the Invention

[0004] Aiming at the technical problems existing in the prior art, the present invention provides a kit for detecting urinary tract infection pathogenic bacteria and a preparation method thereof. The present invention for the first time constructs a multiplex fluorescence quantitative PCR detection method for common urinary tract infection pathogenic bacteria. This method uses the conserved regions of Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Proteus, Klebsiella pneumoniae, and Pseudomonas aeruginosa as detection targets, obtains multiplex fluorescence quantitative PCR primers and probes by using an online primer design software, and thereby realizes the detection and analysis of common urinary tract infection pathogenic bacteria. This method has the advantages of high efficiency, rapidity, high sensitivity, and specificity, can quickly identify urinary tract infection pathogenic bacteria, and has great application prospects.

[0005] The primary object of the present invention is to provide a set of multiplex fluorescence quantitative PCR primer sets for detecting urinary tract infection pathogenic bacteria, and the primer sets include:

[0006] The primer pair for detecting Escherichia coli SEQ ID NO,1-2;

[0007] The primer pair for detecting Staphylococcus aureus SEQ ID NO,4-5;

[0008] Primer pair SEQ ID NO: 7 - 8 for detecting Enterococcus faecalis;

[0009] Primer pair SEQ ID NO: 10 - 11 for detecting Proteus spp.;

[0010] Primer pair SEQ ID NO: 13 - 14 for detecting Klebsiella pneumoniae; and

[0011] Primer pair SEQ ID NO: 16 - 17 for detecting Pseudomonas aeruginosa.

[0012] The present invention also provides a set of multiplex fluorescence quantitative PCR probe sets for detecting pathogenic bacteria causing urinary tract infections, and the probe sets include:

[0013] Probe SEQ ID NO: 3 for detecting Escherichia coli;

[0014] Probe SEQ ID NO: 6 for detecting Staphylococcus aureus;

[0015] Probe SEQ ID NO: 9 for detecting Enterococcus faecalis;

[0016] Probe SEQ ID NO: 12 for detecting Proteus spp.;

[0017] Probe SEQ ID NO: 15 for detecting Klebsiella pneumoniae; and

[0018] Probe SEQ ID NO: 18 for detecting Pseudomonas aeruginosa.

[0019] Preferably, the 5'-end of the probe is labeled with a fluorescent emitting group, and the 3'-end is labeled with a quenching group.

[0020] Preferably, the 5'-ends of the probes for detecting different pathogenic bacteria are labeled with different fluorescent emitting groups.

[0021] Preferably, the fluorescent emitting group is selected from FAM, TET, HEX, Joe, ROX, TAMRA, VIC, CY3 or CY5.

[0022] Preferably, the quenching group is selected from BHQ1, BHQ2, Dabcyl, Eclipse or MGB.

[0023] Another object of the present invention is to provide a kit for detecting pathogenic bacteria causing urinary tract infections, and the kit contains multiplex fluorescence quantitative PCR detection reagents for simultaneously detecting Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Proteus spp., Klebsiella pneumoniae and Pseudomonas aeruginosa.

[0024] Preferably, the multiplex fluorescence quantitative PCR detection reagent includes the above-mentioned multiplex fluorescence quantitative PCR primer set.

[0025] Preferably, the multiplex fluorescence quantitative PCR detection reagent includes the above-mentioned multiplex fluorescence quantitative PCR probe set.

[0026] Preferably, the multiplex fluorescence quantitative PCR detection reagent further includes 2×Taq PCR Mix, template and / or ddH2O.

[0027] Another object of the present invention is to provide the use of the above-mentioned kit for detecting pathogenic bacteria causing urinary tract infections in the preparation of products for detecting pathogenic bacteria causing urinary tract infections.

[0028] Another aspect of the present invention is to further provide a method for detecting pathogenic bacteria causing urinary tract infections, the method comprising the following steps:

[0029] 1) Extract DNA from the sample to be tested;

[0030] 2) Using the DNA as a template, perform multiplex fluorescence quantitative PCR reaction using the above-mentioned kit;

[0031] 3) Use a microplate reader to record the fluorescence signal in the product.

[0032] Preferably, the total reaction system of multiplex fluorescence quantitative PCR in step 2) includes 2×Taq PCR Mix, primer set, probe set, template and / or ddH2O.

[0033] Preferably, the multiplex fluorescence quantitative PCR reaction program: Step 1: Pre-denature at 95°C for 10 minutes;

[0034] Step 2: Denature at 95°C for 10 seconds, anneal at 55°C for 50 seconds, and extend at 70°C for 10 seconds, and cycle step 2 for 35 times;

[0035] Step 3: Extend at 70°C for 10 minutes;

[0036] Step 4: Store at 4°C for low temperature.

[0037] Determination of test results: After the PCR reaction program is completed, record the fluorescence value of each well, and determine whether the test result is positive according to the size of the fluorescence value.

[0038] The advantages of the present invention are as follows: The present invention constructs a multiplex fluorescence quantitative PCR detection method for common urinary tract infection pathogens for the first time. This method uses the conserved regions of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Proteus, Klebsiella pneumoniae, and Pseudomonas aeruginosa as detection targets, obtains multiplex fluorescence quantitative PCR primer probes using an online primer design software, and thereby realizes the detection and analysis of common urinary tract infection pathogens. This method has the advantages of high efficiency, rapidity, high sensitivity, and specificity, can quickly identify urinary tract infection pathogens, and has great application prospects. Brief Description of the Drawings

[0039] Figure 1 : Sensitivity detection of multiplex fluorescence quantitative PCR. Detailed Description of the Invention

[0040] The following further describes the present invention in detail with reference to specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0041] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0042] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well-known to those skilled in the art.

[0043] Example 1

[0044] 1.1 Primer Design and Synthesis

[0045] Taking the clinically common urinary tract infection pathogens: Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Proteus, Klebsiella pneumoniae, and Pseudomonas aeruginosa as the detection objects of the present invention, using a primer design software to design a multiplex PCR amplification primer probe group based on the conserved regions of each pathogen, wherein the 5' end of the probe is labeled with a fluorescent luminescent group and the 3' end is labeled with a quenching group. The detection primer probes for each group are shown in Table 1 below.

[0046] Table 1. Sequence Information of Detection Primer Probes for Urinary Tract Infection Pathogens

[0047]

[0048]

[0049] 1.2 Preparation of Pathogenic Bacteria Standard Samples

[0050] Using the primers in Table 1 above, genomic DNA of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris, Klebsiella pneumoniae, and Pseudomonas aeruginosa was amplified by conventional PCR. After the PCR amplification products were recovered and purified by gel electrophoresis, they were ligated into the pGM-T vector, transfected into DH5α Escherichia coli for culture, and positive recombinant bacteria were screened by blue-white screening and PCR. The recombinant plasmids were extracted using a plasmid extraction kit. The plasmid concentration was quantified using a nucleic acid analyzer Nanodrop2000, and the concentration was converted to the copy number using Avogadro's constant. The recombinant plasmid with the calculated copy number was diluted to a concentration of 1×10 7 copies / μL to prepare a standard for pathogenic bacteria, which was stored at -70°C for later use.

[0051] Example 2

[0052] 2.1 Establishment of multiplex fluorescence quantitative PCR reaction system

[0053] The total reaction system of multiplex fluorescence quantitative PCR provided by the present invention is 50 μL, and the specific reaction system is shown in Table 2 below:

[0054] Table 2. Multiplex fluorescence quantitative PCR reaction system

[0055]

[0056]

[0057] The multiplex fluorescence quantitative PCR reaction program provided by the present invention includes:

[0058] Step 1: Pre-denaturation at 95°C for 10 minutes;

[0059] Step 2: Denaturation at 95°C for 10 seconds, annealing at 55°C for 50 seconds, and extension at 70°C for 10 seconds, and repeat Step 2 for 35 cycles;

[0060] Step 3: Extension at 70°C for 10 minutes;

[0061] Step 4: Preservation at 4°C at low temperature.

[0062] Determination of test results: After the PCR reaction program is completed, record the fluorescence values of each well, and determine whether the test results are positive according to the fluorescence value.

[0063] 2.2 Verification of multiplex fluorescence quantitative PCR reaction

[0064] Using the recombinant plasmid prepared in Section 1.2 as the target, the roots were detected using the multiplex fluorescence quantitative PCR reaction system of the present invention, and the results are shown in Table 3 below:

[0065] Table 3. Verification of multiplex fluorescence quantitative PCR reaction

[0066]

[0067]

[0068] The results show that the multiplex fluorescence quantitative PCR reaction system of the present invention can achieve specific detection and analysis of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Proteus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, and has a higher fluorescence intensity value compared to the blank group.

[0069] 2.3 Specific detection

[0070] Using the established multiplex fluorescence quantitative PCR reaction system, Helicobacter pylori, Streptococcus pneumoniae, Salmonella, and Bacillus subtilis were added as detection targets respectively. The results are shown in Table 4 below. It is confirmed that no fluorescence signal appears in the above-mentioned bacteria, which proves that the multiplex fluorescence quantitative PCR reaction system of the present invention only shows specificity to the target virus and does not cross-react with other detection targets.

[0071] Table 4. Specific detection

[0072] Fluorescence intensity signal (RFU) Helicobacter pylori 36.15±2.21 Streptococcus pneumoniae 35.14±3.12 Salmonella 36.44±1.68 Bacillus subtilis 38.64±2.25

[0073] 2.4 Sensitivity detection

[0074] Taking the pathogen standard product prepared in Example 1 as the object, the pathogen standard product was serially diluted 10-fold with TE (pH 7.0) buffer to prepare 1×10 5 -1×10 -4 copies / μL. Three replicates were set for each concentration gradient, and nuclease-free water was used as the template-free control. Based on the multiplex fluorescence quantitative PCR reaction system, the detection limit of the method for the above-mentioned pathogens was determined to evaluate the sensitivity of the method. The results are as Figure 1 shown that the lowest detection limit for Escherichia coli can reach 1×10 -3 copies / μL, and the other bacteria also have the same detection limit. The results are not repeated here.

[0075] The functions of the above-mentioned examples are to illustrate the substantial content of the present invention, but do not limit the protection scope of the present invention. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solutions of the present invention.

Claims

1. A set of multiplex fluorescence quantitative PCR primer sets for detecting pathogenic bacteria of urinary tract infection, characterized in that, The primer set includes: The primer pair SEQ ID NO: 1-2 for detecting Escherichia coli; The primer pair SEQ ID NO: 4-5 for detecting Staphylococcus aureus; The primer pair SEQ ID NO: 7-8 for detecting Enterococcus faecalis; The primer pair SEQ ID NO: 10-11 for detecting Proteus; The primer pair SEQ ID NO: 13-14 for detecting Klebsiella pneumoniae; and The primer pair SEQ ID NO: 16-17 for detecting Pseudomonas aeruginosa.

2. A multiplex fluorescence quantitative PCR probe set for detecting urinary tract infection pathogenic bacteria, characterized in that, The probe set includes: The probe SEQ ID NO: 3 for detecting Escherichia coli; The probe SEQ ID NO: 6 for detecting Staphylococcus aureus; The probe SEQ ID NO: 9 for detecting Enterococcus faecalis; The probe SEQ ID NO: 12 for detecting Proteus; The probe SEQ ID NO: 15 for detecting Klebsiella pneumoniae; and The probe SEQ ID NO: 18 for detecting Pseudomonas aeruginosa.

3. The multiplex fluorescence quantitative PCR probe set according to claim 2, wherein The 5'-end of the probe is labeled with a fluorescent emitting group, and the 3'-end is labeled with a quenching group.

4. The multiplex fluorescence quantitative PCR probe set according to claim 3, wherein, The 5'-ends of the probes for detecting different pathogenic bacteria are labeled with different fluorescent emitting groups.

5. The multiplex fluorescence quantitative PCR probe set according to claim 3, wherein The fluorescent emitting group is selected from FAM, TET, HEX, Joe, ROX, TAMRA, VIC, CY3 or CY5.

6. The multiplex fluorescence quantitative PCR probe set according to claim 3, wherein The quenching group is selected from BHQ1, BHQ2, Dabcyl, Eclipse or MGB.

7. A kit for detecting pathogenic bacteria causing urinary tract infections, characterized in that, The kit contains a multiplex fluorescence quantitative PCR detection reagent for simultaneously detecting Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Proteus, Klebsiella pneumoniae and Pseudomonas aeruginosa.

8. The kit according to claim 7, wherein The multiplex fluorescence quantitative PCR detection reagent includes the multiplex fluorescence quantitative PCR primer set as claimed in claim 1.

9. The kit according to claim 7, characterized in that The multiplex fluorescence quantitative PCR detection reagent includes the multiplex fluorescence quantitative PCR probe set as claimed in any one of claims 2-6.

10. The kit according to claim 7, characterized in that, The multiplex fluorescence quantitative PCR detection reagent further includes 2×Taq PCR Mix, template and / or ddH2O.

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