Primer probe combinations and kits for detecting three pneumonia pathogens

Abstract

The application belongs to the technical field of biological detection, and specifically discloses a primer probe combination and a kit for detecting three kinds of pneumonia pathogens. The three kinds of pneumonia pathogens are pseudomonas aeruginosa, haemophilus influenzae and escherichia coli. The primer probe and the kit can simultaneously and parallelly detect the infection of the three kinds of pathogenic bacteria of pediatric bacterial pneumonia in one reaction tube, realize the detection of the three kinds of bacteria by single-tube PCR, have high sensitivity, good specificity, short detection time, accurate and reliable results, greatly reduce the pain of patients, save social resources, avoid drug resistance of pathogenic bacteria due to excessive use of antibacterial drugs, and have great clinical practicability.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to primer-probe combinations and kits for detecting three pneumonia pathogens. Background Technology

[0002] Currently used detection methods, such as bacterial isolation and culture, have drawbacks including demanding culture conditions, high difficulty, and long processing time. Immunoassay methods suffer from low sensitivity or specificity, and are prone to missed diagnoses, which can pose challenges to clinical treatment. In recent years, real-time fluorescence PCR technology has become the primary diagnostic tool due to its high specificity, high sensitivity, simple operation, and time-saving characteristics.

[0003] However, in current clinical practice, most pathogen detection methods employ single-target assays. Doctors often rely on experience to start with the most likely pathogens and then rule them out one by one. This approach is time-consuming, costly, exacerbates patient suffering, and delays optimal treatment. Consequently, doctors often resort to empirical medication without a clear etiological diagnosis, leading to drug abuse, increased toxicity and the likelihood of drug resistance, and a waste of medical resources, while failing to improve treatment efficacy.

[0004] For this reason, scholars both domestically and internationally have been dedicated to developing technologies for simultaneous detection of multiple targets for many years, but few similar technologies have yet achieved success. This is because the presence of multiple primer-probe pairs in a single reaction system can lead to unimaginable complexities. First, it is well known that if there are several consecutive complementary bases between primers and probes, they are very likely to bind together under the action of polymerase, resulting in amplification and extension, leading to a large number of non-specific false positive amplification products. Since all DNA consists of only four bases, the probability of three consecutive bases being identical to other sequences is 1 / 4 × 1 / 4 × 1 / 4 = 1 / 64. Clearly, as the number of primers and probes increases, the probability of overlapping or complementary sequences will increase rapidly, even becoming almost unavoidable. Therefore, primers and probes previously used for single-target detection will no longer be applicable.

[0005] The solution to the above problem is to carefully redesign primers and probes, making the differences between primer and probe sequences for each target as large as possible to avoid complementary regions. However, this leads to significant differences in melting temperature (TM value) among the primers and probes. It is well known that the TM value is related to the length of the DNA fragment and the difference in GC / AT content, with GC contributing more to the TM value than AT. Due to the requirements of polymerase recognition ability and amplification specificity, the length of primers and probes is limited to a very small range, such as 20 to 24 nucleotides, thus limiting the ability to regulate the TM value. The differences in the composition of each primer and probe sequence inevitably lead to significant differences in GC / AT content, resulting in large differences in their respective TM values. However, as is generally accepted in PCR theory, good amplification efficiency and specificity are obtained when the annealing / extension temperature is close to the TM value. Therefore, differences in TM values ​​will lead to inconsistent amplification efficiency and specificity for different targets in the same reaction system (i.e., the same temperature cycling parameters), and may even cause false positives for some targets while causing missed detections for others.

[0006] In addition, for many pathogens, there are non-pathogenic bacteria that are very similar to them in terms of species classification widely in the human body and the external environment. The two have a large number of homologous sequences, which also brings difficulties to the selection of specific primers and probes. As the number of primers and probes required for multi-target detection increases dramatically, the difficulty of sequence selection is even greater than that of single-target detection. The risk of false positives caused by cross-reaction due to similar microbial species also increases sharply.

[0007] To address the aforementioned technical problems, this invention utilizes big data and automated computer retrieval technology to repeatedly screen multiple highly specific sequences from a massive global database of known pathogen sequences as preliminary candidate sequences. Then, unique computer software technology is employed to analyze the non-specific complementarity between various sequence combinations and simulate the amplification process, thereby further screening for more suitable secondary candidate sequences. These sequences are then artificially synthesized and subjected to extensive practical testing with actual pathogens and potentially interfering bacterial samples, ultimately resulting in the selection of the following sequences for use in this invention. Therefore, the primer-probe combination included in this invention effectively overcomes the aforementioned problems that existing technologies cannot solve, possessing substantial technological innovation, uniqueness, proprietary nature, and even exclusivity; it is not merely a simple combination of existing single-target detection primers and probes. Summary of the Invention

[0008] In view of this, the present invention provides a primer-probe combination and kit for detecting three pneumonia pathogens in children. The primer-probe combination can achieve rapid, accurate and sensitive identification of PA / HI / Ec. At the same time, the kit containing the primer-probe combination has the advantages of simple and rapid operation and stable storage at 2~8℃.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: Primer-probe assemblies, including: Primer pairs as shown in SEQ ID NO. 1~2 and probe as shown in SEQ ID NO. 3 for detecting Pseudomonas aeruginosa; Primer pairs shown in SEQ ID NO. 4-5 and probe shown in SEQ ID NO. 6 for detecting Haemophilus influenzae; The primer pairs shown in SEQ ID NO. 7-8 and the probe shown in SEQ ID NO. 9 are used to detect Escherichia coli.

[0010] The primers, probes, and kits provided by this invention can easily and rapidly detect three pathogens causing bacterial pneumonia in children simultaneously and in parallel in one reaction tube. This achieves single-tube PCR detection of the three bacteria with high sensitivity, good specificity, short detection time, and accurate and reliable results.

[0011] In this invention, the fragment amplified by the primer pairs shown in SEQ ID NO. 1-2 is shown in SEQ ID NO. 13, which is the conserved OprD gene sequence of *Pseudomonas aeruginosa*. The fragment amplified by the primer pairs shown in SEQ ID NO. 4-5 is shown in SEQ ID NO. 14, which is the omp6 gene of *Haemophilus influenzae*. The fragment amplified by the primer pairs shown in SEQ ID NO. 7-8 is shown in SEQ ID NO. 15, which is the ydiJ gene of *Escherichia coli*. The fragment amplified by the primer pairs shown in SEQ ID NO. 10-11 is shown in SEQ ID NO. 16, which is the β-globin gene.

[0012] In some embodiments, the primer-probe combination further includes: Primer pairs as shown in SEQ ID NO. 10~11 for detecting internal standard genes; The probe shown in SEQ ID NO.12 is used to detect the internal standard gene.

[0013] In this invention, the probe has a fluorescent reporter group attached to its 5' end and a quencher group attached to its 3' end. Specifically: As shown in SEQ ID NO.3, the 5' end of the probe is connected to a FAM fluorescent group; As shown in SEQ ID NO.6, the 5' end of the probe is connected to a ROX fluorescent group; The probe shown in SEQ ID NO.9 has a CY5 fluorescent group attached to its 5' end; The probe shown in SEQ ID NO.12 has a HEX fluorescent group attached to its 5' end.

[0014] The present invention also provides the application of the primer-probe combination in the preparation of a kit for detecting Pseudomonas aeruginosa, Haemophilus influenzae and / or Escherichia coli.

[0015] The present invention also provides a multiplex PCR fluorescent detection kit for detecting Pseudomonas aeruginosa, Haemophilus influenzae and / or Escherichia coli, comprising the primer and probe combination described above.

[0016] The multiplex PCR fluorescence detection kit provided by this invention also includes one or more of Tricine, KOAc, Tween20, glycerol, DMSO, betaine, NaN3, dNTPs, rTth enzyme, UDG enzyme, manganese acetate, NaN3, and purified water.

[0017] In some embodiments, the kit provided by the present invention contains Tricine at a concentration of 1 M / L, KOAc at a concentration of 10 M / L, 10% Tween 20, 80% glycerol, 100% DMSO, dNTPs at a concentration of 25 mM, rTth enzyme at a concentration of 5 u / μL, UDG enzyme at a concentration of 2 u / μL, and manganese acetate at a concentration of 25 mM.

[0018] This invention also provides a method for simultaneously detecting Pseudomonas aeruginosa, Haemophilus influenzae, and Escherichia coli. The method involves using the multiplex PCR fluorescence detection kit described in this invention to perform Real-time PCR detection on the sample, and determining the presence of Pseudomonas aeruginosa, Haemophilus influenzae, or Escherichia coli based on the Ct value.

[0019] In some implementation schemes, the method for making the determination is as follows: The probe channels shown in SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO.9 showed no fluorescence, and the CT value of the probe channel shown in SEQ ID NO.12 was ≤35. Therefore, the test result was reported as negative. If the CT value of the probe channel shown in SEQ ID NO.3 is ≤34, it is reported as positive for Pseudomonas aeruginosa. If the CT value of the probe channel shown in SEQ ID NO.6 is ≤34, it is reported as positive for Haemophilus influenzae. If the CT value of the probe channel shown in SEQ ID NO.9 is ≤33, it is reported as positive for Escherichia coli. The probe channels shown in SEQ ID NO.3 all have CT values ​​≥34, but the probe channel shown in SEQ ID NO.12 has a CT value ≤35, indicating that the concentration of *Pseudomonas aeruginosa* in the sample is below the detection limit; the probe channels shown in SEQ ID NO.6 all have CT values ​​≥34, but the probe channel shown in SEQ ID NO.12 has a CT value ≤35, indicating that the concentration of *Haemophilus influenzae* in the sample is below the detection limit; the probe channels shown in SEQ ID NO.9 all have CT values ​​≥33, but the probe channel shown in SEQ ID NO.12 has a CT value ≤35, indicating that the concentration of *Escherichia coli* in the sample is below the detection limit. When the CT value of the probe channel shown in SEQ ID NO.12 is ≥35, and either the negative control has a CT value or shows a typical S amplification curve, or the positive control has no CT value or no amplification curve, the test result is invalid. The cause should be found and eliminated, and the test should be repeated.

[0020] In some implementations, the sample is sputum; the Real-time PCR detection system includes: 20 μL reaction solution 1, 10 μL reaction solution 2 and 50 μL sample nucleic acid; The reaction solution 1 comprises: .

[0021] In some implementations, the procedure for the Real-time PCR assay includes: .

[0022] This invention optimizes the reaction system based on this sequence combination, adjusting specific components, their concentrations, and parameters to further ensure excellent detection performance. Specifically, the addition of DMSO and its appropriate concentration adjust the thermal conductivity and surface tension of the reaction solution, ensuring the homogeneity of this complex amplification reaction. Betaine, as a mild DNA denaturant, at this concentration appropriately reduces the difference in the contribution of GC and AT to the TM value in the primer-probe combination, making the TM values ​​of these primers and probes tend to be consistent, thus contributing to the coordinated amplification efficiency and specificity of multi-target detection under the same amplification temperature cycle. Furthermore, as an rTth enzyme integrating both reverse transcription and DNA polymerization activities, the conformation of its active site responsible for nucleic acid recognition and replication is unique, resulting in different effects of manganese, magnesium, and potassium ions on activating its activity; that is, the three have different effects on reverse transcription, DNA replication, amplification specificity, and amplification efficiency. Therefore, this invention optimizes their ratio and concentration to balance these characteristics in the primer-probe combination. Meanwhile, the pH value of the reaction system has different effects on the amplification specificity and amplification efficiency of different primers and probes. In this invention, it has also been specifically optimized to adapt it to the above primer and probe combination.

[0023] The primer-probe combination provided by this invention includes specific primer pairs and probes targeting Pseudomonas aeruginosa, Haemophilus influenzae, and Escherichia coli. This primer-probe combination exhibits good specificity, and when combined with real-time PCR detection, it enables rapid, accurate, and sensitive identification of PA / HI / Ec. Experiments show that the detection limits for PA / HI / Ec by the primers and probes of this invention are all 500 copies / ml. Furthermore, the kit containing these primers and probes provided by this invention exhibits good stability; the kit can stably detect samples after storage at 2–8°C for 3, 6, 9, and 12 months. Attached Figure Description

[0024] Figure 1 This shows an amplification diagram of Pseudomonas aeruginosa in Example 5; Figure 2 This shows an amplification diagram of Haemophilus influenzae in Example 5; Figure 3 This shows an amplification diagram of Escherichia coli in Example 5; Figure 4 This shows amplification images of Pseudomonas aeruginosa under different concentrations of samples in Example 6. Figure 4 a represents the detection result using the kit from Example 1 of this invention, and 4b represents the detection result using the control primer probe; Figure 5 This shows amplification images of Haemophilus influenzae at different concentrations in Example 6, where... Figure 5 a represents the detection result using the kit from Example 1 of this invention, and 5b represents the detection result using the control primer probe; Figure 6 This shows amplification images of Escherichia coli at different concentrations in Example 6, where... Figure 6 a represents the detection result using the kit from Example 1 of this invention, and 6b represents the detection result using the control primer probe. Detailed Implementation

[0025] This invention provides primer-probe combinations and kits for detecting three pneumonia pathogens in children. Specific implementation schemes are provided to further illustrate the technical solution of this invention. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention. It should be particularly noted that all similar changes, combinations, substitutions, and modifications are considered part of this invention.

[0026] The consumables and instruments used in this invention are all commercially available products.

[0027] The present invention will be further illustrated below with reference to the embodiments: Example 1: Preparation of a nucleic acid detection kit for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli The primer and probe sequences in the kit are shown in Table 1 below: Table 1 Primer and probe sequences

[0028] The kit also includes: Tricine at a concentration of 1 M / L, KOAc at a concentration of 10 M / L, 10% Tween 20, 80% glycerol, 100% DMSO, dNTPs at a concentration of 25 mM, rTth enzyme at a concentration of 5 u / μL, UDG enzyme at a concentration of 2 u / μL, and manganese acetate at a concentration of 25 mM.

[0029] Example 2: Detection method of the kit of the present invention

[0030] The detection method of this invention is real-time PCR. The real-time PCR reaction process is as follows: (1) Pre-denaturation: the time and length depend on the length and base composition of the target nucleic acid. The pre-denaturation temperature is generally 90℃~105℃ and the time is generally 2~10min. The purpose of pre-denaturation is to completely separate the double-stranded nucleic acid sequence into single strands; (2) Denaturation: the temperature is generally 91℃~105℃ and the time is generally 10s~35s; (3) Annealing: each primer is annealed to the target sequence of Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli or internal standard nucleic acid. The annealing temperature is usually 40℃~60℃ and the annealing time can be 10s~60s; (4) Extension: the primers bind to the template and begin to synthesize new DNA double strands. The extension temperature is generally 40℃~80℃ and the extension time can be 10s~1min.

[0031] The composition of each detection system is shown in Table 2: Table 2

[0032] Table 3

[0033] Fluorescence detection channel selection: (1) Select FAM channel (ReporTer: FAM, QuenCher: none) to detect Pseudomonas aeruginosa; (2) Select ROX channel (ReporTer: ROX, QuenCher: none) to detect Haemophilus influenzae; (3) Select CY5 channel (ReporTer: CY5, QuenCher: none) to detect Escherichia coli; (4) Select HEX channel to detect internal standard; (5) Set reference fluorescence (PAssive ReferenceCe) to none. The real-time reaction conditions for quantitative fluorescence are shown in Table 4 below.

[0034] Table 4: Real-time PCR reaction conditions

[0035] After the reaction, the instrument automatically saves the results. The instrument's built-in software can be used for automatic analysis, or the baseline start, end, and threshold values ​​can be manually adjusted for analysis. Then, the sample CT values ​​and setpoint results are recorded. Specific test results analysis is as follows: The probe channels shown in SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO.9 showed no fluorescence, and the CT value of the probe channel shown in SEQ ID NO.12 was ≤35. Therefore, the test result was reported as negative. If the CT value of the probe channel shown in SEQ ID NO.3 is ≤34, it is reported as positive for Pseudomonas aeruginosa. If the CT value of the probe channel shown in SEQ ID NO.6 is ≤34, it is reported as positive for Haemophilus influenzae. If the CT value of the probe channel shown in SEQ ID NO.9 is ≤33, it is reported as positive for Escherichia coli. The probe channels shown in SEQ ID NO.3 all have CT values ​​≥34, but the probe channel shown in SEQ ID NO.12 has a CT value ≤35, indicating that the concentration of *Pseudomonas aeruginosa* in the sample is below the detection limit; the probe channels shown in SEQ ID NO.6 all have CT values ​​≥34, but the probe channel shown in SEQ ID NO.12 has a CT value ≤35, indicating that the concentration of *Haemophilus influenzae* in the sample is below the detection limit; the probe channels shown in SEQ ID NO.9 all have CT values ​​≥33, but the probe channel shown in SEQ ID NO.12 has a CT value ≤35, indicating that the concentration of *Escherichia coli* in the sample is below the detection limit. When the CT value of the probe channel shown in SEQ ID NO.12 is ≥35, and either the negative control has a CT value or shows a typical S amplification curve, or the positive control has no CT value or no amplification curve, the test result is invalid. The cause should be found and eliminated, and the test should be repeated.

[0036] Example 3: Feasibility test of the kit of the present invention

[0037] 1. Limit of Detection (LOD) Test

[0038] (1) Preparation of nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli: The nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli was prepared by means of the method in Example 1.

[0039] (2) Sample extraction

[0040] Five different concentrations of sputum samples of Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli (treated with sputum pretreatment reagent) were mixed with a pipette and then purified using nucleic acid extraction and purification reagents from Zhengzhou Antu Bioengineering Co., Ltd.

[0041] (3) Sample testing

[0042] Add 50 μL of the processed sample supernatant (nucleic acid) to a reaction tube for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli nucleic acid detection reagent, with 21 replicates for each concentration. At the same time, add 50 μL of purified water to the detection solution as a negative control. Perform the detection according to the detection method in Example 2.

[0043] (4) Results Analysis

[0044] The kit prepared in Example 1 and the detection method in Example 2 were used to detect samples of different concentration gradients of the nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli. The results showed that the detection sensitivity (LOD) of this method for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli was 500 copies / ml. Specific data are shown in Tables 5, 6, and 7.

[0045] Table 5: Confirmation Limit of Detection for Pseudomonas aeruginosa

[0046] Table 6: Confirmatory Limits for Detection of Haemophilus influenzae

[0047] Table 7: Confirmation Limits for Detection of Escherichia coli

[0048] 2. Cross-reactivity with other diseases

[0049] (1) Preparation of nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli: The nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli was prepared by means of the method in Example 1.

[0050] (2) Extraction of cross-pathogen samples: Sputum samples containing Enterococcus faecalis, Serratia marcescens, Staphylococcus epidermidis, Candida tropicalis, Candida krusei, Enterococcus faecalis, Candida albicans, Klebsiella pneumoniae, Streptococcus pyogenes, Candida glabrata, Proteus mirabilis, Streptococcus agalactiae, Enterobacter cloacae, Candida parapneumoniae, Neisseria meningitidis, Micrococcus luteus, Rhodococcus equi, Haemophilus parainfluenzae, Pseudomonas fluorescens, Aeromonas hydrophila, Streptococcus pneumoniae, Legionella pneumophila, Moraxella catarrhalis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, human metapneumovirus, influenza A virus, influenza B virus, respiratory syncytial virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, methicillin-resistant coagulase-negative staphylococci, Acinetobacter baumannii, Stenotrophomonas maltophilia, and Burkholderia cepacia are mixed with a pipette. Nucleic acid DNA was purified using nucleic acid extraction and purification reagents from Zhengzhou Antu Biotechnology Co., Ltd.

[0051] (3) Sample detection: Add 50 μL of the processed sample supernatant (nucleic acid) to the Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli nucleic acid detection reaction tube, and add 50 μL of purified water to the detection solution as a negative control. The extracted Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli is used as a positive control test. The detection is carried out according to the detection method in Example 2.

[0052] (4) Results Analysis: The kit prepared in Example 1 and the detection method in Example 2 were used to detect pathogens other than Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli. The results showed that the kit of the present invention was positive for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli, negative for the positive control, and negative for the negative control. Enterococcus faecalis, Serratia marcescens, Staphylococcus epidermidis, Candida tropicalis, Candida krusei, Enterococcus faecalis, Candida albicans, Klebsiella pneumoniae, Streptococcus pyogenes, Candida glabrata, Proteus mirabilis, Streptococcus agalactiae, Enterobacter cloacae, and Candida glabrata were also detected. Samples infected with the following pathogens showed no cross-reactivity, indicating high specificity. Specific results are shown in Table 8.

[0053] Table 8: Cross-reactivity experiments

[0054] 3. Anti-interference capability

[0055] (1) Preparation of nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli: The nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli was prepared by means of the method in Example 1.

[0056] (2) Sample processing

[0057] A mixed detection limit concentration sample of Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli was selected. Interfering substances were added to the bacterial sample at 3 times the peak concentration (Cmax). The sample was processed using the cross-reaction method in Example 3 and detected according to the detection method in Example 2.

[0058] (3) Results Analysis

[0059] Experiments show that the detection sensitivity of the kit of the present invention is not affected when the sample contains mucin, blood, sodium chloride, dexamethasone, histamine hydrochloride, menthol, oseltamivir, mupirocin, or tobramycin, as detailed in Table 9.

[0060] Table 9: Anti-interference test of exogenous substances

[0061] Example 4: Stability assessment of the reagent kit of the present invention at 2-8℃

[0062] 1. Accelerated stability test

[0063] (1) Preparation of nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli: The nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli was prepared by means of the method in Example 1.

[0064] (2) Sample extraction

[0065] The sputum samples containing the detection limit concentration of Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli in the tube were mixed with a pipette and the nucleic acid DNA was purified using nucleic acid extraction and purification reagents from Zhengzhou Antu Bioengineering Co., Ltd.

[0066] (3) Sample testing

[0067] 50 μL of the extracted sample supernatant (i.e., the nucleic acid in the sample) was added to a Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli nucleic acid detection reagent reaction tube accelerated at 37°C for 7, 14, and 21 days. 20 positive samples were used for each target. At the same time, 50 μL of purified water was added to the detection solution as a negative control. The detection was carried out according to the detection method in Example 2.

[0068] (4) Results Analysis

[0069] The nucleic acid detection reagents for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli were tested using the kit prepared in Example 1 and the detection method in Example 2 at 37°C for 7, 14, and 21 days. Specific data are shown in Table 10.

[0070] Table 10: Accelerated Stability Test

[0071] 2. Real-time stability test

[0072] (1) Preparation of nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli: The nucleic acid detection reagent for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli was prepared by means of the method in Example 1.

[0073] (2) Sample extraction

[0074] The sputum samples containing the detection limit concentration of Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli in the tube were mixed with a pipette and the nucleic acid DNA was purified using nucleic acid extraction and purification reagents from Zhengzhou Antu Bioengineering Co., Ltd.

[0075] (3) Sample testing

[0076] Add 50 μL of the processed specimen supernatant (nucleic acid) to a reaction tube containing Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli nucleic acid detection reagent, which can be transported at 2-8℃ for 7 days and stored at 2-8℃ for 3, 6, 9, or 12 months. For each target, there are 20 positive samples. At the same time, add 50 μL of purified water to the detection solution as a negative control. The detection is performed according to the detection method in Example 2.

[0077] (4) Results Analysis

[0078] The nucleic acid detection reagents for Pseudomonas aeruginosa / Haemophilus influenzae / Escherichia coli were tested using the kit prepared in Example 1 and the detection method in Example 2. The results are shown in Table 11.

[0079] Table 11: Real-time stability test

[0080] Example 5: Validation of the concordance rate of positive and negative clinical samples

[0081] The kit, having passed quality control, was used to validate collected clinical samples. Nucleic acid testing was performed on 10 clinically positive samples each of Pseudomonas aeruginosa, Haemophilus influenzae, and Escherichia coli, as well as 20 negative samples. To ensure the reliability of the kit's results, positive and negative control samples were included. The results showed a 100% concordance rate between positive and negative results for the clinical samples. Specific data... Figure 1 , Figure 2 , Figure 3 The test results from the reagent kit are as follows: Table 12: Clinical positive / negative concordance rate trial

[0082] Example 6: Comparative Test of Sensitivity Detection

[0083] Mix reaction solution 1 and reaction solution 2, and prepare the system using primers and probes corresponding to other bacteria (see Table 13). Then, add 50 μL of the corresponding sample nucleic acid to each system, with a sample nucleic acid concentration of 5 × 10⁻⁶. 6 copies / ml, 5×10 4 copies / ml, 5×10 3 copies / ml, 5×10 2 Copies / ml were used together for analysis. Results are as follows: Figures 4-6 As shown, where, Figure 4 a, 5a, and 6a are the results of detection using the kit of Example 1 of the present invention, and 4b, 5b, and 6b are the results of detection using the control primer probe.

[0084] Table 13 Control primer and probe sequences

[0085] The results showed that the three bacteria could be detected at four different concentration gradients using the system of this invention, while the systems using other primers and probes could not be detected at 5 × 10⁻⁶ concentrations. 2 The copies / ml were undetectable, and their effect was far inferior to that of the present invention. The detection sensitivity of the present invention is higher than that of other primer probes.

[0086] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer-probe combination, characterized in that, include: Primer pairs as shown in SEQ ID NO. 1~2 and probe as shown in SEQ ID NO. 3 for detecting Pseudomonas aeruginosa; Primer pairs shown in SEQ ID NO. 4-5 and probe shown in SEQ ID NO. 6 for detecting Haemophilus influenzae; The primer pairs shown in SEQ ID NO. 7-8 and the probe shown in SEQ ID NO. 9 are used to detect Escherichia coli.

2. The primer-probe combination according to claim 1, characterized in that, Also includes: Primer pairs as shown in SEQ ID NO. 10~11 for detecting internal standard genes; The probe shown in SEQ ID NO.12 is used to detect the internal standard gene.

3. The primer-probe combination according to claim 2, characterized in that, The probe has a fluorescent reporter group attached to its 5' end and a quencher group attached to its 3' end.

4. The primer-probe combination according to claim 3, characterized in that, As shown in SEQ ID NO.3, the 5' end of the probe is connected to a FAM fluorescent group; As shown in SEQ ID NO.6, the 5' end of the probe is connected to a ROX fluorescent group; The probe shown in SEQ ID NO.9 has a CY5 fluorescent group attached to its 5' end; The probe shown in SEQ ID NO.12 has a HEX fluorescent group attached to its 5' end.

5. The use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of a kit for detecting Pseudomonas aeruginosa, Haemophilus influenzae and Escherichia coli.

6. A kit for detecting Pseudomonas aeruginosa, Haemophilus influenzae, and Escherichia coli, characterized in that, Includes the primer-probe combination as described in claim 1 or 2.

7. The reagent kit according to claim 6, characterized in that, It also includes Tricine, KOAc, Tween20, glycerol, DMSO, betaine, NaN3, dNTPs, rTth enzyme, UDG enzyme, manganese acetate, NaN3, and one or more of the purified water.

8. A method for simultaneously detecting Pseudomonas aeruginosa, Haemophilus influenzae, and Escherichia coli for non-diagnostic purposes, characterized in that, The sample is subjected to Real-time PCR detection using the kit described in claim 6 or 7, and the presence of Pseudomonas aeruginosa, Haemophilus influenzae, or Escherichia coli is determined based on the fluorescence signal.

9. The method according to claim 8, characterized in that, The Real-time PCR detection system includes: 20 μL reaction solution 1, 10 μL reaction solution 2, and 50 μL sample nucleic acid; 。 10. The method according to claim 8, characterized in that, The procedure for the Real-time PCR detection includes: 。

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