Primer group for identifying multiple genes of 10 bacteria related to lower respiratory tract infection and application of primer group

Abstract

The invention provides a primer group for identifying multiple genes of ten bacteria related to lower respiratory tract infection and application of the primer group, and belongs to the technical field of gene detection, the sequence of the primer group is shown as SEQ ID NO.1-20, the primer group designs specific primers for ten bacteria such as pseudomonas aeruginosa and streptococcus pyogenes, and the specific primers of ten genes of a 5'end primer are labeled by FAM fluorescence. 10 target gene loci and 2 internal reference loci can be detected in one PCR system, the traditional PCR deviation is overcome, and gene expression is accurately quantified. The reagent and the kit developed on the basis have the advantages of simplicity and convenience in operation, low cost, high accuracy and the like. When the kit is used, DNA of a sample is extracted for multiple PCR, and pathogen load is determined through electrophoresis and software analysis. Experiments prove that the method has strong specificity and high sensitivity to 10 bacteria, and has important application value in LRTI bacterial identification.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and particularly relates to a multiplex gene identification primer set for 10 bacteria related to lower respiratory tract infection and its application. Background Art

[0002] Respiratory tract infection is the most common infectious disease seriously threatening human health clinically, and can be divided into upper respiratory tract infection and lower respiratory tract infection. LRTI is an infection in the area below the larynx, often causing serious symptoms such as acute and chronic tracheitis, acute and chronic pneumonia, bronchitis, etc.

[0003] The most common pathogens of LRTI are bacteria (including Mycobacterium tuberculosis and Legionella), and can also be caused by fungi, chlamydia, mycoplasma and other atypical microorganisms. Some viruses can also cause LRTI. Based on a comprehensive statistical analysis of a large number of literature reports, the most common pathogens causing LRTI in China include Klebsiella pneumoniae (Kpn), Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hin), Pseudomonas aeruginosa (Pau), Legionella pneumophila (Leg), Staphylococcus aureus (Sau), Stenotrophomonas maltophilia (Sma), Mycobacterium tuberculosis (TB), Escherichia coli (E. coli), Acinetobacter baumannii (Aba), Enterobacter cloacae (Ecl), Serratia marcescens (SM), Haemophilus parainfluenzae (Hpi), Streptococcus pyogenes (SPY), Cryptococcus neoformans (Cn), Aspergillus, Candida albicans (Cal), Mycoplasma pneumoniae (Mpn), Chlamydia pneumoniae (Cpn), Chlamydia trachomatis (Ctr), Adenovirus (HAdV), Parainfluenza virus (HPIV), Respiratory Syncytial Virus (RSV) and Novel Coronavirus-19 (COVID-19), covering more than 95% of the pathogens.

[0004] At present, the tests for LRTI pathogens in clinical laboratories mainly include smear microscopy and isolation culture methods for bacteria, fungi, and mycoplasma. For viruses, it mainly relies on immunological detection and molecular biology methods, etc. Common sample types include sputum, bronchoalveolar lavage fluid, peripheral blood, and serous cavity effusion.

[0005] The conventional techniques for detecting LRTI pathogens at home and abroad are as follows:

[0006] (1) Smear microscopy: Smear microscopy is a classic method for etiological diagnosis, mainly used for acid-fast staining microscopy of Mycobacterium tuberculosis infection and India ink staining microscopy of Cryptococcus neoformans. The advantages of smear microscopy are simplicity, economy, and the fastest detection speed, which can quickly provide a reference basis for the diagnosis of LRTI. The disadvantage is poor specificity, and requires experimental personnel to have rich experience. Clinically, the isolation culture method and smear microscopy are combined to improve the diagnostic accuracy and reduce false negatives and false positives.

[0007] (2) Pathogen isolation culture method: The isolation culture method is the gold standard for diagnosing LRTI caused by bacteria and fungi, and is a routine method for detecting respiratory secretions. The advantage of the culture method is that a positive culture can identify the pathogen type, and at the same time, in vitro drug sensitivity tests can be performed. The results of the drug sensitivity tests can provide accurate and effective reference basis for clinical treatment of LRTI and control of bacterial drug resistance, etc. The disadvantages are high nutritional requirements, low positive detection rate (about 30%), long culture cycle, and high biosafety requirements. The long culture cycle and low positive detection rate of the culture method cannot provide a rapid and accurate etiological basis for the diagnosis and treatment of clinical LRTI.

[0008] (3) Immunological methods: Pathogens such as viruses and chlamydia cannot be detected by the isolation culture method. The types of pathogens can be identified by detecting their specific antigens or specific antibodies (IgM and IgG antibodies). Commonly used immunological detection methods include enzyme-linked immunosorbent assay, immunofluorescence assay, colloidal gold immunochromatography, fluorescence immunochromatography, electrochemiluminescence immunoassay, etc. The advantages of immunological methods are rapidity and non-invasiveness, but the disadvantages are limited by factors such as the specificity of the selected protein and the patient's immune status, relatively low specificity, long window period, and IgG-type antibodies cannot be used to judge vaccination, post-infection, or latent infection, and can only be used as auxiliary diagnostic indicators.

[0009] (4) Molecular biology methods: Include sequencing, single PCR, nested PCR, line probe, and next-generation sequencing (NGS), etc. The advantages of molecular biology methods are rapidity, high sensitivity, and strong specificity. The disadvantages are that methods such as ordinary PCR or qPCR have few detection sites, low throughput, high price, long detection cycle, and cannot distinguish infection, colonization, or contamination.

[0010] Therefore, there is an urgent need for a multiplex gene detection kit product for the identification of 10 bacteria related to LRTI, which has high throughput, low cost, high sensitivity and excellent specificity, and has important clinical guiding significance. Summary of the Invention

[0011] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a primer set for multiplex gene identification of 10 bacteria related to lower respiratory tract infection and its application. The sequences of the primer set are shown in SEQ ID NO.1-20. The primer set designs specific primers for 10 bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes, and uses FAM fluorescence to label the specific primers of 10 genes at the 5′ end of the primer. It can detect 10 target gene loci and 2 internal reference loci in one PCR system, overcome traditional PCR deviation, and accurately quantify gene expression. Based on this, the developed reagents and kits have the advantages of simple operation, low cost and high accuracy. When in use, the sample DNA is extracted for multiplex PCR, and then the pathogen load is determined by electrophoresis and software analysis. Experiments have proved that this method has strong specificity and high sensitivity for 10 bacteria and has important application value in the identification of LRTI bacteria.

[0012] To achieve the above object, the present invention provides the following solutions:

[0013] One object of the present invention is to provide a primer set for multiplex gene identification of 10 bacteria related to lower respiratory tract infection, and the nucleotide sequences of the primer set are shown in SEQ ID NO.1-20.

[0014] Preferably, the 10 bacteria detected by the primer set include Pseudomonas aeruginosa, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Stenotrophomonas maltophilia, Legionella pneumophila, Escherichia coli, and Acinetobacter baumannii.

[0015] Another object of the present invention is to provide a reagent for differentiating 10 bacteria related to lower respiratory tract infection, and the primer set described above is in the reagent.

[0016] Preferably, the reagent also contains primers for detecting human internal reference gene locus Hum DNA and internal reference IC in the system, and the nucleotide sequences are shown in SEQ ID NO.21-24.

[0017] Another object of the present invention is to provide a kit for differentiating 10 bacteria related to lower respiratory tract infection, and the primer set described above is contained in the kit.

[0018] Preferably, the kit also contains a primer set with nucleotide sequences shown in SEQ ID NO.21-24.

[0019] A fourth object of the present invention is to provide an application of the above primer set in the preparation of a product for identifying 10 bacteria related to lower respiratory tract infection.

[0020] A fifth object of the present invention is to provide an application of a primer set with nucleotide sequences shown in SEQ ID NO.1-24 in the preparation of a product for identifying 10 bacteria related to lower respiratory tract infection.

[0021] Preferably, the product is a reagent or a kit.

[0022] More preferably, the usage method of the kit is as follows:

[0023] (1) Obtain the DNA of the sample to be tested;

[0024] (2) Based on the DNA in step (1), perform multiplex PCR using the primer set with nucleotide sequences shown in SEQ ID NO.1-20 or the primer set with nucleotide sequences shown in SEQ ID NO.21-24;

[0025] (3) Perform capillary electrophoresis on the multiplex PCR product, detect the FAM fluorescence signal, and use the ID-X system software to analyze the peak intensity of the target gene locus, and obtain the relative load of each pathogen.

[0026] According to the specific embodiments provided by the present invention, the following technical effects are disclosed:

[0027] (1) The present invention uses specific primers for 8 genes with FAM fluorescently labeled 5′-ends, optimizes the reaction system, realizes the simultaneous systematic detection of 10 target gene loci in one PCR system, overcomes the deviation caused by unequal amplification in traditional PCR, can accurately and sensitively quantify the expression of a group of target genes, saves costs, and improves efficiency;

[0028] (2) In addition to detecting the above 10 gene loci at one time, the present invention can also simultaneously detect 1 human internal reference gene locus Hum DNA and 1 system internal reference IC.

[0029] (3) The capillary electrophoresis fragment analysis technology applied in the fragment analysis of the kit provided by the present invention is different from the traditional gel electrophoresis analysis mode, making the analysis of PCR results more intuitive, concise, reliable, easy to identify and judge, and more conducive to standardized operation.

[0030] (4) The detection kit provided by the present invention has a simple structure, convenient operation, low detection cost, high accuracy, good repeatability, and the usage conditions can be patterned, which is convenient for large-scale popularization and application. Description of the Drawings

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0032] Figure 1 Detection results of 10 standard strains provided in Embodiment 1 of the present invention; from left to right are Pseudomonas aeruginosa (122.5 ± 1.5 bp), Streptococcus pyogenes (144.5 ± 1.5 bp), Streptococcus pneumoniae (161.5 ± 1.5 bp), Haemophilus influenzae (171.5 ± 1.5 bp), Klebsiella pneumoniae (184.5 ± 1.5 bp), Staphylococcus aureus (208.6 ± 1.5 bp), Stenotrophomonas maltophilia (228.8 ± 1.5 bp), Legionella pneumophila (234.8 ± 1.5 bp), Escherichia coli (251.2 ± 1.5 bp), Acinetobacter baumannii (270.9 ± 1.5 bp), huDNA (293.9 ± 1.5 bp), and IC (346.4 ± 1.5 bp).

[0033] Figure 2 LOD detection results of Acinetobacter baumannii provided in Embodiment 1 of the present invention; where (a) is the detection result corresponding to 1×10 6 CFU / mL, (b) is the detection result corresponding to 5×10 5 CFU / mL, (c) is the detection result corresponding to 2.5×10 5 CFU / mL, and (d) is the detection result of the negative control;

[0034] Figure 3 LOD detection results of Haemophilus influenzae provided in Embodiment 1 of the present invention; where (a) is the detection result corresponding to 8×10 5 CFU / mL, (b) is the detection result corresponding to 4×10 5 CFU / mL, (c) is the detection result corresponding to 2×10 5 CFU / mL, and (d) is the detection result of the negative control;

[0035] Figure 4 LOD detection results of Klebsiella pneumoniae provided in Embodiment 1 of the present invention; where (a) is the detection result corresponding to 9×10 4 CFU / mL, (b) is the detection result corresponding to 4.5×10 4 CFU / mL, (c) is the detection result corresponding to 2.25×10 4 CFU / mL, and (d) is the detection result of the negative control;

[0036] Figure 5 This is the LOD detection result of Legionella pneumophila provided in Example 1 of the present invention; among them, (a) is the detection result corresponding to 6×10 4 CFU / mL, (b) is the detection result corresponding to 3×10 4 CFU / mL, (c) is the detection result corresponding to 1.5×10 4 CFU / mL, and (d) is the detection result of the negative control;

[0037] Figure 6 This is the LOD detection result of Pseudomonas aeruginosa provided in Example 1 of the present invention; among them, (a) is the detection result corresponding to 3×10 4 CFU / mL, (b) is the detection result corresponding to 1.5×10 4 CFU / mL, (c) is the detection result corresponding to 7.5×10 3 CFU / mL, and (d) is the detection result of the negative control;

[0038] Figure 7 This is the LOD detection result of Staphylococcus aureus provided in Example 1 of the present invention; among them, (a) is the detection result corresponding to 4×10 6 CFU / mL, (b) is the detection result corresponding to 2×10 6 CFU / mL, (c) is the detection result corresponding to 1×10 6 CFU / mL, and (d) is the detection result of the negative control;

[0039] Figure 8 This is the LOD detection result of Stenotrophomonas maltophilia provided in Example 1 of the present invention; among them, (a) is the detection result corresponding to 1×10 6 CFU / mL, (b) is the detection result corresponding to 5×10 5 CFU / mL, (c) is the detection result corresponding to 2.5×10 5 CFU / mL, and (d) is the detection result of the negative control;

[0040] Figure 9 This is the LOD detection result of Streptococcus pneumoniae provided in Example 1 of the present invention; among them, (a) is the detection result corresponding to 1.2×10 4 CFU / mL, (b) is the detection result corresponding to 6×10 3 CFU / mL, (c) is the detection result corresponding to 3×10 3 CFU / mL, and (d) is the detection result of the negative control;

[0041] Figure 10LOD test results of Streptococcus pyogenes provided in Example 1 of the present invention; where (a) is the test result corresponding to 1×10 4 CFU / mL, (b) is the test result corresponding to 5×10 4 CFU / mL, (c) is the test result corresponding to 5×10 3 CFU / mL, and (d) is the test result of the negative control;

[0042] Figure 11 LOD test results of Escherichia coli provided in Example 1 of the present invention; where (a) is the test result corresponding to 1×10 6 CFU / mL, (b) is the test result corresponding to 5×10 5 CFU / mL, (c) is the test result corresponding to 2.5×10 5 CFU / mL, and (d) is the test result of the negative control;

[0043] Among them, Figures 1 to 11 the abscissa in both is Size (bp), and the ordinate in both is Dye Signal (rfu). Detailed implementation manners

[0044] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0045] To make the above objects, features, and advantages of the present invention more obvious and understandable, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners.

[0046] Example 1

[0047] This example provides a multiplex gene detection kit, which includes 4×Reaction Buffer (SPE) (Ningbo Haiyi Gene Technology Co., Ltd.), Enzyme Mix (SPE) (Ningbo Haiyi Gene Technology Co., Ltd.), template sequences, and primers pool.

[0048] Primer design principle: Download the gene sequences of 10 target pathogens from Genebank, perform multiple sequence alignment using Vector NTI software, select highly conserved and single-copy specific sequences to design primers, and label the 5′ end of the primers with FAM fluorescent dye. Use Oligo7 to design primers and perform sequence alignment at the National Center for Biotechnology Information (NCBI) in the United States to prove the specificity of each primer. In addition to the basic principles of PCR primer design, the main design principles of the multiple primers in the present invention also include: ① Limit the amplified product fragment lengths of all primers between 100 - 400 bp, and the difference in the amplified product lengths of different primers > 3 bp; ② Try to avoid the formation of dimer structures between different primers; ③ Avoid non-specific amplification of each pair of primers and any two non-paired primers. The primers were synthesized by Shanghai Bioengineering Co., Ltd. The sequences of each primer are shown in Table 1.

[0049] Table 1 Primer sequences and final reaction concentrations of target sites

[0050]

[0051]

[0052] The usage steps of the detection kit in this example include:

[0053] A. Collect samples and extract DNA according to conventional experimental methods.

[0054] B. Add the primer pool for identification and perform multiplex PCR.

[0055] C. The multiplex PCR products are subjected to capillary electrophoresis using a 3500Dx genetic analyzer, and the PCR products of different fragment sizes are separated. The universal primers are labeled with FAM fluorescence, so that the PCR products are labeled with fluorescence, and the fluorescence intensity values of each PCR fragment can be obtained. Use ID-X system software to analyze the peak intensity of the target gene target, and further obtain the relative load of each pathogen.

[0056] The detection kit includes 4×Reaction Buffer (SPE) (Ningbo Haiyi Gene Technology Co., Ltd.), Enzyme Mix (SPE) (Ningbo Haiyi Gene Technology Co., Ltd.), template sequences, and primers pool fixed in a paper packaging box.

[0057] Example 2

[0058] Detect 10 bacteria related to LRTI using the detection kit of Example 1: Pseudomonas aeruginosa (122.5±1.5bp), Streptococcus pyogenes (144.5±1.5bp), Streptococcus pneumoniae (161.5±1.5bp), Haemophilus influenzae (171.5±1.5bp), Klebsiella pneumoniae (184.5±1.5bp), Staphylococcus aureus (208.6±1.5bp), Stenotrophomonas maltophilia (228.8±1.5bp), Legionella pneumophila (234.8±1.5bp), Escherichia coli (251.2±1.5bp), Acinetobacter baumannii (270.9±1.5bp), huDNA (293.9±1.5bp) and IC (346.4±1.5bp), and establish LRTI-HMGS. The implementation plan is as follows:

[0059] (1) Extract the genomic DNA of the standard strain pathogens.

[0060] Vigorously shake and mix the suspension of 10 standard strain bacteria related to LRTI, pipette 300 μL into the Bacterial Genomic DNA Rapid Extraction Kit, and use Smart Lab Assist (Dot Nano Co., Ltd., Taiwan, China) to extract the genomic DNA of the pathogens. After measuring the concentration with an ultraviolet spectrophotometer, store it at 4°C or -20°C for later use.

[0061] (2) Perform multiplex PCR reaction using DNA as a template.

[0062] Use the DNA obtained in the previous step as a template, and use forward and reverse primers labeled with FAM to amplify the PCR products. The reaction solution in the same PCR reaction tube is prepared as follows:

[0063] Table 2

[0064]

[0065]

[0066] The prepared system runs the following program on a PCR instrument:

[0067] Table 3

[0068]

[0069] (3) Use a 3500Dx genetic analyzer to perform capillary electrophoresis, detect the FAM fluorescence signal, and perform data analysis.

[0070] Add 9 μL of HiDi and 1 μL of multiplex PCR product to each well of the 96-well sample plate.

[0071] According to the operation manual of the 3500Dx genetic analyzer, install the capillary and gel. Place the sample plate into the machine, run the separation program, execute the default 3500Dx analysis method, and finally save the data.

[0072] Further use The ID-X software was used to analyze the results of capillary electrophoresis. The PCR product fragment sizes of each gene were different. The capillary electrophoresis peak diagram obtained from the experiment was as Figure 1 shown. The abscissa represents the fragment length, and the ordinate represents the peak height.

[0073] (4) LRTI-HMGS is specific for all target detection targets

[0074] Ten specific fluorescence primers for specific identification genes of target pathogens were designed and optimized. The standard strains corresponding to 10 pathogens were synchronously detected using LRTI-HMGS. The results showed that all targets produced specific peaks at the target positions, that is, at the positions of the amplified product fragment length ±1.5 bp, as Figure 1 shown.

[0075] (5) LRTI-HMGS has high sensitivity for the detection of 10 pathogen targets

[0076] The standard strains of 10 pathogens were respectively mixed with negative sputum, and the sputum was gradient-diluted to simulate positive samples. LRTI-HMGS was used for detection respectively (the reaction system and procedure were the same as those in Table 2 and Table 3), and each gradient was repeated 20 times. The determination criterion for the lowest detection limit was the concentration corresponding to a positive detection rate ≥90% in 20 repeated tests, and at the same time, the positive detection rate of the lower concentration of the next gradient <10%. Referring to the Figures 2 to 11 shown results, the LOD detection result of Acinetobacter baumannii was 5×10 5 CFU / mL, the LOD detection result of Haemophilus influenzae was 4×10 5 CFU / mL, the LOD detection result of Klebsiella pneumoniae was 4.5×10 5 CFU / mL, the LOD detection result of Legionella pneumophila was 3×10 4 CFU / mL, the LOD detection result of Pseudomonas aeruginosa was 7.5×10 3 CFU / mL, the LOD detection result of Staphylococcus aureus was 1×10 6 CFU / mL, the LOD detection result of Stenotrophomonas maltophilia was 5×10 5 CFU / mL, the LOD detection result of Streptococcus pneumoniae was 6×10 3 CFU / mL, the LOD detection result of Streptococcus pyogenes was 1×10 4 CFU / mL, and the LOD detection result of Escherichia coli was 5×105 CFU / mL, further indicating that LRTI-HMGS has high sensitivity.

[0077] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0078] Specific examples are used in this article to elaborate on the principles and implementation manners of the present invention. The descriptions of the above embodiments are only used to help understand the method of the present invention and its core idea. At the same time, for those of ordinary skill in the art, based on the idea of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A primer set for multiple gene identification of 10 bacteria related to lower respiratory tract infection, characterized in that: The nucleotide sequences of the primer set are shown in SEQ ID NO.1-20.

2. A primer set for multiple gene identification of 10 bacteria related to lower respiratory tract infection according to claim 1, characterized in that: The primer set is used to detect 10 bacteria including Pseudomonas aeruginosa, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Stenotrophomonas maltophilia, Legionella pneumophila, Escherichia coli, and Acinetobacter baumannii.

3. A reagent for distinguishing 10 types of bacteria related to lower respiratory tract infection, characterized in that: The reagent contains the primer set described in claim 1.

4. The reagent according to claim 3, characterized in that The reagent also contains primers for detecting human internal reference gene site Hum DNA and system internal reference IC, and the nucleotide sequences are shown in SEQ ID NO.21-24.

5. A kit for distinguishing 10 types of bacteria related to lower respiratory tract infection, characterized in that: The kit contains the primer set according to claim 1.

6. The kit according to claim 5, characterized in that The kit also contains a primer set with nucleotide sequences as shown in SEQ ID NO. 21-24.

7. Use of the primer set as claimed in claim 1 in preparing a product for identifying 10 bacteria related to lower respiratory tract infection.

8. Use of a primer set having nucleotide sequences as shown in SEQ ID NO. 1-24 in preparing a product for identifying 10 bacteria related to lower respiratory tract infection.

9. The use according to claim 7 or 8, characterized in that: The product is a reagent or a kit.

10. The use according to claim 9, characterized in that: The method of using the kit is as follows: (1) Obtaining DNA from the sample to be tested; (2) Based on the DNA in step (1), multiplex PCR is performed using a primer set with nucleotide sequences such as SEQ ID NOs. 1-20 or a primer set with SEQ ID NOs. 21-24; (3) Perform capillary electrophoresis on the multiplex PCR products, detect the FAM fluorescence signal, and use The ID-X system software analyzes the peak intensity of the target gene target point to obtain the relative load of each pathogen.

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