Primer group for identifying multiple genes of mycoplasma and chlamydia related to lower respiratory tract infection and application of primer group
By designing a multi-gene identification primer set and optimizing the PCR reaction system, combined with capillary electrophoresis technology, the problems of low sensitivity, insufficient specificity, high cost and long detection cycle when detecting lower respiratory tract infection-related mycoplasma and chlamydia in the prior art are solved, and efficient, accurate and economical multi-gene detection effect is achieved.
Patent Information
- Application Number
- CN202510102766.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-22
- Publication Date
- 2025-04-18
AI Technical Summary
When detecting mycoplasma and Chlamydia related to lower respiratory tract infection, the prior art has problems such as low sensitivity, insufficient specificity, high cost and long detection cycle, which is difficult to meet the clinical needs for rapid, accurate and economical testing.
A multi-gene identification primer set was designed, including specific primers for Mycoplasma pneumoniae, Chlamydia pneumoniae, huDNA and IC. The PCR reaction system was also labeled FAM fluorescently and optimized. Two target gene loci, one human internal reference gene loci and one system internal reference were detected in one PCR system at the same time, and fragment analysis was performed using capillary electrophoresis.
It realizes efficient and accurate detection of multiple gene loci in one PCR system, overcomes the uneven amplification deviation of traditional PCR, improves the sensitivity and specificity of detection, reduces the cost and detection cycle, and is suitable for large-scale promotion and application.
Smart Images
Figure CN119842938A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of gene detection, and in particular to a primer set for multiple gene identification of mycoplasma and chlamydia related to lower respiratory tract infection and application thereof. Background Art
[0002] Respiratory tract infection is the most common infectious disease that seriously threatens human health in clinical practice. It can be divided into upper respiratory tract infection and lower respiratory tract infection (LRTI). LRTI is an infection below the larynx, which often causes severe symptoms such as acute and chronic tracheitis, acute and chronic pneumonia, and bronchitis.
[0003] The main pathogens of LRTI are bacteria (including Mycobacterium tuberculosis and Legionella), and can also be caused by fungi and atypical microorganisms such as Chlamydia and Mycoplasma. Some viruses can also cause LRTI. According to 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), Legionnaire (Leg), Staphylococcus aureus (Sau), Stenotrophomonas maltophilia (Sma), Mycobacterium tuberculosis (TB), Escherichia coli (E.coli), Acinetobacter baumannii (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 (Novel Coronavirus-19, COVID-19), which covers more than 95% of pathogens.
[0004] Currently, clinical laboratory tests for LRTI pathogens mainly include smear microscopy and isolation culture methods for bacteria, fungi and mycoplasma, while viruses mainly rely on immunological testing and molecular biology methods. Common sample types include sputum, alveolar lavage fluid, peripheral blood and serosal cavity effusion.
[0005] At present, the conventional technologies 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 ink staining microscopy of Cryptococcus neoformans. The advantages of smear microscopy are simplicity, economy, and the fastest detection, which can quickly provide a reference for LRTI diagnosis. The disadvantage is that the specificity is poor and requires the experimenter to have rich experience. In clinical practice, the isolation culture method and smear microscopy are combined to improve the accuracy of diagnosis and reduce false negatives and false positives.
[0007] (2) Pathogen isolation and culture method: Isolation and culture method is the gold standard for diagnosing LRTI caused by bacteria and fungi, and is a routine method for testing respiratory secretions. The advantage of the culture method is that a positive culture can identify the type of pathogen, and at the same time, an in vitro drug sensitivity test can be performed. The results of the drug sensitivity test can provide an accurate and effective reference for the clinical treatment of LRTI and the control of bacterial resistance. The disadvantages are high nutritional requirements, low positive detection rate (about 30%), long culture cycle, and high biosafety requirements. The culture method has a long cycle and a low positive detection rate, and 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 isolation and culture, but the type of pathogen can be determined 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, fluorescent immunochromatography, electrochemiluminescence immunoassay, etc. The advantages of immunological methods are rapid and non-destructive, but the disadvantages are that they are limited by factors such as the specificity of the selected protein and the patient's immune status. The specificity is relatively low, the window period is long, and IgG antibodies cannot be judged as vaccination, post-infection or latent infection, and are only used as auxiliary diagnostic indicators.
[0009] (4) Molecular biology methods: including sequencing, single PCR, nested PCR, linear probes, and high-throughput sequencing (NGS). The advantages of molecular biology methods are rapidity, high sensitivity, and strong specificity. The disadvantages are that common PCR or qPCR methods have few detection sites, low throughput, high price, long detection cycle, and cannot determine site infection, colonization, or contamination.
[0010] Therefore, there is an urgent need for a multiplex gene detection kit product with high throughput, low cost, high sensitivity and excellent specificity for the identification of LRTI-related mycoplasma and chlamydia, which has important clinical guidance significance. Summary of the invention
[0011] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a primer set for multiple gene identification of mycoplasma and chlamydia associated with lower respiratory tract infection and its application, the primer set comprises specific primers for mycoplasma pneumoniae, chlamydia pneumoniae, huDNA and IC, and the reaction system is optimized by FAM fluorescent labeling of the 5′ end primer, and two target gene sites and one human internal reference gene site and one system internal reference can be detected simultaneously in one PCR system, overcoming the unequal amplification bias of traditional PCR, and can accurately and sensitively quantify the expression of target genes. The reagents and kits developed based on this primer set, in combination with specific use methods, have the advantages of simple structure, convenient operation, low cost, high accuracy, good repeatability, etc., and the fragment analysis thereof adopts capillary electrophoresis technology, and the result analysis is intuitive and reliable, which is convenient for large-scale promotion and application, and has great significance for the clinical detection of mycoplasma and chlamydia associated with LRTI.
[0012] To achieve the above object, the present invention provides the following solutions:
[0013] One of the purposes of the present invention is to provide a primer set for multiple gene identification of mycoplasma and chlamydia associated with lower respiratory tract infection, the nucleotide sequence of the primer set is shown in SEQ ID NO.1-4.
[0014] Preferably, the primer set comprises primers for detecting Mycoplasma pneumoniae and Chlamydia pneumoniae
[0015] A second object of the present invention is to provide a reagent for distinguishing mycoplasma and chlamydia associated with lower respiratory tract infection, wherein the reagent contains the above-mentioned primer set.
[0016] Preferably, the reagent also contains primers for detecting human internal reference gene loci Hum DNA and system internal reference IC, and the nucleotide sequences are shown in SEQ ID NO.5-8.
[0017] A third object of the present invention is to provide a kit for distinguishing mycoplasma and chlamydia associated with lower respiratory tract infection, wherein the kit contains the above-mentioned primer set.
[0018] Preferably, the kit further comprises primers with nucleotide sequences as shown in SEQ ID NO.5-8.
[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 mycoplasma and chlamydia associated with lower respiratory tract infection.
[0020] A fifth object of the present invention is to provide a primer set such as a nucleotide sequence as shown in SEQ ID NO.1-8 for use in preparing a product for identifying mycoplasma and chlamydia associated with lower respiratory tract infection.
[0021] Preferably, the product is a reagent or a kit.
[0022] More preferably, the method for using the kit is:
[0023] (1) Obtaining DNA from the sample to be tested;
[0024] (2) Based on the DNA in step (1), multiplex PCR is performed using a primer set with nucleotide sequences such as SEQ ID NOs. 1-4 or a primer set with SEQ ID NOs. 5-8;
[0025] (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.
[0026] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects:
[0027] (1) The present invention uses two gene-specific primers with FAM fluorescently labeled 5′-end primers to optimize the reaction system, realize the simultaneous and systematic detection of two target gene loci in one PCR system, overcome the deviation caused by unequal amplification in traditional PCR, and can accurately and sensitively quantify the expression of a group of target genes, saving costs and improving efficiency;
[0028] (2) In addition to detecting the above two gene loci at one time, the present invention can also simultaneously detect one human internal reference gene locus Hum DNA and one system internal reference IC.
[0029] (3) The capillary electrophoresis fragment analysis technology used in the fragment analysis of the kit of the present invention is different from the traditional gel electrophoresis analysis mode, making the PCR result analysis 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 use conditions can be standardized, which is convenient for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.
[0032] Figure 1 The test results of the two standard strains provided in Example 1 of the present invention are as follows; from left to right, they are Mycoplasma pneumoniae (112.0±1.5 bp), Chlamydia pneumoniae (133.7±1.5 bp), huDNA (293.9±1.5 bp), and IC (346.4±1.5 bp);
[0033] Figure 2 The LOD test result of Mycoplasma pneumoniae provided in Example 1 of the present invention; wherein (a) is 4×10 3 The test result corresponding to CFU / mL, (b) is 2×10 3 The test result corresponding to CFU / mL, (c) is 1×10 3 The test results corresponding to CFU / mL, (d) is the test result of the negative control;
[0034] Figure 3 The LOD detection result of Chlamydia pneumoniae provided in the embodiment of the present invention; wherein (a) is 1×10 4 The test results corresponding to CFU / mL, (b) is 5×10 3 The test result corresponding to CFU / mL, (c) is 2.5×10 3 The test results corresponding to CFU / mL, (d) is the test result of the negative control;
[0035] in, Figure 1 to Figure 3 The horizontal axis is Size (bp), and the vertical axis is Dye Signal (rfu). DETAILED DESCRIPTION
[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.
[0038] Example 1
[0039] This embodiment 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 sequence and primers pool.
[0040] Primer design principles: Download the gene sequences of 10 target pathogens from Genebank, use Vector NTI software for multiple sequence alignment, select highly conserved, single-copy specific sequences to design primers, and label the 5′ end of the primers with FAM fluorescent dye. Oligo7 was used to design primers, and sequence alignment was performed at the National Center for Biotechnology Information (NCBI) to prove the specificity of each primer. In addition to the basic principles of PCR primer design, the main design principles of multiple primers in the present invention also include: ① Limit the length of the amplification product fragment of all primers to between 100-400bp, and the difference in the length of the amplification product of different primers is greater than 3bp; ② Try to avoid the formation of dimer structures between different primers; ③ Avoid non-specific amplification of any two primers in each pair of primers and non-paired primers. Primer synthesis was completed by Shanghai Bioengineering Co., Ltd. The sequences of each primer are shown in Table 1.
[0041] Table 1 Target primer sequences and final reaction concentrations
[0042]
[0043] The steps of using the detection kit of this embodiment include:
[0044] A. Collect samples and extract DNA according to routine experimental methods.
[0045] B. Add the primer pool used for identification and perform multiplex PCR.
[0046] C. Multiple PCR products are subjected to capillary electrophoresis by 3500Dx Genetic Analyzer, and PCR products of different fragment sizes are separated. The universal primer is labeled with FAM fluorescence, so that the PCR product is fluorescently labeled, and the fluorescence intensity value of each PCR fragment can be obtained. The ID-X system software analyzes the peak intensity of the target gene target point and further obtains the relative load of each pathogen.
[0047] The detection kit includes 4×Reaction Buffer (SPE) (Ningbo Haiyi Gene Technology Co., Ltd.), Enzyme Mix (SPE) (Ningbo Haiyi Gene Technology Co., Ltd.), template sequence and primers pool fixed in a paper packaging box.
[0048] Example 2
[0049] The detection kit of Example 1 was used to detect LRTI-related mycoplasmas and chlamydia: Mycoplasma pneumoniae (112.0±1.5bp), Chlamydia pneumoniae (133.7±1.5bp), huDNA (293.9±1.5bp), huDNA (293.9±1.5bp) and IC (346.4±1.5bp), and LRTI-HMGS was established. The implementation plan is as follows:
[0050] (1) Extract the pathogen genome of the standard strain.
[0051] The suspension of LRTI-related mycoplasma and chlamydia standard strains was thoroughly shaken and mixed, and 300uL was pipetted into the bacterial genome rapid extraction kit, and the pathogen genome was extracted using Smart Lab Assist (Dot Nano, Taiwan, China). After the concentration was measured by UV spectrophotometer, it was stored at 4°C or -20°C for future use.
[0052] (2) Perform multiple PCR reactions using DNA as a template.
[0053] Using the DNA obtained in the previous step as a template, use FAM-labeled forward and reverse primers to amplify the PCR product. The reaction solution in the same PCR reaction tube is prepared as follows:
[0054] Table 2
[0055]
[0056]
[0057] After the system is prepared, run the following program on the PCR instrument:
[0058] Table 3
[0059]
[0060] (3) Use 3500Dx genetic analyzer to perform capillary electrophoresis, detect FAM fluorescence signals, and perform data analysis.
[0061] Add 9 μL of HiDi and 1 μL of multiplex PCR product to each well of a 96-well sample plate.
[0062] According to the operating manual of the 3500Dx Genetic Analyzer, install the capillaries and gel, put the sample plate into the machine, run the separation program, execute the default 3500Dx analysis method, and finally save the data.
[0063] Further Use ID-X software analyzes the results of capillary electrophoresis, and the PCR product fragments of each gene are different in size. The capillary electrophoresis peak diagram obtained by the experiment is as follows Figure 1 As shown, the abscissa represents the fragment length, and the ordinate represents the peak height.
[0064] (4) LRTI-HMGS is specific for all target detection targets
[0065] Specific fluorescent primers for specific identification genes of two target pathogens were designed and optimized, and the standard strains corresponding to the two pathogens were detected simultaneously using LRTI-HMGS. The results showed that all targets produced specific peaks at the target position, that is, the position of the amplified product fragment length ±1.5bp, such as Figure 1 shown.
[0066] (5) LRTI-HMGS has high sensitivity in detecting two pathogen targets
[0067] The standard strains of the two pathogens were mixed with negative sputum, and the sputum was diluted in a gradient to simulate positive samples. The LRTI-HMGS was used for detection (the reaction system and procedure were the same as those in Tables 2 and 3), and each gradient was repeated 20 times. The criterion for the minimum detection limit was the concentration corresponding to the positive detection rate ≥ 90% in the 20 repeated tests, and the positive detection rate of the next lower concentration in the gradient was < 10%. Figure 2 and Figure 3 The results shown in the figure show that the LOD of Mycoplasma pneumoniae is 5×10 3 The LOD test results of CFU / mL and Chlamydia pneumoniae were 2×10 3 CFU / mL, indicating that LRTI-HMGS has high sensitivity.
[0068] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.
[0069] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of the present invention. At the same time, for those skilled in the art, according to the idea of the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.
Claims
1. A primer set for multiple gene identification of mycoplasma and chlamydia associated with lower respiratory tract infection, characterized in that: The nucleotide sequences of the primer set are shown in SEQ ID NO.1-4.
2. A multiple gene identification primer set for mycoplasma and chlamydia associated with lower respiratory tract infection according to claim 1, characterized in that: The primer set comprises primers for detecting Mycoplasma pneumoniae and Chlamydia pneumoniae.
3. A reagent for distinguishing mycoplasma and chlamydia associated with 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.5-8.
5. A kit for distinguishing mycoplasma and chlamydia associated with 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 primers with nucleotide sequences as shown in SEQ ID NO.5-8.
7. Use of the primer set as claimed in claim 1 in preparing a product for identifying mycoplasma and chlamydia associated with lower respiratory tract infection.
8. Use of a primer set having nucleotide sequences as shown in SEQ ID NO. 1-8 in preparing a product for identifying mycoplasma and chlamydia associated with 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. 1-4 or a primer set with nucleotide sequences such as SEQ ID NOs. 5-8; (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.