Specific detection target and molecular marker primer for detecting legionella pneumophila
By designing PCR molecular marker primers and RAA-LFD detection systems for Legionella pneumophila-specific detection targets, the problems of long detection cycle and low sensitivity in existing technologies were solved, and rapid and sensitive Legionella pneumophila detection was achieved, with the amplified product sequence having a high similarity with the expected sequence.
Patent Information
- Application Number
- CN202211233265.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-10-10
- Publication Date
- 2025-10-17
- Estimated Expiration
- 2042-10-10
AI Technical Summary
Existing Legionella pneumophila detection technology has problems such as long detection cycle, cumbersome operation, insufficient sensitivity and specificity, making it difficult to effectively distinguish Legionella pneumophila from its closely related species.
Using the PCR molecular marker primers LpnF/LpnR and RAA-LFD detection system, which are specific detection targets of Legionella pneumophila, specific primers and probes were designed to achieve rapid and sensitive detection through PCR amplification and RAA-LFD reaction.
Rapid and sensitive detection of Legionella pneumophila was achieved, which could effectively distinguish Legionella pneumophila from other microorganisms. The similarity between the amplified product sequence and the expected sequence reached 100%, and the detection limit reached 0.4 fg/μL.
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Figure CN115851997B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of molecular biology, and relates to a Legionella pneumophila detection technology, in particular to a specific detection target for detecting Legionella pneumophila, a PCR molecular marker of the detection target, and a method for detecting Legionella pneumophila using the molecular marker; and further relates to a RAA-LFD detection system of the detection target and a kit thereof. BACKGROUND
[0002] Legionella pneumophila is a gram-negative opportunistic pathogen, a facultative intracellular bacterium, and is easily transmitted through central air conditioning system cooling water and aerosol. The bacterium can invade human macrophages, and the infection process is fast and the mortality rate is high.
[0003] Among the pathogenic bacteria in air conditioning refrigeration devices, Legionella pneumophila is the most pathogenic (accounting for 85% to 90% of cases), which can cause serious respiratory diseases such as pneumonia in humans. At present, researchers at home and abroad have developed various detection technologies, among which the traditional separation and identification methods include bacterial culture, biochemical and serological identification, etc. These methods mainly rely on bacterial morphology and physiological and biochemical differences to separate and identify bacteria, and the operation process is complicated, the detection period is long, and it is difficult to deal with acute food poisoning events. Immunological detection method is faster, but the detection sample has higher requirements, and it is difficult to apply to samples such as food and water source which are difficult to enrich antigens and antibodies; in addition, the immunological detection method usually has low sensitivity and specificity, and has certain limitations for pathogenic bacteria detection.
[0004] In recent years, with the rapid development of molecular biology technology, detection technologies based on molecular biology have been widely used. According to 5S rDNA and mip gene fragments, Lv Qin-feng designed primers and established a Legionella pneumophila detection system; Zhang Bao-ying et al. established a nested PCR technology based on the mip gene to detect Legionella pneumophila; Guo Shou established a PCR technology for detecting 16S rDNA specific target in the genome of Legionella pneumophila; Zhang Bing et al. designed primers based on the specific mip gene of Legionella pneumophila and established a Legionella pneumophila loop-mediated isothermal amplification system.
[0005] The key to the sensitivity and specificity of molecular detection technology lies in the selection of target sequences. The above-mentioned molecular detection technology for Legionella pneumophila commonly uses molecular targets such as mip gene, 16S rDNA gene and 5S rDNA. However, these target genes are usually difficult to effectively distinguish the target microorganism from its close relatives. Among them, 16S rDNA and 5S rDNA can only identify Legionella, and cannot distinguish Legionella pneumophila; using the mip gene as a detection target also cannot effectively distinguish close relatives.
[0006] Therefore, screening new molecular detection targets with species specificity is very important for accurate identification of Legionella pneumophila. SUMMARY
[0007] In view of this, one of the purposes of the present application is to provide a specific detection target of Legionella pneumophila, which is mined by taking ex vivo Legionella pneumophila as an object.
[0008] The second purpose of the present application is to provide a PCR molecular marker for detecting Legionella pneumophila.
[0009] The third purpose of the present application is to provide a rapid and sensitive detection method for Legionella pneumophila.
[0010] The fourth purpose of the present application is to provide a RAA-LFD detection system for detecting Legionella pneumophila.
[0011] The fifth purpose of the present application is to provide a method for detecting Legionella pneumophila based on the RAA-LFD detection system.
[0012] The sixth purpose of the present application is to provide a kit for detecting Legionella pneumophila.
[0013] The inventors, through long-term exploration and attempts, as well as multiple experiments and efforts, and continuous reform and innovation, provide a specific detection target of Legionella pneumophila to solve the above technical problems, and the nucleotide sequence of the detection target is shown as SEQ ID NO: 1.
[0014] The present application also provides a PCR molecular marker primer for detecting the specific detection target of Legionella pneumophila, and the molecular marker primer is Lpn primer, which includes a forward primer LpnF and a reverse primer LpnR.
[0015] The sequence of the forward primer LpnF is shown as SEQ ID NO: 2.
[0016] The sequence of the reverse primer LpnR is shown as SEQ ID NO: 3.
[0017] The present application also provides a method for detecting Legionella pneumophila using the Lpn primer, which performs PCR amplification by taking Legionella pneumophila genomic DNA as a template.
[0018] The amplification procedure is as follows: 95℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 58.4℃ annealing for 30s, 72℃ extension for 20s, a total of 25-35 cycles; and finally 72℃ extension for 5min.
[0019] Further, the total system of the PCR reaction is 20μL, in which 2×Easy PCR SuperMix 10.0 μL, 1 μL of each of the upstream and downstream primers, 1.0 μL of template DNA, 7 μL of ddH2O;
[0020] The application also provides a RAA-LFD detection system for detecting the specific detection target of the Legionella pneumophila, characterized in that the RAA-LFD detection system comprises RAA-LFD primers and a specific probe, the RAA-LFD primers comprise forward primers RAA-LF and reverse primers RAA-LR,
[0021] The sequence of the forward primers RAA-LF is shown as SEQ ID NO: 4,
[0022] The sequence of the reverse primers RAA-LR is shown as SEQ ID NO: 5, and the 5' end is modified by Bio;
[0023] The sequence of the probe is shown as SEQ ID NO: 6, the 5' end is labeled by FAM, the 3' end is modified by C3Spacer, and THF modification is performed at a position 30 bp away from the 5' end in the middle of the sequence of the probe.
[0024] The application also provides a method for detecting the Legionella pneumophila by using the RAA-LFD detection system, wherein the amplification is performed by taking the genomic DNA of the Legionella pneumophila as a template, the amplification temperature is 37℃, and the amplification time is 15-25 min.
[0025] The application also provides a kit for detecting the Legionella pneumophila, which comprises at least one dose of a detection solution containing the RAA-LFD primers and the specific probe.
[0026] Compared with the prior art, one of the above technical solutions has the following advantages:
[0027] The sensitivity and specificity of the pathogenic bacteria molecular detection system depend on the selection of the molecular target. According to the mined one DNA fragment with species specificity, the applicant designs one pair of PCR molecular marker primers and one RAA-LFD detection system. The length of the amplified fragment of the PCR molecular marker primers is 396 bp, and the length of the amplified fragment of the RAA-LFD detection system is 186 bp. The sequence of the amplification product is analyzed by sequencing, and the similarity with the expected sequence reaches 100%, and there is no amplification band when the genomic DNA of harmful microorganisms such as Streptococcus equi and Streptococcus agalactiae is taken as a template. The designed primers are specific. BRIEF DESCRIPTION OF DRAWINGS
[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as a limitation to the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.
[0029] Figure 1 is the electrophoresis detection result of the PCR amplification product of the Legionella pneumophila genomic DNA template in Example 2 under different annealing temperature conditions.
[0030] Figure 2 is the electrophoresis detection result of the PCR amplification product of different microbial genomic DNA templates in Example 2.
[0031] Figure 3 is the electrophoresis detection result of the PCR amplification product of the Legionella pneumophila genomic DNA template in Example 2 under different cycle numbers.
[0032] Figure 4 is the test strip detection result of the amplification product of the RAA-LFD detection system of the Legionella pneumophila genomic DNA template in Example 3 under different amplification temperature conditions.
[0033] Figure 5 is the electrophoresis chart of the amplification product of the RAA-LFD detection system of the Legionella pneumophila genomic DNA template in Example 3 under different amplification temperature conditions.
[0034] Figure 6 is the test strip detection result of the amplification product of the RAA-LFD detection system of different microbial genomic DNA templates in Example 3.
[0035] Figure 7 is the electrophoresis detection result of the amplification product of the RAA-LFD detection system of different microbial genomic DNA templates in Example 3.
[0036] Figure 8 is the test strip detection result of the amplification product of the RAA-LFD detection system of the Legionella pneumophila genomic DNA template in Example 3 under different amplification time conditions. DETAILED DESCRIPTION
[0037] The following will be described in combination with the drawings and specific embodiments.
[0038] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application.
[0039] Embodiment 1
[0040] At present, the molecular detection technology system of Legionella pneumophila often takes mip gene, 16S rRNA gene and 5S rRNA gene as detection targets, and these detection targets are not high in sequence species specificity. In the research work, the inventors found a DNA fragment with species specificity in the genome sequence of Legionella pneumophila by bioinformatics method, the fragment is 951 bp in size, and the nucleotide sequence is shown as SEQ ID NO: 1. NT database is the database with the most and the most complete nucleic acid sequence data in the world today. After sequence alignment with the NT database of NCBI, the most complete existing nucleic acid sequence data, the fragment can only be aligned with the DNA of Legionella pneumophila (including different strains), and cannot be aligned with the DNA sequence of any other species, so the sequence has good species specificity.
[0041] Based on this, the inventors take the sequence shown as SEQ ID NO: 1 as the specific detection target of Legionella pneumophila.
[0042] Embodiment 2
[0043] This embodiment is a specific embodiment of the PCR detection system based on the Legionella pneumophila specific detection target described in embodiment 1. The Legionella pneumophila in this embodiment is derived from the air conditioning and refrigeration device in Sichuan Normal University.
[0044] For the Legionella pneumophila specific detection target described in embodiment 1, PCR molecular marker primers LpnF1 / R1 are designed, and the upper and lower primers are shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively:
[0045] The upper primer LpnF1 is 5'-TAGTCTTGGTGGTGCGGCTGT-3';
[0046] The lower primer LpnR1 is 5'-GCGGCGTATTGTGAGTCCCT-3'.
[0047] After optimization of the PCR amplification system, a rapid molecular detection reaction system of Legionella pneumophila was obtained.
[0048] 20 μL reaction system: 2x Easy PCR SuperMix 10.0 μL, 1 μL of each of the upstream and downstream primers, 1.0 μL of template DNA, and 7 μL of ddH2O.
[0049] The amplification reaction was as follows: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58.4℃ annealing for 30 s, 72℃ extension for 20 s, a total of 35 cycles; and finally 72℃ extension for 5 min.
[0050] In the process of realizing the present application, the inventors optimized the annealing temperature. Eight annealing temperatures were set, and the PCR amplification efficiency under different annealing temperatures was analyzed with the genomic DNA of Legionella pneumophila as the template. The results showed that good amplification effects were obtained at the eight annealing temperatures, as shown in Figure 1 , Figure 1 In the figure, the annealing temperatures corresponding to lanes 1-8 were as follows: 1, 52.0℃; 2, 52.5℃; 3, 53.5℃; 4, 55.1℃; 5, 56.9℃; 6, 58.4℃; 7, 59.4℃; and 8, 60.0℃. In combination with the evaluation results of the primers by the DNAMAN software, 58.4℃ was finally selected as the optimal annealing temperature.
[0051] With 58.4℃ as the annealing temperature, PCR amplification was performed with the genomic DNA of seven microorganisms, i.e., Amycolatopsis orientalis, Escherichia coli, Bacillus amyloliquefaciens, Bacillus subtilis, Streptococcus agalactiae, Streptococcus equi, and Legionella pneumophila, as the templates, and the amplified sequence was SEQ ID NO: 4, with a length of 396 bp. The electrophoretic detection results of the amplified products are shown in Figure 2 . The results showed that the primer LpnF1 / R1 could only obtain a 396 bp positive amplification band when the DNA of Legionella pneumophila was used as the template, and the similarity of the sequence to the expected sequence reached 100%. However, no amplification band was obtained when the genomic DNA of harmful microorganisms such as Streptococcus equi and Streptococcus agalactiae was used as the template, indicating that the specificity of the primer LpnF1 / R1 was good.
[0052] In the process of realizing the present application, the inventors also optimized the number of PCR cycles. The detection sensitivity was analyzed, and it was found that the detection sensitivity was different at different numbers of PCR cycles, as shown in Figure 3 ; Figure 3 In the figure, A, B, and C were 25, 30, and 35 PCR cycles, respectively; the template concentration of lane 1 was 400 fg / uL (1.17x10 5 copies / μL), and the 10-fold gradient dilutions of lane 1 were sequentially added to lanes 2-8.
[0053] See also Figure 3 Figure A, after 25 PCR cycles, the detection sensitivity was 40 fg / μL (1.17×10 4 copies / μL);
[0054] See also Figure 3 In Figure B, at 30 cycles, the detection sensitivity was 4 fg / μL (1.17×10 3 copies / μL);
[0055] See also Figure 3 Figure C, at 35 cycles, the detection sensitivity reached 0.4 fg / μL (1.17×10 2 copies / μL).
[0056] Example 3
[0057] This example is a specific implementation of the RAA-LFD detection system based on the specific detection target of Legionella pneumophila described in Example 1. The Legionella pneumophila in this example was derived from an air-conditioning refrigeration device on the campus of Sichuan Normal University.
[0058] The inventors designed RAA-LFD primers and specific probes for the specific detection target of Legionella pneumophila described in Example 1, as shown in Table 1 and SEQ ID NOs: 5 to 7 in the sequence listing. The sequence of the amplified product is shown in SEQ ID NO: 8.
[0059] The RAA-LFD 50 μL amplification reaction system is shown in Table 2.
[0060] Table 1. Primers and probes specific for Legionella pneumophila RAA-LFD
[0061]
[0062] Note: RAA-LF and RAA-LR are the upstream and downstream primers of the RAA-LFD detection system for Legionella pneumophila; Lpn-Probe: RAA-LFD detection system probe; Biotin: biotin label; 6-Carboxyfluorescein: 6-carboxyfluorescein group; THF: abasic site; Spacer: C3-spacer.
[0063] Table 2 RAA-LFD 50μL amplification reaction system
[0064]
[0065]
[0066] Note: RAA-nfo nucleic acid amplification kit (test strip type, item number: S005ZC), disposable lateral flow chromatography test strip (item number: R103ZC) purchased from Hangzhou Zhongce Biotechnology Co., Ltd.
[0067] The amplification temperature of the RAA-LFD detection system (Table 2) was optimized, and it was found that the amplification products under the conditions of 35-39℃ all had detection lines on the test strip. Among them, the detection line of the amplification product under the condition of 35℃ was weak, while the detection line under the condition of 36-38℃ was the most obvious, see Figure 4 . From the practicability (human body temperature is about 37℃), 37℃ was finally selected as the subsequent amplification temperature. The electrophoresis detection results are shown in Figure 5 .
[0068] The RAA-LFD primer and probe were subjected to specific detection, and the results showed that: the primer could only appear a clear detection line on the disposable lateral flow chromatography test strip when using Legionella pneumophila DNA as the template (see Figure 6 ), a positive amplification band could appear on the agarose gel electrophoresis (see Figure 7 ), and the amplification product was 100% similar to the expected sequence after sequencing analysis, while no amplification band was obtained when using harmful microorganism genomic DNA such as Streptococcus equi and Streptococcus agalactiae as the template, and the primer had good specificity.
[0069] The amplification time of the RAA-LFD detection system (Table 2) was optimized, and the sensitivity of the RAA-LFD reaction system under different amplification times was different, and the results are shown in Figure 8 .
[0070] When the amplification condition was 37℃, 5min, the minimum detection limit was 400fg / μL (1.26×10 5 copies / μL);
[0071] When the amplification condition was 37℃, 10min, the detection limit was 40fg / μL (1.26×10 4 copies / μL);
[0072] When the amplification condition was 37℃, 15min, the detection limit was 4×10 -2 fg / μL (1.26×10 1 copies / μL);
[0073] When the amplification condition was 37℃, 20min, the detection limit was 4×10 -2 fg / μL (1.26×10 1 copies / μL);
[0074] When the amplification condition was 37℃, 25min, the minimum detection limit was 4×10 -2fg / μL (1.26 x 10 1 copies / μL).
[0075] The above merely is the preferred embodiment of the present application, it should be pointed out that the above preferred embodiment should not be regarded as a limitation to the present application, the protection scope of the present application should be limited by the scope defined by the claims. For the ordinary skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.
Claims
1. A primer for detecting a specific target of Legionella pneumophila, characterized in that: The nucleotide sequence of the detection target is shown in SEQ ID NO: 1; the primers are Lpn primers, including a forward primer LpnF and a reverse primer LpnR; The forward primer LpnF sequence is shown in SEQ ID NO: 2; The reverse primer sequence LpnR is shown in SEQ ID NO:
3.
2. A RAA-LFD detection system for detecting the Legionella pneumophila specific detection target described in claim 1, characterized in that: The method comprises RAA-LFD primers and a specific probe, wherein the RAA-LFD primers comprise a forward primer RAA-LF and a reverse primer RAA-LR. The forward primer RAA-LF sequence is shown in SEQ ID NO: 4, The reverse primer RAA-LR sequence is shown in SEQ ID NO: 5, and its 5' end is modified with Bio; The sequence of the specific probe is shown in SEQ ID NO: 6, the 5' end of the probe is labeled with FAM, the 3' end is modified with C3 Spacer, and the position 30 bp away from the 5' end in the probe sequence is modified with THF.
3. A kit for detecting Legionella pneumophila, characterized in that: The method comprises a detection solution containing the RAA-LFD primer and a specific probe according to claim 2.