Primer probe group, kit and method for detecting legionella pneumophila

Abstract

The invention belongs to the field of digital PCR (polymerase chain reaction) detection of legionella pneumophila, and relates to a primer probe group, a kit and a detection method for detecting legionella pneumophila, primers comprise an upstream primer for detecting legionella pneumophila, a downstream primer for detecting legionella pneumophila, an internal control upstream primer and an internal control downstream primer; the probes comprise a legionella pneumophila detection probe and an internal control gene probe. According to the application, a sample with the concentration as low as 4000 copies / mL can be stably detected, the operation is simple, the detection efficiency is high, meanwhile, the legionella pneumophila is qualitative and quantitative, and secondly, the primer probe combination characteristic is high, and the detection sensitivity and the detection accuracy of the legionella pneumophila are high.

Description

Technical Field

[0001] The present application relates to the technical field of digital PCR for detecting Legionella pneumophila, and particularly to a primer-probe set, a kit and a detection method for detecting Legionella pneumophila. Background Art

[0002] Legionella pneumophila (LP) is a small, Gram-negative, aerobic, non-spore-forming, non-capsulated bacillus with catalase and oxidase, which is commonly present in the environment or parasitizes in a variety of protozoan hosts and is the main pathogen causing human Legionnaires' disease pneumonia.

[0003] The first outbreak of Legionnaires' disease pneumonia occurred in 1976. It is an acute systemic disease mainly characterized by lung lesions, which spreads from the lungs into the blood, causing damage to multiple systems of the human body. It has a rapid onset and a high fatality rate, and is a disease widely concerned by the current medical community. Legionnaires' disease has always been one of the three most common respiratory pathogens in community-acquired pneumonia and is also one of the reported causes of hospital-acquired pneumonia. Its main transmission routes are droplet and aerosol transmission.

[0004] At present, the domestic standard methods for detecting Legionella pneumophila include standards at different levels such as national standards, industry standards and local standards. The detection methods involved include bacterial isolation and culture method, colloidal gold immunochromatography method and fluorescence quantitative PCR detection method, etc. Most of them adopt the bacterial isolation and culture method, and biochemical culture and serological experiments are used to identify whether the colonies are Legionella pneumophila. However, this method has low detection sensitivity, long culture time, and cannot detect viable non-culturable Legionella pneumophila, so there are certain limitations; the colloidal gold immunochromatography method uses colloidal gold as a tracer marker, and uses the principle of chromatography and antigen-antibody reaction to qualitatively analyze Legionella pneumophila in the sample. It is simple to operate and fast to detect, and is suitable for on-site use. However, the accuracy of the detection depends on the specificity of the antibody. If the antibody specificity is poor, cross-reaction is likely to occur; the fluorescence quantitative PCR detection method additionally adds specific probes designed for the conserved region of Legionella pneumophila. These probes are linked with fluorescent groups, so that the detection and analysis of Legionella pneumophila can be realized by monitoring the fluorescence signal of the PCR reaction process. However, it depends on the standard curve and has poor resolution for low-copy target gene molecules, and the sensitivity, precision and resolution are limited. Summary of the Invention

[0005] The purpose of the present application is to provide a primer-probe set, a kit and a detection method for detecting Legionella pneumophila, so as to solve the technical problems in the prior art that Legionella pneumophila cannot be quantitatively detected quickly and accurately, and at the same time, the detection sensitivity, precision and resolution are relatively low.

[0006] To solve the above technical problems, the embodiments of the present application provide a primer-probe set for detecting Legionella pneumophila, and adopt the following technical solutions:

[0007] The primers include an upstream primer for detecting Legionella pneumophila, a downstream primer for detecting Legionella pneumophila, an internal control (an upstream primer for internal control and a downstream primer for internal control); the probes include a probe for detecting Legionella pneumophila and an internal control gene probe;

[0008] Among them, the nucleotide sequence of the upstream primer for detecting Legionella pneumophila is as shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer for detecting Legionella pneumophila is as shown in SEQ ID NO: 2; the nucleotide sequence of the upstream primer for internal control is as shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer for internal control is as shown in SEQ ID NO: 4;

[0009] The nucleotide sequence of the probe for detecting Legionella pneumophila is as shown in SEQ ID NO: 5, and the nucleotide sequence of the internal control gene probe is as shown in SEQ ID NO: 6.

[0010] Further, the 5' end of the nucleotide sequences of the probe for detecting Legionella pneumophila and the internal control gene probe is labeled with a fluorescent group; the 3' end of the nucleotide sequences of the probe for detecting Legionella pneumophila and the internal control gene probe is labeled with a quenching group;

[0011] Among them, the fluorescent group is selected from FAM, HEX or VIC; the quenching group is selected from MGB, BHQ series, TAMRA or Eclipse.

[0012] Further, the 5' end of the nucleotide sequence of the probe for detecting Legionella pneumophila is labeled with a FAM fluorescent group, and the 3' end of the nucleotide sequence of the probe for detecting Legionella pneumophila is labeled with an MGB quenching group; the 5' end of the nucleotide sequence of the internal control gene probe is labeled with a VIC fluorescent group, and the 3' end of the nucleotide sequence of the internal control gene probe is labeled with an MGB quenching group.

[0013] To solve the above technical problems, the embodiments of the present application also provide a kit for detecting Legionella pneumophila, and adopt the following technical solutions:

[0014] It includes a primer-probe mixture, a premix, an LP positive control and an LP negative control; among them, the primer-probe mixture includes the primer-probe set for detecting Legionella pneumophila as described above.

[0015] Further, the premix includes Tris-HCl buffer, dNTP, and PCR reaction enzyme.

[0016] Further, the final concentrations of the upstream primer for detecting Legionella pneumophila and the downstream primer for detecting Legionella pneumophila in the PCR reaction system are 0.400 - 0.500 μmol / L respectively; the final concentration of the probe for detecting Legionella pneumophila in the PCR reaction system is 0.220 - 0.230 μmol / L;

[0017] The final concentrations of the internal control upstream primer and the internal control downstream primer in the PCR reaction system are 0.400 - 0.500 μmol / L respectively; the final concentration of the internal control gene probe in the PCR reaction system is 0.220 - 0.230 μmol / L.

[0018] Further, the components of the LP positive control product include Legionella pneumophila pseudovirus and pseudovirus containing internal standard fragment; the components of the LP negative control product include pseudovirus containing internal standard fragment and TE buffer.

[0019] To solve the above technical problems, the embodiment of the present application also provides a detection method for detecting Legionella pneumophila, which uses the kit for detecting Legionella pneumophila as described above for non - diagnostic purpose detection, and adopts the following technical solutions:

[0020] Collect a sample to be tested for Legionella pneumophila, extract nucleic acid from the sample to be tested to obtain the nucleic acid of the sample to be tested;

[0021] Take out the primer - probe mixture and the premix from the kit, melt them at room temperature and mix well, prepare the PCR reaction system, and dispense the PCR reaction system into a preset number of PCR reaction tubes;

[0022] Correspondingly add the nucleic acid of the sample to be tested, the LP positive control product and the LP negative control product into the PCR reaction tubes, centrifuge instantaneously and then transfer them to the droplet preparation area;

[0023] Perform droplet preparation on the liquid in the PCR reaction tubes, and perform PCR reaction on the prepared droplets according to the PCR amplification conditions to obtain amplification products;

[0024] Perform quantitative analysis on the amplification products to obtain the content of Legionella pneumophila in the sample to be tested.

[0025] Further, the sample to be tested is human sputum.

[0026] Further, the PCR amplification conditions are as follows:

[0027] Hot start the enzyme at 95°C for 10 min; denature at 94°C for 30 s, anneal at 58°C for 1 min, for a total of 45 cycles; finally inactivate the enzyme at 98°C for 10 min.

[0028] Compared with the prior art, the present application mainly has the following beneficial effects:

[0029] The present application provides a primer-probe set, a kit and a detection method for detecting Legionella pneumophila. Based on digital PCR technology, the nucleic acid of Legionella pneumophila in a sample is quantitatively detected, and a quantitative detection result can be obtained in as fast as 3 hours. Samples with a stable detection concentration as low as 4000 copies / mL can be detected. The operation is simple, the detection efficiency is high, and qualitative and quantitative detection of Legionella pneumophila can be carried out simultaneously. Secondly, the primer-probe combination of the present application has high characteristics, and the detection sensitivity and detection accuracy for Legionella pneumophila are high. In addition, the present application does not need to set a standard curve. According to the results of fluorescence type and fluorescence microdroplet number, the positive and negative of Legionella pneumophila and the copy number can be directly judged, greatly simplifying the operation steps. Description of the Drawings

[0030] In order to more clearly illustrate the solutions in the present application, the following will briefly introduce the drawings required for the description of the embodiments of the present application. Obviously, the following described drawings are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 is the PCR reaction result of the kit of the present application for detecting the LP negative control product;

[0032] Figure 2 is the PCR reaction result of the kit of the present application for detecting the LP positive control product;

[0033] Figures 3 to 7 are the PCR reaction results of the kit of the present application for detecting the enterprise reference product S1 of Legionella pneumophila without dilution, diluted 1:10, diluted 1:100, diluted 1:1000, and diluted 1:10000 in sequence;

[0034] Figures 8 to 12 are the PCR reaction results of the kit of the present application for detecting the enterprise reference product S2 of Legionella pneumophila without dilution, diluted 1:10, diluted 1:100, diluted 1:1000, and diluted 1:10000 in sequence;

[0035] Figures 13 to 17 are the PCR reaction results of the kit of the present application for detecting the enterprise reference product S3 of Legionella pneumophila without dilution, diluted 1:10, diluted 1:100, diluted 1:1000, and diluted 1:10000 in sequence;

[0036] Figure 18 is the PCR reaction result of the kit of the present application for detecting the enterprise reference products PC01 - PC05 of Legionella pneumophila;

[0037] Figure 19The PCR reaction results of the enterprise reference products NC01 - NC10 for detecting Legionella pneumophila by the kit of the present application;

[0038] Figure 20 The detection results of the clinical samples by the kit of the present application and the third - party kit. Detailed implementation manners

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above - mentioned drawings are intended to cover non - exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above - mentioned drawings are used to distinguish different objects and not to describe a specific order.

[0040] Reference to "embodiment" herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0041] To enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0042] The primer - probe set, kit, and detection method for detecting Legionella pneumophila provided by this application use droplet digital PCR (hereinafter referred to as "ddPCR") technology to quantitatively detect the nucleic acid of Legionella pneumophila in the extracted sputum.

[0043] Among them, digital PCR (dPCR) is a test method that can be used to absolutely quantify the target fragment in the sample to be tested. Digital PCR will disperse a fluorescent PCR reaction system into tens of thousands of micro-reaction systems before performing PCR, and each micro-reaction is parallel. The target fragment template of the sample to be tested will also be dispersed into each micro-reaction system. If the number of dispersed micro-reaction systems is large enough, then in the end there will be only 1 or 0 fragment templates in each micro-reaction system, thus realizing "single-molecule template PCR amplification". Micro-reactions with 1 or more target fragments will produce fluorescence after PCR, which is a positive unit, and micro-reactions without target fragments will not produce fluorescence after PCR, which is a negative unit. By calculating the number of positive and negative units, combined with statistical formulas, the number of copies of the target fragment in the original sample can be calculated.

[0044] Based on digital PCR technology, the present application proposes a primer probe set for detecting Legionella pneumophila. The primers in the primer probe set include an upstream primer for detecting Legionella pneumophila, a downstream primer for detecting Legionella pneumophila, an internal control upstream primer and an internal control downstream primer; the probes include a Legionella pneumophila detection probe and an internal control gene probe.

[0045] The design of primers and probes should first follow the general design principles of primers and probes, that is, the GC% of primers and probes is required to be between 30% and 70%, the theoretical Tm>48℃, and the Tm value of probes is usually 5-10℃ higher than the Tm value of primers; the 3' ends of primers and probes themselves and between primers and probes should avoid base pairing as much as possible; at the same time, the 3' end of primers should avoid 3 or more consecutive identical bases, and the probes should avoid 4 consecutive Gs, or 6 consecutive A. At the same time, the principle of specific design should be followed. The selected target gene primers and probes are required to be designed in the conserved region of the target gene, which is specific only to the target gene and has no cross-reaction with other species and human genes. The probe is located in the region between primers. After the primers are designed according to the above principles, a large number of experiments and screening are carried out on the designed specific primers and probes, and they are combined, optimized and verified. By exploring the reaction conditions and optimizing the methods, the advantages and disadvantages of linearity, specificity, sensitivity and repeatability between different primers and probes are compared, and finally a set of optimal primer-probe combinations with no mutual interference, high amplification efficiency and good specificity are selected.

[0046] Among them, the nucleotide sequence of the upstream primer for detecting Legionella pneumophila (LP upstream primer) is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer for detecting Legionella pneumophila (LP downstream primer) is shown in SEQ ID NO: 2; the nucleotide sequence of the internal control upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the internal control downstream primer is shown in SEQ ID NO: 4; the nucleotide sequence of the probe for detecting Legionella pneumophila (LP probe) is shown in SEQ ID NO: 5, and the nucleotide sequence of the internal control gene probe is shown in SEQ ID NO: 6.

[0047] The nucleotide sequence information of the primer-probe group for detecting Legionella pneumophila in this application is shown in Table 1.

[0048] Table 1 Nucleotide sequence information of primer-probes for detecting Legionella pneumophila MIP region detection sites and internal controls

[0049]

[0050]

[0051] For the nucleotide sequences corresponding to the above primer-probe combinations, according to the gene detection sites, this application is designed for the detection sites of the Legionella pneumophila MIP region, so that the detection sensitivity and detection efficiency of the primer-probe combination at these gene detection sites are optimal.

[0052] Both ends of the above probes are labeled with a fluorescent group and a quenching group. Among them, the 5' ends of the probe for detecting Legionella pneumophila and the internal control gene probe are respectively labeled with a fluorescent group; the 3' ends of the probe for detecting Legionella pneumophila and the internal control gene probe are respectively labeled with a quenching group.

[0053] Among them, the fluorescent group is selected from FAM, HEX or VIC; the quenching group is selected from MGB, BHQ series, TAMRA or Eclipse.

[0054] The fluorescent groups labeled at the 5' ends of the probe for detecting Legionella pneumophila and the internal control gene probe are different, so as to obtain detection results according to different fluorescence channels. Among them, the 5' end of the probe for detecting Legionella pneumophila is labeled with the FAM fluorescent group, and the 3' end of the probe for detecting Legionella pneumophila is labeled with the MGB quenching group; the 5' end of the internal control gene probe is labeled with the VIC fluorescent group, and the 3' end of the internal control gene probe is labeled with the MGB quenching group.

[0055] The primers for detecting Legionella pneumophila and the probe for detecting Legionella pneumophila labeled with FAM fluorophore can be used for the quantitative detection of Legionella pneumophila. According to the presence or absence of positive droplets in the FAM channel and the number of positive and negative droplets, it can be determined whether Legionella pneumophila is contained in the sample and the concentration of the target fragment of Legionella pneumophila in the sample can be calculated. Among them, the target fragment is the DNA of LP virus or a DNA fragment of LP virus.

[0056] The primer-probe set for detecting Legionella pneumophila in this application has been specifically optimized to reduce the misdiagnosis probability caused by non-specific binding, and an internal control gene detection system has been designed for monitoring and correction.

[0057] This application also provides a kit for detecting Legionella pneumophila, which includes a primer-probe mixture, a premix, an LP positive control, and an LP negative control; among them, the primer-probe mixture includes the primer-probe set for detecting Legionella pneumophila as described above.

[0058] In some optional implementation manners, the final concentrations of the upstream primer for detecting Legionella pneumophila and the downstream primer for detecting Legionella pneumophila in the PCR reaction system are 0.400 - 0.500 μmol / L respectively; the final concentration of the probe for detecting Legionella pneumophila in the PCR reaction system is 0.220 - 0.230 μmol / L; the final concentrations of the upstream primer for the internal control and the downstream primer for the internal control in the PCR reaction system are 0.400 - 0.500 μmol / L respectively; the final concentration of the internal control gene probe in the PCR reaction system is 0.220 - 0.230 μmol / L.

[0059] Preferably, the final concentration of the upstream primer for detecting Legionella pneumophila in the PCR reaction system is 0.455 μmol / L, and the final concentration of the downstream primer for detecting Legionella pneumophila in the PCR reaction system is 0.455 μmol / L; the final concentration of the probe for detecting Legionella pneumophila in the PCR reaction system is 0.227 μmol / L; the final concentration of the upstream primer for the internal control in the PCR reaction system is 0.455 μmol / L, and the final concentration of the downstream primer for the internal control in the PCR reaction system is 0.455 μmol / L; the final concentration of the internal control gene probe in the PCR reaction system is 0.227 μmol / L.

[0060] The premix can be the ddPCR Supermix for Probes (No dUTP) for DNA sample droplet digital PCR provided by Bio-rad, with the product numbers 1863023, 1863024, or 1863025.

[0061] The kit of the present application further includes reference substances, which include LP positive control and LP negative control. Among them, the components of the LP positive control include Legionella pneumophila pseudovirus and pseudovirus containing internal standard fragments, and the components of the LP negative control include pseudovirus containing internal standard fragments and TE buffer.

[0062] The detection sample applicable to the kit of the present application is human sputum.

[0063] The criteria for the kit of the present application to determine the detection effectiveness are as follows:

[0064] In each detection, only the reaction wells with the number of droplets ≥ 10,000 in all reaction wells are valid reaction wells;

[0065] Among the valid reaction wells, negative control and positive control should be included. When the detection result of the positive control is positive, the detection result of the negative control is negative, and the detection result of the concentration of the positive control is 10 5 ±15% copies / mL, the detection result of this time is valid.

[0066] Based on the above kit for detecting Legionella pneumophila, the present application also provides a detection method for detecting Legionella pneumophila using the above kit, and the method includes the following steps:

[0067] Step S10, collect the sample to be detected for Legionella pneumophila, extract nucleic acid from the sample to be detected to obtain the nucleic acid of the sample to be detected.

[0068] Any one of the samples to be detected is human sputum.

[0069] Among them, before collecting the sputum sample, the patient needs to perform thorough oral cleaning, cough out the sputum forcefully and deeply, and collect it into a sterile cup for subsequent detection.

[0070] Use a qualified human sputum extraction kit to extract the sampling solution. For example, use the nucleic acid extraction or purification reagent (registration number for medical device in Guangdong Province: 20170583) produced by Daan Gene Co., Ltd. to extract and purify the nucleic acid of the collected sputum sample.

[0071] Step S20, take out the primer-probe mixture and the premix from the above kit, melt them at room temperature and mix well, prepare a PCR reaction system, and dispense the PCR reaction system into a preset number of PCR reaction tubes.

[0072] Take out the primer-probe mixture and the premix from the kit, melt them at room temperature and mix well with a vortex oscillator. After centrifuging at 8000 rpm - 10000 rpm for 10 - 20 s, prepare a PCR reaction system, and dispense the PCR reaction system into a preset number of PCR reaction tubes.

[0073] Among them, the preset quantity N = the number of samples to be tested + LP negative control product + LP positive control product

[0074] The single - person PCR reaction system is shown in Table 2.

[0075] Table 2 Single - person PCR reaction system

[0076] Component Volume Primer-probe mixture 5 μL Premix 10 μL

[0077] In step S30, add the nucleic acid of the sample to be tested, LP positive control product, and LP negative control product to the PCR reaction tube respectively. After instantaneous centrifugation, transfer it to the droplet preparation area.

[0078] Taking the above single - person PCR reaction system as an example, add 5 μL of the nucleic acid of the sample to be tested, LP positive control product, and LP negative control product to the PCR reaction tube after adding the PCR reaction system respectively. Tighten the tube cap, and transfer it to the droplet preparation area after instantaneous centrifugation for 15 seconds.

[0079] In step S40, perform droplet preparation on the liquid in the PCR reaction tube, and perform PCR reaction on the prepared droplets according to the PCR amplification conditions to obtain amplification products.

[0080] Perform droplet preparation according to the instruction manual of Bio - rad's droplet digital PCR platform. Transfer the prepared droplets to the special 96 - well plate for Bio - rad's droplet digital PCR.

[0081] Specifically, mix the PCR reaction system with the added samples using a vortex oscillator. Take out a droplet preparation chip, transfer one column (8 reaction systems) of the reaction system to the sample wells of the chip, add 70 μL of droplet preparation oil to the droplet preparation oil wells, and send it into the droplet preparation instrument for droplet preparation. After the instrument reports that the droplet preparation is completed, carefully transfer the prepared droplets to the special 96 - well plate using a pipette, discard the droplet preparation chip, and then take another droplet preparation chip to process the next column of reaction systems until all the reaction systems to be tested are processed. Then seal the 96 - well plate with an aluminum mold on a heat sealer.

[0082] Transfer the sealed 96 - well plate to the qualitative PCR, and set the cycles according to Table 3 for PCR reaction. Specifically, perform enzyme heat activation at 95 °C for 10 min; denature at 94 °C for 30 s, anneal at 58 °C for 1 min, for a total of 45 cycles; finally, inactivate the enzyme at 98 °C for 10 min.

[0083] Table 3 PCR amplification conditions

[0084]

[0085] After PCR is completed, take out the 96 - well plate containing the amplification products and transfer it to the droplet reader.

[0086] Step S50, perform quantitative analysis on the amplification product to obtain the content of Legionella pneumophila in the sample to be tested.

[0087] Set the droplet reading parameters according to the instruction manual. Pay attention to the selection of the fluorescence channel: select the FAM channel to detect the nucleic acid of Legionella pneumophila and the VIC channel to detect the internal standard (internal control gene). After setting, start the droplet reading program.

[0088] After the droplet reading is completed, the results are automatically saved and enter the result analysis interface. Check the total number of droplets in each reaction well. If the number of droplets in a certain reaction well < 10,000, it is regarded as an invalid reaction well. Then check whether the positive and negative quality control products of this test are included in the valid reaction wells. If so, this test is valid.

[0089] See Figure 1 and Figure 2 the PCR reaction results of the LP negative quality control product and the LP positive quality control product detected by the kit of the present application shown in. Among them, Ch1 corresponds to the FAM fluorescence channel and Ch2 corresponds to the VIC fluorescence channel. As can be seen from the figure, in Figure 1 , no signal is detected through the Ch1 channel, and the internal control gene fragment is detected through the Ch2 channel; Figure 2 , the Legionella pneumophila gene fragment and the internal control gene fragment are detected through the Ch1 channel and the Ch2 channel respectively.

[0090] If the test is valid, check the fluorescence scatter plot of Ch1 (FAM channel). After drawing the threshold line, check the concentration of Ch1, in units of copies / μL. Finally, the concentration of the original sample can be calculated by combining the sample dilution factor.

[0091] It should be noted that the detection principle of the present application is as follows:

[0092] The kit of the present application adopts the droplet digital PCR technology of Bio-rad. Specific primers and probes are designed in the conserved region of the Legionella pneumophila genome. Legionella pneumophila is labeled with the FAM fluorescent group, and the internal standard gene is labeled with the VIC fluorescent group. After preparing the PCR reaction system and loading the samples, it is dispersed into tens of thousands of droplets. Each droplet can perform PCR simultaneously. After the PCR is completed, a droplet reader is used to detect the fluorescence level of each droplet to judge the positivity or negativity of the droplet. After counting the positivity and negativity of all droplets, it can be judged whether there is Legionella pneumophila nucleic acid in the sample. Further, according to the statistical results of the positive and negative droplets and combined with the statistical formula, the copy number of Legionella pneumophila nucleic acid in the sample can be calculated.

[0093] The following will further illustrate the content of the present application in more detail with specific embodiments and further elaborate on the present application. However, these embodiments are by no means a limitation to the present application.

[0094] Example 1

[0095] This example provides the composition, packaging, and quantity (96 tests per box) of a kit for detecting Legionella pneumophila, as shown in Table 4.

[0096] Table 4 Composition, packaging, and quantity of a kit for detecting Legionella pneumophila (96 tests per box)

[0097]

[0098] Example 2

[0099] In this example, a detection experiment was conducted on the sample detection range and sensitivity of the kit of the present application. The specific process is as follows:

[0100] Take an appropriate amount of LP primer-probe mixture and ddPCR Supermix for Probes (No dUTP), prepare the PCR reaction system according to the method of step S20, and transfer it into a PCR reaction tube; detect the enterprise reference products S1-S3 of Legionella pneumophila, use RNase-free deionized water, dilute them at 1:10, 1:100, 1:1000, and 1:10000. After nucleic acid extraction, take 5 μL of the extraction solution and add it to the octuplet PCR reaction system prepared in step S20 to make the total volume 20 μL. Tighten the lids of the eight tubes, shake vigorously with an oscillator for 15 seconds, centrifuge instantaneously for 15 seconds, and then transfer to the droplet preparation area.

[0101] After preparing the droplets according to the droplet preparation method of step S40 above, seal the 96-well plate with an aluminum mold on a heat sealer, transfer the sealed 96-well plate to a qualitative PCR, and perform a PCR reaction on the prepared droplets according to the PCR amplification conditions in Table 3.

[0102] After the reaction, the reaction results are shown in Table 5 and Figures 3 to 17 as shown, Figures 3 to 17 which are the PCR reaction results of different dilution concentrations of S1-S3 in sequence.

[0103] Table 5 Detection results of sample detection range and sensitivity

[0104]

[0105] It can be seen from the above table results that when the sample concentration is between 4000 and 4000000 copies / mL, the quantitative test results of this kit have a difference of <15% from the true concentration. The results show that the lowest detection limit of the kit of the present application is 4000 copies / mL, and the kit has high sensitivity.

[0106] Example 3

[0107] This example detects the accuracy of the kit of the present application.

[0108] The kit of the present application is used to detect the enterprise reference products PC01 - PC05 and NC01 - NC10 containing Legionella pneumophila. Take 5 μL of the extraction solution and add it to the octuplet tube of the PCR reaction system prepared in step S20 to make the total volume 20 μL. Tighten the lids of the eight tubes, vigorously shake and mix evenly with a shaker for 15 seconds, transfer it to the droplet preparation area after centrifuging instantaneously for 15 seconds.

[0109] After preparing the droplets according to the droplet preparation method in step S40 above, seal the 96 - well plate with an aluminum mold on a heat sealer, transfer the sealed 96 - well plate to a qualitative PCR, and perform a PCR reaction on the prepared droplets according to the PCR amplification conditions in Table 3.

[0110] After the reaction ends, the reaction results are shown in Table 6 and Figure 18 、 Figure 19 as shown.

[0111] Table 6 Results of accuracy detection

[0112]

[0113] According to the detection results in the above table, the coincidence rate of negative reference products is 100%, the coincidence rate of positive reference products is 100%, and the positive - negative coincidence rate of the detection results of the accuracy of each reference product is 100%. This shows that the detection accuracy is high and the quantitative results are accurate, indicating that the accuracy detection of the kit of the present application meets the requirements.

[0114] Example 4

[0115] This example is a clinical application experiment, using the kit of the present application and a third - party influenza A detection kit to detect the positive and negative of the tested clinical samples.

[0116] Respectively select 10 clinically positive samples and 10 negative samples of Legionella pneumophila. After nucleic acid extraction, mark the samples well and ensure that the label information is correct, and store them at - 80 °C. During the experiment, take 5 μL of each sample and add it to the reaction tube containing the PCR reaction system prepared according to step S20 to make the total volume 20 μL. Vigorously shake and mix evenly with a shaker for 15 seconds, transfer it to the droplet preparation area after centrifuging instantaneously for 15 seconds.

[0117] After preparing the droplets according to the droplet preparation method in step S40 above, seal the 96 - well plate with an aluminum mold on a heat sealer, transfer the sealed 96 - well plate to a qualitative PCR, and perform a PCR reaction on the prepared droplets according to the PCR amplification conditions in Table 3.

[0118] After the reaction ends, the reaction results are shown in Table 7 and Figure 20 as shown.

[0119] Table 7 Detection Results of Clinical Samples of the Kit of the Present Application and Third-Party Kits

[0120]

[0121]

[0122] The above detection results show that among the 20 samples, 10 are positive for Legionella pneumophila and 10 are negative. The consistency of the detection results of positive and negative clinical samples with those of the third-party kit reaches 100%. Through the detection of the kit of the present application, it is possible to identify whether the tested population carries Legionella pneumophila and conduct quantitative detection on it, which is worthy of popularization and application.

[0123] Obviously, the embodiments described above are only a part of the embodiments of the present application, rather than all the embodiments. The preferred embodiments of the present application are given in the drawings, but do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing specific embodiments, or perform equivalent replacements on some of the technical features. Any equivalent structure made by using the specification and drawings of the present application, directly or indirectly applied in other related technical fields, is similarly within the scope of the patent protection of the present application.

Claims

1. A primer-probe set for detecting Legionella pneumophila, characterized in that, Comprising primers and probes: The primers include an upstream primer for detecting Legionella pneumophila, a downstream primer for detecting Legionella pneumophila, an internal control upstream primer, and an internal control downstream primer; the probes include a probe for detecting Legionella pneumophila and an internal control gene probe; Among them, the nucleotide sequence of the upstream primer for detecting Legionella pneumophila is shown as SEQ ID NO: 1, and the nucleotide sequence of the downstream primer for detecting Legionella pneumophila is shown as SEQ ID NO: 2; the nucleotide sequence of the internal control upstream primer is shown as SEQ ID NO: 3, and the nucleotide sequence of the internal control downstream primer is shown as SEQ ID NO: 4; The nucleotide sequence of the probe for detecting Legionella pneumophila is shown as SEQ ID NO: 5, and the nucleotide sequence of the internal control gene probe is shown as SEQ ID NO:

6.

2. The primer-probe set for detecting Legionella pneumophila according to claim 1, wherein The 5'-end of the nucleotide sequences corresponding to the probe for detecting Legionella pneumophila and the internal control gene probe is labeled with a fluorescent group; the 3'-end of the nucleotide sequences corresponding to the probe for detecting Legionella pneumophila and the internal control gene probe is labeled with a quenching group; Among them, the fluorescent group is selected from FAM, HEX or VIC; the quenching group is selected from MGB, BHQ series, TAMRA or Eclipse.

3. The primer-probe set for detecting Legionella pneumophila according to claim 1, characterized in that, The 5'-end of the nucleotide sequence of the probe for detecting Legionella pneumophila is labeled with a FAM fluorescent group, and the 3'-end of the nucleotide sequence of the probe for detecting Legionella pneumophila is labeled with an MGB quenching group; the 5'-end of the nucleotide sequence of the internal control gene probe is labeled with a VIC fluorescent group, and the 3'-end of the nucleotide sequence of the internal control gene probe is labeled with an MGB quenching group.

4. A kit for detecting Legionella pneumophila, characterized in that, Comprising a primer-probe mixture, a premix, an LP positive control, and an LP negative control; among them, the primer-probe mixture includes the primer-probe group for detecting Legionella pneumophila according to any one of claims 1 to 3.

5. The kit for detecting Legionella pneumophila according to claim 4, wherein The premix includes Tris-HCl buffer, dNTP, and PCR reaction enzyme.

6. The kit for detecting Legionella pneumophila according to claim 4, wherein The final concentrations of the upstream primer for detecting Legionella pneumophila and the downstream primer for detecting Legionella pneumophila in the PCR reaction system are 0.400 - 0.500 μmol / L respectively; the final concentration of the probe for detecting Legionella pneumophila in the PCR reaction system is 0.220 - 0.230 μmol / L; The final concentrations of the internal control upstream primer and the internal control downstream primer in the PCR reaction system are 0.400 - 0.500 μmol / L respectively; the final concentration of the internal control gene probe in the PCR reaction system is 0.220 - 0.230 μmol / L.

7. The kit for detecting Legionella pneumophila according to claim 4, wherein, The components of the LP positive control include Legionella pneumophila pseudovirus and pseudovirus containing an internal standard fragment; the components of the LP negative control include pseudovirus containing an internal standard fragment and TE buffer.

8. A detection method for detecting Legionella pneumophila, which uses the kit for detecting Legionella pneumophila according to any one of claims 4 to 7 for non-diagnostic purposes, characterized in that, Comprising the following steps: Collect a sample to be tested for Legionella pneumophila, extract nucleic acid from the sample to be tested to obtain the nucleic acid of the sample to be tested; Take out the primer-probe mixture and the premix from the kit, melt them at room temperature and mix them evenly to prepare a PCR reaction system, and divide the PCR reaction system into a preset number of PCR reaction tubes; Add the nucleic acid of the sample to be tested, the LP positive control product, and the LP negative control product into the PCR reaction tube respectively, and transfer it to the droplet preparation area after instantaneous centrifugation; Prepare droplets from the liquid in the PCR reaction tube, and perform PCR reaction on the prepared droplets according to the PCR amplification conditions to obtain amplification products; Perform quantitative analysis on the amplification products to obtain the content of Legionella pneumophila in the sample to be tested.

9. The method for detecting Legionella pneumophila according to claim 8, wherein The sample to be tested is human sputum.

10. The method for detecting Legionella pneumophila according to claim 8, characterized in that, The PCR amplification conditions are as follows: Enzyme heat activation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing at 58°C for 1 min, for a total of 45 cycles; finally, enzyme inactivation at 98°C for 10 min.

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