Multiple fluorescent probe primers and kit for simultaneous detection of multiple central nervous system infection pathogens

Abstract

The present invention provides multiple fluorescent probe primers and kits for simultaneously detecting multiple pathogens of central nervous system infection. Amplification primers and different fluorescent labeled Taqman fluorescent probes are designed for 9 pathogenic bacteria, including Listeria monocytogenes, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus agalactiae, Neisseria meningitidis, coagulase-negative Staphylococcus, Staphylococcus aureus, Acinetobacter baumannii and Klebsiella pneumoniae. At the same time, human-derived internal references are added as internal quality control of the kit, so that the reagent can distinguish 3 kinds of target targets and endogenous internal reference genes in a single tube. In addition, probes and primers for coagulase-negative Staphylococci in cerebrospinal fluid can effectively detect Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus saprophyticus, and Staphylococcus hemolyticus, and can be effectively distinguished from coagulase-positive Staphylococci such as Staphylococcus aureus, Staphylococcus dolphinus, Staphylococcus suis, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of medical biological detection, relates to the rapid detection of pathogenic bacteria of central nervous system infection, and specifically relates to multiple fluorescent probe primers and a kit for simultaneously detecting multiple pathogenic bacteria of central nervous system infection. Background Art

[0002] Central nervous system infections (CNSIs) refer to acute or chronic inflammatory (or non-inflammatory) diseases caused by various pathogenic microorganisms invading the parenchyma, membrane or blood vessels of the central nervous system. Pathogenic microorganisms include but are not limited to viruses, bacteria, fungi, spirochetes, parasites, rickettsia, prions, etc. According to the site of infection, they can be divided into: (1) encephalitis, myelitis or encephalomyelitis, which mainly invade the brain and (or) spinal cord parenchyma; (2) meningitis, meningitis or cerebrospinal meningitis: mainly invade the brain and (or) spinal cord pia mater; (3) meningoencephalitis: brain parenchyma and meninges are affected together. According to the onset and course of the disease, they can be divided into acute, subacute and chronic infections. The symptoms of central nervous system infection can be varied, depending on the site and severity of the infection. Some common symptoms include fever, headache, vomiting, insomnia, hallucinations, coma, etc. These symptoms may affect the patient's ability to move, cognitive function and sensory ability. Severe central nervous system infections may lead to death.

[0003] According to the type of pathogenic pathogen, it can also be generally divided into viral meningitis, bacterial meningitis, fungal meningitis, etc. Among them, fungal meningitis is relatively rare; bacterial meningitis is more critical than viral meningitis and has a worse prognosis. It is one of the infectious diseases with a higher mortality rate in children. 30% to 50% of survivors of bacterial meningitis have permanent neurological sequelae. Therefore, early intervention can alleviate the condition and reduce the risk of complications. Therefore, for patients with suspected central nervous system infection, healthcare professionals usually take emergency measures, such as cerebrospinal fluid testing, prophylactic use of antibiotics, empirical medication, etc.

[0004] Traditional cerebrospinal fluid routine, biochemical, staining and culture examinations are insufficient in terms of diagnostic timeliness, sensitivity and specificity, which brings difficulties to the early diagnosis and rapid differential diagnosis of the disease. With the improvement of molecular diagnostic technology, it is possible to perform specific detection of pathogen genes, making the test more direct and accurate. For molecular detection of meningitis bacteria, many useful explorations have been made in the prior art: for example, the Chinese invention patent with patent number CN201610296666 uses a multiplex ordinary PCR method to detect 6 pathogens at the same time, which is complicated to operate and cannot be quantified; the Chinese invention patent with patent number CN108085408A expands the detection types to 12, using fluorescent PCR technology, but it is a single tube double-plex, and 6 tubes of reaction solution are required for detection, which is relatively complicated to operate, and there is no internal reference control, and the entire reaction system cannot be monitored.

[0005] In addition, most diagnostic techniques do not include coagulase-negative Staphylococci, which have the highest positive rate in cerebrospinal fluid culture, in the detection range. Coagulase-negative Staphylococci (CoNS) are the pathogens with the highest positive culture rate in cerebrospinal fluid samples. They are part of Staphylococci that do not produce plasma coagulase. Plasma coagulase is a virulence factor of Staphylococci and an enzyme substance that can coagulate human or rabbit plasma containing sodium citrate or heparin anticoagulants. Therefore, coagulase is an important indicator for distinguishing whether Staphylococci are pathogenic. In the past, it was believed that CoNS was non-pathogenic and was a normal bacterium commonly found in the skin, oral cavity and intestines of healthy people. However, clinical and experimental test results in recent years have confirmed that it has become a common pathogen of nosocomial infection, especially the emergence of drug-resistant strains. Because it is widely present in the environment and colonized in human skin, it is easy to be infected through wounds, such as after craniocerebral injury, craniocerebral surgery and lumbar puncture.

[0006] Cerebrospinal fluid is a sterile body fluid without colonized bacteria, but when CSF is collected by percutaneous puncture, CoNS colonized in the epidermis may still contaminate the CSF specimen; when there is a central drainage tube, the CSF flowing out of the catheter may be contaminated with colonized bacteria, so strict aseptic operation is required, and whether it is contaminated by CoNS is determined based on the test results and clinical conditions. Since there are many types of coagulase-negative Staphylococcus aureus, and they belong to the same genus as coagulase-positive Staphylococcus aureus and have a high degree of homology in gene sequence, how to accurately distinguish the two through fluorescent PCR is also a recognized difficulty, and there are no reports on the simultaneous detection of them and other common CSF pathogens. Summary of the invention

[0007] The present invention is to solve the above technical problems. Amplification primers and different fluorescent-labeled Taqman fluorescent probes are designed for the specific conservative sequences of the most common community-acquired Listeria monocytogenes, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus agalactiae, Neisseria meningitidis and hospital-acquired common coagulase-negative Staphylococcus, Staphylococcus aureus, Acinetobacter baumannii and Klebsiella pneumoniae. At the same time, a human internal reference is added as the internal quality control of the kit, which can distinguish 3 target targets and endogenous internal reference genes in a single tube.

[0008] After analyzing and comparing a large number of gene sequences, the present invention successfully designed a set of primer probes for coagulase-negative staphylococci in cerebrospinal fluid, which can effectively detect coagulase-negative staphylococci such as Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus saprophyticus, and Staphylococcus haemolyticus, and can effectively distinguish them from coagulase-positive staphylococci such as Staphylococcus aureus, Staphylococcus dolphinus, and Staphylococcus hyosus.

[0009] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a probe primer composition for detecting coagulase-negative staphylococci, wherein the upstream and downstream primer sequences are shown in SEQ ID NO.19 and 20 respectively, and the probe sequence is shown in SEQ ID NO.21.

[0011] In a second aspect, the present invention provides a probe primer composition for multiple pathogenic bacteria causing central nervous system infection: (1) the upstream and downstream primer sequences for detecting Neisseria meningitidis are shown in SEQ ID NOs. 1 and 2, respectively, and the probe sequence is shown in SEQ ID NO. 3; (2) the upstream and downstream primer sequences for detecting Haemophilus influenzae are shown in SEQ ID NOs. 4 and 5, respectively, and the probe sequence is shown in SEQ ID NO. 6; (3) the upstream and downstream primer sequences for detecting Streptococcus agalactiae are shown in SEQ ID NOs. 7 and 8, respectively, and the probe sequence is shown in SEQ ID NO. 9; (4) the upstream and downstream primer sequences for detecting Listeria monocytogenes are shown in SEQ ID NOs. 10 and 11, respectively, and the probe sequence is shown in SEQ ID NO. 12; (5) the upstream and downstream primer sequences for detecting Staphylococcus aureus are shown in SEQ ID NOs. 13 and 14, respectively, and the probe sequence is shown in SEQ ID NO. 15; (6) the upstream and downstream primer sequences for detecting Streptococcus pneumoniae are shown in SEQ ID NOs. 16 and 17, respectively, and the probe sequence is shown in SEQ ID NO. NO.18; (7) the upstream and downstream primer sequences for detecting coagulase-negative Staphylococcus are shown in SEQ ID NO.19 and 20, respectively, and the probe sequence is shown in SEQ ID NO.21; (8) the upstream and downstream primer sequences for detecting Klebsiella pneumoniae are shown in SEQ ID NO.22 and 23, respectively, and the probe sequence is shown in SEQ ID NO.24; (9) the upstream and downstream primer sequences for detecting Acinetobacter baumannii are shown in SEQ ID NO.25 and 26, respectively, and the probe sequence is shown in SEQ ID NO.27. For specific sequences, see Table 1.

[0012] Preferably, the probe primer composition for multiple pathogens of central nervous system infection provided by the present invention also includes internal reference universal primers and probes: the internal reference universal upstream and downstream primer sequences are shown in SEQ ID NO. 28 and 29, respectively, and the internal reference universal probe sequence is shown in SEQ ID NO. 30. See Table 1 for specific sequences.

[0013] Table 1 Summary of pathogenic bacteria and internal reference upstream primers, downstream primers and probe sequences

[0014]

[0015]

[0016] In a third aspect, the present invention provides a multiplex fluorescent PCR kit for multiple pathogens of central nervous system infection, comprising a PCR reaction solution, a detection solution, a positive quality control product and a negative quality control product. The detection solution contains the above-mentioned probe primer combination.

[0017] The PCR reaction solution includes PCR Buffer, dNTP, dUTP, Mg 2+ , UDG enzyme and Taq hot start enzyme;

[0018] There are three test solutions, each containing a probe-primer combination for detecting three pathogens and an internal reference universal primer and probe; the above pathogens are randomly divided into three groups, each group corresponding to a test solution. In each test solution, the four probes are labeled with different fluorescent groups. For example, the FAM, CY5, and ROX probes in each tube of test solution correspond to three detection targets, respectively, and the VIC probe corresponds to one internal reference target, so it is possible to detect nine pathogens using only three PCR reaction wells, and each well has an effective internal reference control quality control.

[0019] The negative quality control product is normal saline; the positive quality control product contains recombinant plasmids of 9 pathogen detection target sequences, the sequences are shown in Table 2:

[0020] Table 2 Recombinant plasmid sequences of 9 pathogen detection target sequences in positive quality control products

[0021]

[0022]

[0023]

[0024] The fourth aspect of the present invention provides a multiplex fluorescent PCR detection method for multiple pathogens of central nervous system infection using the above kit, which is as follows:

[0025] 1. Reaction mixture configuration

[0026] Since there are three tubes of detection solution, each tube contains three primer probes for detection targets and internal references, the PCR reaction mixture needs to be prepared according to the number of samples when used, with 30μL of reaction mixture in each tube. Add 20μL of the extracted template from each sample to the PCR reaction tubes containing the reaction mixture 1, 2, and 3, respectively, that is, 50μL of the reaction system is amplified on the machine. The instrument selects FAM, VIC / HEX, ROX, and CY5 channels, and the volume is 50μL.

[0027] 2. PCR reaction

[0028] The PCR reaction program was set up as follows:

[0029] (1) Cycle number 1, temperature 50°C, 2 min, no fluorescence signal collection;

[0030] (2) Cycle number 1, temperature 95°C, 2 min, no fluorescence signal collection;

[0031] (3) Cycle number 40, temperature 95°C, 5 s, 63°C, 31 s, collect fluorescence signals.

[0032] The detection fluorescence channels of the target are FAM, ROX, and CY5 channels, and VIC / HEX is the internal reference detection channel. The baseline is generally set for 3-15 cycles, and the threshold line is set just above the negative control amplification curve.

[0033] 3. Interpretation of results

[0034] The threshold of Ct value for Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, coagulase-negative Staphylococcus, Klebsiella pneumoniae, and Acinetobacter baumannii was 38; the test was considered positive when Ct≤38 and there was an obvious amplification curve.

[0035] The threshold of the Ct value of the internal reference is 35, and the test result is judged to be valid when the Ct value is ≤35.

[0036] The beneficial protection and effects of the present invention are as follows:

[0037] The present invention designs a multiple fluorescent probe primer and a kit for simultaneously detecting multiple pathogens of central nervous system infection. Amplification primers and Taqman fluorescent probes with different fluorescent labels are designed for the specific conservative sequences of 9 pathogens, including Listeria monocytogenes, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus agalactiae, Neisseria meningitidis, coagulase-negative Staphylococci, Staphylococcus aureus, Acinetobacter baumannii and Klebsiella pneumoniae. At the same time, a human internal reference is added as the internal quality control of the kit, so that the reagent can distinguish 3 target genes and endogenous internal reference genes in a single tube. After experimental verification, the positive coincidence rate of the kit of the present invention is 100%, the negative coincidence rate is 100%, and when the precision detection concentration is 1.0E+04CFU / mL, the CV value of the Ct value is ≤5; the sensitivity confirmation experiment shows that the detection rate is not less than 95%

[0038] In addition, probes and primers targeting coagulase-negative staphylococci in cerebrospinal fluid can effectively detect Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus saprophyticus, and Staphylococcus haemolyticus, and can effectively distinguish them from coagulase-positive staphylococci such as Staphylococcus aureus, Staphylococcus dolphinus, and Staphylococcus hyosus. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the test result of the test solution 1 in Example 1;

[0040] Figure 2 is the test result of the test solution 2 in Example 1;

[0041] Figure 3 is the test result of test solution 3 in Example 1;

[0042] Figure 4 These are the results of cross-amplification detection of coagulase-negative staphylococci and different concentrations of Staphylococcus aureus. DETAILED DESCRIPTION

[0043] The implementation of the present invention is described in detail below in conjunction with the embodiments of the present invention. The following embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operating processes, but the protection scope of the present invention is not limited to the following embodiments.

[0044] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0045] Example 1. Composition of the kit and detection method

[0046] 1. Kit Composition

[0047] A multiplex fluorescent PCR kit for multiple pathogens of central nervous system infection, including PCR reaction solution, detection solution, positive quality control product and negative quality control product.

[0048] The PCR reaction solution is 1.2 mL / tube, 1 tube / box, including PCR Buffer, dNTP, dUTP, Mg 2+ , UDG enzyme and Taq hot start enzyme.

[0049] The specification of the detection liquid is 200 μL / tube, with a total of 3 tubes / box, each of which contains a probe primer composition for detecting three pathogens and an internal reference universal primer and probe. The above pathogens are randomly divided into three groups, each group corresponding to a detection liquid. The grouping is not limited to the following combinations: detection liquid 1 contains Neisseria meningitidis primer probe, Haemophilus influenzae primer probe, Streptococcus agalactiae primer probe, and internal reference primer probe; detection liquid 2 contains Listeria monocytogenes primer probe, Streptococcus pneumoniae primer probe, Staphylococcus aureus primer probe, and internal reference primer probe; detection liquid 3 contains Acinetobacter baumannii primer probe, coagulase negative primer probe, Klebsiella pneumoniae primer probe, and internal reference primer probe.

[0050] In each test solution, the four probes are labeled with different fluorescent groups. For example, the FAM, CY5, and ROX probes in each tube of test solution correspond to three detection targets, and the VIC probe corresponds to one internal reference target, so it is possible to detect nine pathogens using only three PCR reaction wells, and each well has an effective internal reference quality control.

[0051] The negative quality control product is normal saline, with a specification of 1mL / tube, 1 tube / box; the positive quality control product contains recombinant plasmids of 9 pathogen detection target sequences, with a specification of 1mL / tube, 1 tube / box.

[0052] 2. Detection Method

[0053] 1. Extraction of nucleic acid components

[0054] The nucleic acid extraction component in the cerebrospinal fluid uses the nucleic acid extraction kit (magnetic bead method) (Suchang Food Equipment No. 20200138) of Jifan Biotechnology (Changzhou) Co., Ltd. The extraction reagent uses magnetic bead method for nucleic acid extraction, and the nucleic acid loading volume is 200μL per test, including lysis, washing and elution steps. Cerebrospinal fluid nucleic acids can be extracted manually with a magnetic rack, or they can be extracted using a commercially available fully automatic nucleic acid extraction instrument. The recommended elution volume is 80μL.

[0055] 2. Reaction mixture configuration

[0056] The nucleic acid amplification part is divided into PCR reaction solution and detection solution. The PCR reaction solution is produced by Nearshore Protein Company. High-Specificity Probe qPCR SuperMix (UDG), the primers and Taqman probes in the test solution are synthesized by Suzhou Zixi Biotechnology Co., Ltd. The probes are labeled with four fluorescent groups, namely FAM, CY5, VIC, and ROX. The FAM, CY5, and ROX probes in each tube of the test solution correspond to three detection targets, and the VIC probe corresponds to one internal reference target, so it is possible to detect nine pathogens using only three PCR reaction wells, and each well has an effective internal reference control quality control.

[0057] Since there are three tubes of detection solution, each tube contains three primer probes for detection targets and internal references, the PCR reaction mixture needs to be prepared according to the number of samples when used. The preparation method is shown in Table 3 above, n = number of samples + negative control + positive control, and the final reaction mixture is 30 μL in the PCR reaction tube.

[0058] Table 3 Configuration of three-tube reaction mixture

[0059]

[0060] Add 20 μL of the extracted template from each sample to the PCR reaction tubes containing the reaction solution mixture 1, 2, and 3, respectively, and amplify the 50 μL reaction system on the instrument. Select the FAM, VIC / HEX, ROX, and CY5 channels on the instrument, and select the volume of 50 μL.

[0061] 3. PCR reaction

[0062] The PCR reaction program settings are shown in Table 4:

[0063] Table 4 PCR reaction program

[0064]

[0065] The detection fluorescence channels of the target are FAM, ROX, and CY5 channels, and VIC / HEX is the internal reference detection channel. The baseline is generally set for 3-15 cycles, and the threshold line is set just above the negative control amplification curve.

[0066] 4. Interpretation of results

[0067] The interpretation criteria are shown in Table 5:

[0068] Table 5 Results interpretation criteria

[0069]

[0070]

[0071] The test results of the three tubes of mixed reaction solution are as follows Figure 1 to Figure 3 As shown; the cross-amplification test results of coagulase-negative staphylococci and different concentrations of Staphylococcus aureus are shown in Figure 4 The results show that the method of the present invention can effectively detect nine pathogenic bacteria, including Listeria monocytogenes, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus agalactiae, Neisseria meningitidis, coagulase-negative Staphylococcus, Staphylococcus aureus, Acinetobacter baumannii and Klebsiella pneumoniae; and can effectively distinguish coagulase-negative Staphylococcus from positive Staphylococcus aureus.

[0072] Example 2 Methodological verification of the detection kit of the present invention

[0073] The research objects of the 9 pathogenic bacteria detection involved in the present invention are all first-generation culture strains of ATCC strains of the American Collection Center, and the ATCC strain codes are as follows: Listeria monocytogenes ATCC19115, Haemophilus influenzae ATCC10211, Streptococcus pneumoniae ATCC49619, Streptococcus agalactiae ATCC13813, Neisseria meningitidis: ATCC 43744, coagulase-negative Staphylococcus ATCC12228 (Staphylococcus epidermidis), Staphylococcus aureus ATCC25923, Acinetobacter baumannii ATCC19606 and Klebsiella pneumoniae ATCC700603.

[0074] The above bacterial solution was diluted with negative human cerebrospinal fluid clinical samples to simulate real clinical samples, and the sensitivity, precision and specificity of the test were tested. The positive coincidence rate was 100%, and the negative coincidence rate was 100%. When the precision test concentration was 1.0E+04CFU / mL, the CV value of the Ct value was ≤5.

[0075] The detection sensitivity of the present invention was determined by extracting and testing 9 kinds of bacterial liquid diluted with negative cerebrospinal fluid, as shown in Table 6 below, and the sensitivity confirmation experiment: 20 wells were repeatedly tested, and the detection rate was not less than 95%.

[0076] Table 6 Summary of detection limits of 9 pathogens

[0077]

[0078]

[0079] The embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A probe primer composition for multiple pathogenic bacteria of central nervous system infection, characterized in that: The probe primer composition comprises upstream and downstream primers and probes for detecting Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, coagulase-negative Staphylococcus, Klebsiella pneumoniae and Acinetobacter baumannii. The upstream and downstream primer sequences for detecting Neisseria meningitidis are shown in SEQ ID NO. 1 and 2, respectively, and the probe sequence is shown in SEQ ID NO. 3; The upstream and downstream primer sequences for detecting Haemophilus influenzae are shown in SEQ ID NO. 4 and 5, respectively, and the probe sequence is shown in SEQ ID NO. 6; The upstream and downstream primer sequences for detecting Streptococcus agalactiae are shown in SEQ ID NO.7 and 8 respectively, and the probe sequence is shown in SEQ ID NO.9; The upstream and downstream primer sequences for detecting Listeria monocytogenes are shown in SEQ ID NO. 10 and 11, respectively, and the probe sequence is shown in SEQ ID NO. 12; The upstream and downstream primer sequences for detecting Staphylococcus aureus are shown in SEQ ID NO. 13 and 14, respectively, and the probe sequence is shown in SEQ ID NO. 15; The upstream and downstream primer sequences for detecting Streptococcus pneumoniae are shown in SEQ ID NO. 16 and 17, respectively, and the probe sequence is shown in SEQ ID NO. 18; The upstream and downstream primer sequences for detecting coagulase-negative staphylococci are shown in SEQ ID NO. 19 and 20, respectively, and the probe sequence is shown in SEQ ID NO. 21; The upstream and downstream primer sequences for detecting Klebsiella pneumoniae are shown in SEQ ID NO. 22 and 23, respectively, and the probe sequence is shown in SEQ ID NO. 24; The upstream and downstream primer sequences for detecting Acinetobacter baumannii are shown in SEQ ID NO. 25 and 26, respectively, and the probe sequence is shown in SEQ ID NO.

27.

2. The probe primer composition for multiple pathogenic bacteria of central nervous system infection according to claim 1, characterized in that: It also includes internal reference universal upstream and downstream primers and probes. The sequences of the internal reference universal upstream and downstream primers are shown in SEQ ID NOs. 28 and 29, respectively, and the sequence of the internal reference universal probe is shown in SEQ ID NO.

30.

3. A multiplex fluorescent PCR kit for multiple pathogens of central nervous system infection, characterized in that: It comprises a PCR reaction solution, a detection solution, a positive quality control product and a negative quality control product, wherein the detection solution contains the probe primer combination according to claim 1 or 2.

4. The multiplex fluorescent PCR kit for multiple pathogens of central nervous system infection according to claim 3, characterized in that: in, The PCR reaction solution includes PCR Buffer, dNTP, dUTP, Mg 2+ , UDG enzyme and Taq hot start enzyme; There are three test solutions in total. Test solution 1 contains primer probes for detecting Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae and an internal reference; test solution 2 contains primer probes for detecting Listeria monocytogenes, Streptococcus pneumoniae, Staphylococcus aureus and an internal reference; test solution 3 contains primer probes for detecting Acinetobacter baumannii, coagulase-negative Staphylococcus, Klebsiella pneumoniae and an internal reference. The negative quality control product is normal saline, and the positive quality control product is a recombinant plasmid containing 9 pathogen detection target sequences.

5. The multiplex fluorescent PCR kit for multiple pathogens of central nervous system infection according to claim 4, characterized in that: in, In each detection solution, the four probes are labeled with different fluorescent groups.

6. The multiplex fluorescent PCR kit for multiple pathogens of central nervous system infection according to claim 3, characterized in that: in, The PCR reaction program was set up as follows:

7. The multiplex fluorescent PCR kit for multiple pathogens of central nervous system infection according to claim 3, characterized in that: The threshold of Ct value for Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, coagulase-negative Staphylococcus, Klebsiella pneumoniae and Acinetobacter baumannii was 38; the test was considered positive when Ct≤38 and there was an obvious amplification curve. The threshold of the Ct value of the internal reference is 35, and the test result is judged to be valid when the Ct value is ≤35.

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