Composition, kit, method and application for rapidly detecting meningitis pathogens

Abstract

The invention relates to the field of molecular biological detection, in particular to joint detection of meningitis pathogens, and more particularly relates to detection of streptococcus viridis, group B streptococcus, neisseria meningitidis, Elizabeth anopheles and Listeria monocytogenes. A real-time fluorescent quantitative PCR technology is used, TaqMan fluorescent probes specifically aiming at streptococcus viridis, group B streptococcus, neisseria meningitidis, Elizabeth anopheles and Listeria monocytogenes are used as a basis, and a fluorescent molecule is generated every time a DNA chain is amplified, so that accumulation of a fluorescent signal is completely synchronized with formation of a PCR product; therefore, the specific recognition of streptococcus viridis, group B streptococcus, neisseria meningitidis, Elizabeth anopheles and Listeria monocytogenes is realized. The method not only can improve the early diagnosis rate of streptococcus viridis, group B streptococcus, neisseria meningitidis, Elizabeth anopheles and Listeria monocytogenes, but also provides certain guidance for accurate medication.

Description

Technical Field

[0001] The present invention relates to the field of molecular biological detection, in particular, to the joint detection of meningitis pathogens, and more particularly, to the detection of viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria monocytogenes. Background Art

[0002] Meningitis is a type of disease that causes diffuse inflammatory changes in the meninges. The meninges are the lining covering the brain. Meningitis occurs when other parts of the body are infected. Infectious microorganisms can move through the blood to cover the brain, and meningitis can occur. Viridans Streptococcus, Group B Streptococcus, Neisseria meningitidis, Anopheles Elizabethella and Listeria are pathogens that cause meningitis.

[0003] Viridans streptococci (SVG) are a group of heterogeneous pathogens. They are gram-positive bacteria that are usually arranged in spheres or chains. They are widely found on the mucosal surfaces of the human oral cavity, respiratory tract, and digestive tract, especially on the gums, back of the tongue, and buccal mucosa in the oral cavity. They can help us digest food, resist the invasion of foreign pathogens, and maintain the balance of the oral microecology with other microorganisms. At the same time, they can also cause oral diseases such as caries and myringitis. In the case of low immunity, they may also cause serious infections such as endocarditis and meningitis. Viridans streptococci can also be isolated from secretion specimens of sinusitis, brain abscess, liver abscess, lung abscess, etc. They are often part of mixed flora and are often accompanied by mixed infections with anaerobic bacteria.

[0004] Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a Gram-positive coccus that can colonize in healthy humans. It is a conditionally pathogenic bacterium, with a gastrointestinal and / or urogenital tract carriage rate of about 30% in healthy adults and a vaginal carriage rate of 25% to 40% in pregnant women. 10% of newborns can acquire GBS through vertical transmission through the birth canal, of which about 50% to 70% are colonized, and 1% to 2% develop invasive GBS infection, leading to sepsis and / or bacterial meningitis. GBS is one of the common pathogens of bacterial meningitis in children abroad, with a high incidence of neurological sequelae and a poor prognosis.

[0005] Neisseria meningitidis (NM), also known as meningococcus or meningococcus, is a Gram-negative bacterium and the causative agent of epidemic cerebrospinal meningitis (abbreviated as meningococcus). N. meningitidis is best known for the meningitis it causes, and can also cause meningococcemia (a fatal sepsis). It only infects humans and has no parasitic animals. It is the only bacteria that has caused bacterial infection meningitis to become an epidemic. About 10% of adults have traces of it in their nasopharynx.

[0006] Elizabethkingia anophelis is an aerobic, non-motile, oxidase-positive, indole-positive, non-fermenting Gram-negative bacillus that is widely distributed in various natural environments, including water, soil, sediment, plants, and the digestive tract of animals. Elizabethkingia anophelis is a relatively newly discovered pathogen, and neonates, postoperative patients, or elderly people with underlying diseases are susceptible to it. Conventional identification methods or biochemical detection methods often fail to distinguish them from other bacteria, and Elizabethkingia anophelis is often mistakenly identified as Elizabethkingia meningoseptica in clinical practice.

[0007] Listeria monocytogenes (LM) is a facultative intracellular Gram-positive bacterium, a psychrophilic bacterium, and a zoonotic pathogen with a high mortality rate. LM mainly causes meningitis and sepsis in adults. LM can cause early-onset and late-onset neonatal diseases. The early-onset disease is an intrauterine infection that often leads to fetal sepsis, while the late-onset disease causes meningitis, meningoencephalitis, and sepsis 2-3 days after birth.

[0008] In terms of pathogenic bacteria identification, multi-gene sequence analysis is currently used in fungal classification, molecular typing and identification. The β-tubulin protein gene sequence combined with ITS (Internal Transcribed Spacer) intergenic region sequence analysis can assist in the identification of bacterial species. The qPCR (Real-time Quantitative PCR) real-time fluorescence quantitative nucleic acid amplification detection system can quickly and accurately detect pathogenic fungi in formalin-fixed paraffin-embedded tissues and identify them to the species level by analyzing the ITS2 intergenic region of ribosomal DNA. Bacterial culture takes a long time, making early diagnosis difficult.

[0009] Therefore, there is a need in the art for a product that can simply and quickly detect the above pathogens so as to provide targeted treatment strategies with high sensitivity and good specificity. Summary of the invention

[0010] In view of this, the present invention, in a first aspect, provides a composition for rapid detection of meningitis pathogens, comprising primer pairs and probes for detecting at least three pathogens of viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria monocytogenes:

[0011] Primer pairs and probes for detecting viridans streptococci as shown in SEQ ID NOs: 1 to 3;

[0012] Primer pairs and probes for detecting Group B Streptococcus as shown in SEQ ID NOs: 4 to 6;

[0013] Primer pairs and probes for detecting Neisseria meningitidis as shown in SEQ ID NOs: 7 to 9;

[0014] The primer pair and probe for detecting Anopheles elizabethii as shown in SEQ ID NOs: 10 to 12; and

[0015] The primer pair and probe for detecting Listeria are shown in SEQ ID NOs: 13-15.

[0016] The present invention uses real-time fluorescence quantitative PCR technology, based on TaqMan fluorescent probes specific for viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria, and generates a fluorescent molecule for each amplified DNA chain, so that the accumulation of fluorescent signals is completely synchronized with the formation of PCR products, thereby achieving specific identification of viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria. This can not only improve the early diagnosis rate of viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria, but also provide certain guidance for accurate medication.

[0017] Fluorescence quantitative PCR (polymerase chain reaction), in this reaction system, contains a pair of specific primers and a TaqMan probe, which is a specific oligonucleotide sequence, with a reporter fluorescent group and a quencher fluorescent group labeled at both ends. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; if there is a target sequence in the reaction system, the probe binds to the template during the PCR reaction, and the DNA polymerase uses the exonuclease activity of the enzyme to enzymatically degrade the probe along the template, and the reporter group is separated from the quencher group, emitting fluorescence. The fluorescence quantitative PCR instrument can monitor the number of cycles (Ct value) at which the fluorescence reaches the pre-set threshold value, which is related to the concentration of pathogen nucleic acid. The higher the concentration of pathogen nucleic acid, the smaller the Ct value. In the end, the infection can be judged based on the Ct value.

[0018] Furthermore, the composition also includes a primer pair and a probe for the housekeeping gene RnaseP as shown in SEQ ID NOs: 16-18.

[0019] In some embodiments, the composition of the present invention may include one or more sets of the above primer pairs and probes. In this article, "set" refers to upstream primers, downstream primers and probes that match each other to detect a target.

[0020] Furthermore, the composition of the present invention can be arbitrarily combined into any combination form for detecting corresponding targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe sets corresponding to the targets. These combinations are all included in the present invention.

[0021] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0022] In some specific embodiments, the probes in the compositions of the present invention are labeled with a fluorescent reporter group at the 5' end.

[0023] Furthermore, the fluorescent reporter group of the probe for detecting viridans streptococci as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting group B streptococci as shown in SEQ ID NO:6 is HEX or VIC; the fluorescent reporter group of the probe for detecting Neisseria meningitidis as shown in SEQ ID NO:9 is ROX.

[0024] Furthermore, the fluorescent reporter group of the probe for detecting Anopheles elizabethii as shown in SEQ ID NO:12 is CY5; the fluorescent reporter group of the probe for detecting Listeria as shown in SEQ ID NO:15 is QUASAR 705; and the fluorescent reporter group of the probe for detecting the housekeeping gene RnaseP as shown in SEQ ID NO:18 is ATTO 425.

[0025] In some specific embodiments, the 3' end of the probe further has a fluorescence quenching group, such as MGB, BHQ1 or BHQ2.

[0026] Furthermore, the components of the composition exist in a mixed form.

[0027] In a second aspect, there is provided a use of the composition of the present invention in preparing a kit for detecting meningitis pathogens, wherein the meningitis pathogens are viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria monocytogenes.

[0028] In a third aspect, a kit for rapid detection of meningitis pathogens is provided, the kit comprising the composition of the present invention.

[0029] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0030] Furthermore, the kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, a UNG enzyme, a DNA polymerase, dNTPs (U) and Mg 2+ At least one of .

[0031] In some specific embodiments, the DNA polymerase is Taq enzyme.

[0032] In a specific embodiment, the kit of the present invention comprises PCR buffer, UNG enzyme, Taq enzyme, dNTPs (U), primers, probe and Mg 2+ .

[0033] Common PCR buffers are composed of Tris-HCl, KCl, MgCl 2 Equal buffer system composition.

[0034] In a fourth aspect, a method for rapid detection of meningitis pathogens for non-diagnostic purposes is provided, the method comprising the following steps:

[0035] (1) extracting or releasing nucleic acid from the sample to be tested;

[0036] (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition of the present invention or the kit of the present invention;

[0037] (3) Obtain and analyze the results.

[0038] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0039] UNG enzyme reaction, temperature is 50℃, time is 2min, 1 cycle; Taq enzyme activation, temperature is 94℃, time is 2min, 1 cycle; denaturation, temperature is 94℃, time is 15s, annealing, extension and fluorescence collection, temperature is 60℃, time is 20s, 45 cycles.

[0040] The above scheme of the present invention has the following beneficial effects:

[0041] The present invention uses real-time fluorescence quantitative PCR technology, based on TaqMan fluorescent probes specific for viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria, and generates a fluorescent molecule for each amplified DNA chain, so that the accumulation of fluorescent signals is completely synchronized with the formation of PCR products, thereby achieving specific identification of viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria. This can not only improve the early diagnosis rate of viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria, but also provide certain guidance for accurate medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figures 1 to 6It is an amplification curve diagram of the embodiment of the present invention for sequentially detecting viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii, Listeria monocytogenes, and a five-combination test;

[0044] Figures 7 to 11 It is an amplification curve diagram of detecting viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria at different concentrations in the embodiment of the present invention;

[0045] Fig.12 is a graph showing the specificity detection results of the composition of an embodiment of the present invention;

[0046] Figures 13 to 17 is an amplification curve diagram of detecting viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria monocytogenes in the presence of interfering substances in an embodiment of the present invention;

[0047] Fig.18 It is an amplification curve diagram of the comparative example composition of the present invention for joint detection of viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria monocytogenes. DETAILED DESCRIPTION

[0048] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0049] The disclosed embodiments are described to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

[0050] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0051] In view of the existing problems, the present invention provides a composition, a kit, a method and a use for rapidly detecting meningitis pathogens.

[0052] Example 1: Primer pairs and probes used in the present invention

[0053] The primer pairs and probes used in the present invention are shown in Table 1 below.

[0054] Table 1

[0055]

[0056]

[0057] Among them, the fluorescent reporter group of the probe for detecting viridans streptococci as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting group B streptococci as shown in SEQ ID NO:6 is HEX or VIC; the fluorescent reporter group of the probe for detecting Neisseria meningitidis as shown in SEQ ID NO:9 is ROX. The fluorescent reporter group of the probe for detecting Anopheles elizabethii as shown in SEQ ID NO:12 is CY5; the fluorescent reporter group of the probe for detecting Listeria as shown in SEQ ID NO:15 is QUASAR 705; the fluorescent reporter group of the probe for detecting the housekeeping gene RnaseP as shown in SEQ ID NO:18 is ATTO 425.

[0058] Example 2: Method for detecting pathogens

[0059] (1) Reagent preparation

[0060] (1.1) Take out each component from the box, place it at room temperature, wait for the temperature to equilibrate to room temperature, mix well, centrifuge briefly, and set aside.

[0061] (1.2) According to the number of samples to be tested, negative controls, and positive controls, take the corresponding amount of components according to the ratio (PCR reaction solution 44 μL / person + enzyme mixture 1 μL / person), mix them thoroughly to form a PCR mixture, centrifuge it briefly, and set aside.

[0062] (1.3) Transfer the prepared reagents to the sample processing area for later use.

[0063] (2) Sample processing and sample addition

[0064] The sample tested in the present invention is sputum. The viral nucleic acid is extracted by magnetic bead method, and the following operations are performed in the sample processing room:

[0065] (2.1) Take a 1.5mL cryotube and add glass beads. Add 300uL of the treated sample and 300uLTE to the 1.5mL cryotube containing the mixed glass beads and grind them using a grinder. After the grinding is completed, take out the cryotube and centrifuge it instantly. Take out the supernatant liquid as the sample to be tested for later use.

[0066] (2.2) Take a number of 1.5 mL centrifuge tubes according to the number of samples to be tested, and add 300 μL of the sample to be tested to each tube;

[0067] (2.3) Add 500 μL of extraction solution 1 and 50 μL of proteinase K-magnetic bead mixture; cover the tube, shake and mix for 30 seconds, and heat at 60°C for 10 minutes.

[0068] (2.4) Let stand at room temperature for 1 min, centrifuge at low speed, place the centrifuge tube on a magnetic separator, and slowly discard the waste liquid after 5 min (*Be careful not to touch the magnetic beads adsorbed on the inner side of the tube wall);

[0069] (2.5) Add 200 μL of washing solution 1 and 600 μL of washing solution 2, shake and mix for 30 seconds, centrifuge at low speed, and place the centrifuge tube in a magnetic separator. Magnetic suction for 3 minutes, completely aspirate and discard the liquid.

[0070] (2.6) Place the centrifuge tube in a centrifuge and centrifuge it at a low speed for a short time. Place the centrifuge tube in a magnetic separator again and magnetically absorb it for 3 minutes to completely remove the liquid at the bottom of the tube.

[0071] (2.7) Add 30-100 μL of elution buffer S (80 μL is recommended), shake and mix for 30 seconds to elute the magnetic beads on the wall of the centrifuge tube to the bottom of the tube, and let it stand at room temperature for 3 minutes; centrifuge at a low speed, place the centrifuge tube on the magnetic separator again for 3 minutes, and then transfer the eluted nucleic acid to a clean 1.5 mL centrifuge tube.

[0072] (2.9) Pipette 5 μL of the above-treated samples, positive controls and negative controls into the corresponding 0.2 mL PCR reaction tubes, add 45 μL of PCR mixture to each tube, and cover the tubes.

[0073] The real-time fluorescence PCR reaction system is configured according to Table 2 below:

[0074] Table 2

[0075]

[0076]

[0077] (3) PCR amplification was performed on PCR instruments such as ABI7500 fluorescence quantitative PCR instrument, Life Technologies QuantStudioTM5 fluorescence PCR instrument, SLAN-96P fully automatic medical PCR analysis system according to certain temperature and time settings. The amplification reaction program is shown in Table 3.

[0078] Table 3

[0079]

[0080] (*Note: Due to the ABI7500 instrument, the shortest annealing, extension, and fluorescence acquisition time can be set to 31 seconds.)

[0081] (4) Explanation of the test results is shown in Table 4 below.

[0082] If the sample FAM, HEX (VIC), ROX, CY5, QUASAR 705, ATTO 425 channels have an obvious S-shaped amplification curve and the Ct value is ≤40, it is judged as positive; if the sample FAM, HEX (VIC), ROX, CY5, QUASAR 705, ATTO 425 channels have no amplification curve (NoCt) or the Ct value is greater than 40, it is judged as negative.

[0083] 1. For FAM, HEX (VIC), ROX, CY5, QUASAR 705, if a typical S-shaped amplification curve is detected and Ct≤40, the sample is reported as positive; for FAM, HEX (VIC), ROX, CY5, QUASAR 705 channels, if a typical S-shaped amplification curve is not detected or Ct>40, the sample is reported as negative.

[0084] 2. For ATTO 425 channels, no typical S-type amplification curve value is detected, or Ct>40, which means that the cell content of the sample tested is too low or there are interfering substances that inhibit the reaction. The test result of the sample is invalid. The cause should be found and eliminated, and the sample should be resampled and repeated.

[0085] Note: For pathogen cultures, internal standard test results are not required.

[0086] Table 4

[0087]

[0088]

[0089] Example 3: Test results of the test samples of the composition of the present invention

[0090] The primer pairs and probes shown in Example 1 were used in turn according to the method of Example 2 to perform PCR detection on samples of viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii, Listeria monocytogenes and the above five pathogens on a Macrostone fluorescent quantitative PCR instrument. The results are shown in FIG. Figures 1 to 6 The composition of the present invention can perform joint detection and differentiation of the above pathogens.

[0091] Example 4: Sensitivity of the composition of the present invention

[0092] The primer pairs and probes shown in Example 1 were verified according to the method of Example 2 on samples of viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii, and Listeria monocytogenes. The gradient dilution plasmid was taken, and the concentrations were 5000000, 500000, 50000, 5000, 5000, and 500 copies / mL, respectively. Five concentrations were tested, and 5uL was taken as a template. The test results are shown in FIG. Figures 7 to 11 , which shows that the method of the present invention has high sensitivity and the detection concentration can reach 500 copies / mL.

[0093] Example 5. Specificity of the composition of the present invention

[0094] Using the fluorescent PCR reaction system in Example 2 of the present invention, according to the detection method in Example 2, specificity tests were performed in reaction systems containing other relevant pathogens such as Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, Bordetella pertussis, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Mycobacterium tuberculosis, nontuberculous mycobacteria, Acinetobacter baumannii, Acinetobacter junii, Escherichia coli, Rhodococcus equi, Candida albicans, Candida tropicalis, Candida krusei, influenza A virus, influenza B virus, respiratory syncytial virus, etc. The test results are as follows: Fig.12 As shown, each target channel has no non-specific amplification and no cross reaction, which shows that the composition provided by the present invention has good specificity.

[0095] Example 6: Anti-interference performance of the composition of the present invention

[0096] Using the fluorescent PCR reaction system in Example 2 of the present invention, according to the detection method in Example 2, an anti-interference test was performed in a reaction system containing interfering substances such as 10 μg / mL mucin, 20 μg / mL heme, 0.5 mg / mL meropenem, 20 μg / mL trimethoprim, 0.44 mg / mL sulfamethoxazole, 0.8 mg / mL amphotericin B, 0.1 g / mL itraconazole, 0.5 mg / mL fluconazole, 0.1 g / ml azithromycin, 1 mg / ml epinephrine, and 4 mg / mL lidocaine hydrochloride, and the test results are as follows: Figures 13 to 17 The above interfering substances have no obvious effect on the composition provided in Example 1, and the repeatability is good. This shows that the composition provided by the present invention has a strong anti-interference ability.

[0097] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0098] Based on the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be ruled out at the beginning of the design. However, when multiple pathogens are detected together, there are many primers and probes, and dimers are likely to occur between primers and primers, probes and probes, or primers and probes. To ensure the conservatism of the design (conservatism is crucial to the accuracy of the test), and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed and verified.

[0099] The inventors also designed other primer pairs and probes (sequences not shown) to form different detection systems for detecting viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii and Listeria monocytogenes. Fig.18 As shown, the results show that only part of the amplification curve appears in the detection, and the amplification curve has a low increase and poor repeatability, and missed detection occurs, and the overall detection effect is poor. This fully demonstrates the superiority of the composition of the present invention, and the mutual interference between different primer pairs and probes is small. In other words, the combination of primers and probes in the present invention is irreplaceable and irreplaceable.

[0100] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition for rapid detection of meningitis pathogens, characterized in that: Includes primer pairs and probes for detection of at least 3 pathogens among viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii, and Listeria monocytogenes: Primer pairs and probes for detecting viridans streptococci as shown in SEQ ID NOs: 1 to 3; Primer pairs and probes for detecting Group B Streptococcus as shown in SEQ ID NOs: 4 to 6; Primer pairs and probes for detecting Neisseria meningitidis as shown in SEQ ID NOs: 7 to 9; The primer pair and probe for detecting Anopheles elizabethii as shown in SEQ ID NOs: 10 to 12; and The primer pair and probe for detecting Listeria are shown in SEQ ID NOs: 13-15.

2. The composition according to claim 1, characterized in that The fluorescent reporter group of the probe for detecting viridans streptococci as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting group B streptococci as shown in SEQ ID NO:6 is HEX or VIC; the fluorescent reporter group of the probe for detecting Neisseria meningitidis as shown in SEQ ID NO:9 is ROX.

3. The composition according to any one of claims 1, characterized in that The fluorescent reporter group of the probe for detecting Anopheles elizabethii as shown in SEQ ID NO:12 is CY5; the fluorescent reporter group of the probe for detecting Listeria as shown in SEQ ID NO:15 is QUASAR 705.

4. The composition according to any one of claims 1 to 3, characterized in that The composition also includes a primer pair and a probe for the housekeeping gene RnaseP as shown in SEQ ID NOs: 16-18.

5. The composition according to claim 4, characterized in that The fluorescent reporter group of the probe for detecting the housekeeping gene RnaseP as shown in SEQ ID NO:18 is ATTO 425.

6. Use of the composition according to any one of claims 1 to 5 in preparing a kit for detecting meningitis pathogens, wherein: The pathogens of meningitis are viridans streptococci, group B streptococci, Neisseria meningitidis, Anopheles elizabethii, and Listeria monocytogenes.

7. A kit for rapid detection of meningitis pathogens, characterized in that: The kit comprises the composition according to any one of claims 1 to 5.

8. The kit according to claim 7, characterized in that The kit also includes a negative quality control product and a positive quality control product.

9. The kit according to claim 7 or 8, characterized in that The kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, a UNG enzyme, a DNA polymerase, dNTPs (U) and Mg 2+ At least one of .

10. A method for rapid detection of meningitis pathogens for non-diagnostic purposes, characterized in that: The method comprises the following steps: (1) extracting or releasing nucleic acid from the sample to be tested; (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition according to any one of claims 1 to 5 or the kit according to any one of claims 7 to 9; (3) Obtain and analyze the results.

Images