Multi-droplet type digital PCR (polymerase chain reaction) detection kit for central nervous system infection pathogens and application of multi-droplet type digital PCR detection kit

Abstract

The invention belongs to the technical field of medical biological detection, and particularly relates to a multiple microdroplet type digital PCR detection kit for central nervous system infection pathogens and application of the multiple microdroplet type digital PCR detection kit. The kit is based on the digital PCR technology, the detection range of the kit covers 23 kinds of clinically common pathogens of central nervous system infection of bacteria, fungi, viruses and mycoplasmas, and pathogen coverage is more complete. Compared with a real-time fluorescent PCR quantitative method, accurate absolute quantification can be realized without setting a standard curve. The kit has the advantages of wide detection range, accurate quantification, high sensitivity, simplicity and convenience in operation, controllable cost and the like, the detection rate of the central nervous system infection pathogens is greatly increased, and an accurate and rapid judgment basis is provided for early diagnosis and early treatment of central nervous system infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical biological detection, and in particular relates to a multiple droplet digital PCR detection kit for central nervous system infection pathogens and its application. Background Art

[0002] Central nervous system infections (CNSIs) can be caused by a variety of known and unknown biological pathogens, including bacteria, fungi, viruses, spirochetes, rickettsiae, parasites, and prions. They are among the most severe clinically transmitted infections, with the primary symptoms being meningitis and encephalitis. CNSI infections are primarily bacterial and viral. Bacterial central nervous system infections (BCNIs) are among the top 10 most fatal infectious diseases worldwide, with a mortality rate of 10% to 30%, exceeding 35% in some African countries. The proportion of BCNI survivors who develop permanent neurological sequelae is also as high as 30% to 50%. According to statistics, the incidence of bacterial meningitis (BM) in children under five years of age in my country ranges from 6.95 to 22.30 per 100,000 children. 30% to 50% of children with BM develop neurological complications. These complications include acute complications during BM diagnosis and treatment, such as subdural effusion, brain abscess, and cranial nerve palsy, as well as long-term complications (or sequelae) that persist or develop during recovery, such as hearing loss, epilepsy, cognitive impairment, and hydrocephalus. Furthermore, viral meningitis is a common clinical infectious disease of the central nervous system. According to WHO statistics, the global annual incidence of viral CNS infections is approximately 3.5 to 7.4 per 100,000 children. Based on this, it is estimated that the annual number of cases of viral meningitis in my country is between 50,000 and 100,000.

[0003] Currently, cerebrospinal fluid (CSF) smears and bacterial cultures are still the most commonly used methods for diagnosing CNSI in clinical laboratories. However, obtaining CSF requires invasive procedures such as lumbar puncture and EVD. Furthermore, CSF smears have a low positive rate, while bacterial cultures have disadvantages such as being time-consuming, having a low positive rate, and being unable to confirm sterile CNSI. These disadvantages cannot meet the clinical need for rapid and accurate diagnosis of CNSI pathogens.

[0004] Molecular diagnostic technology has freed CNSI diagnosis from traditional testing methods, but each molecular diagnostic technique has its limitations. Real-time fluorescence quantitative PCR can rapidly and qualitatively detect one or several known pathogens in a short period of time, but the number of pathogens that can be detected at one time is relatively small. Pathogen detection based on next-generation high-throughput sequencing technology can significantly improve pathogen screening and detection capabilities, but it requires specialized testing platforms and personnel and is relatively expensive. Therefore, the development of a low-cost, simple-to-use, broad-coverage, highly sensitive, and specific multiplexed genetic testing kit for the identification and quantitative analysis of pathogens that cause central nervous system infections has important clinical implications.

[0005] Digital PCR (digital PCR), a newly emerging technology, does not rely on standard curve quantification and is unaffected by PCR amplification efficiency, offering higher sensitivity and accuracy. The principle of digital PCR is to distribute a standard PCR reaction across a large number of tiny reactors, enabling single-molecule template PCR amplification and detection. Finally, based on the principles of Poisson distribution, the absolute copy number of the target nucleic acid molecule is calculated from the proportion of positive droplets. Therefore, digital PCR is particularly suitable for the detection of trace nucleic acids, offering wide coverage, high sensitivity, simple operation, short detection times, and a lower price than next-generation high-throughput sequencing technologies.

[0006] Currently, the reagents disclosed in Chinese patent literature for nucleic acid detection of meningitis mainly include:

[0007] Chinese patent document CN113151610B discloses a central nervous system infection pathogen detection kit and its application, including 18 pathogens including human enterovirus, Haemophilus influenzae, Epstein-Barr virus, Streptococcus pneumoniae, human cytomegalovirus, Streptococcus agalactiae, herpes simplex virus type 1, Listeria monocytogenes, herpes simplex virus type 2, Neisseria meningitidis, varicella-zoster virus, double echovirus, Cryptococcus gattii, mumps virus, Cryptococcus neoformans, human herpes virus type 6, Escherichia coli K1 type and Staphylococcus aureus. The detection method adopted by this kit is the melting curve method, which can only perform qualitative detection. In addition, the nucleic acid needs to be reverse transcribed and amplified first, and then a secondary amplification is performed to detect fluorescence. The experimental steps are many and time-consuming, which is not conducive to actual clinical application.

[0008] Chinese patent document CN 118028532A discloses a digital PCR detection system for pathogens that cause encephalitis and meningitis, including cytomegalovirus, Epstein-Barr virus, herpes simplex virus type 1 and type 2, human herpes virus type 6, varicella virus, enterovirus, Japanese encephalitis virus, and mumps virus. The test kit uses digital PCR, but it requires pre-amplification of nucleic acids before digital PCR detection. This involves multiple experimental steps and makes it impossible to accurately quantify the original sample after pre-amplification. Furthermore, the kit covers a limited range of pathogens (all viruses) and lacks detection of other pathogens related to central nervous system infections, such as bacteria, fungi, and mycoplasmas.

[0009] Chinese patent document CN103074448B discloses a kit and a detection method for the simultaneous detection of 23 encephalitis and meningitis pathogens. The kit can detect Japanese encephalitis virus, Powassen virus, West Nile virus, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, African lymphoblastic leukemia virus, human cytomegalovirus, coxsackievirus A16, enterovirus 71, mumps virus, Nipah virus, Neisseria meningitidis, Haemophilus influenzae type b, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus suis, Escherichia coli, Cryptococcus, Toxoplasma gondii, Cysticercus cervix, Plasmodium falciparum, and Mycoplasma pneumoniae. The kit covers a wide range of pathogens, but uses capillary electrophoresis, which can only perform qualitative detection. The operation is complicated, the detection sensitivity is low, and expensive equipment is required.

[0010] In clinical practice, early diagnosis and early treatment are key measures for the prevention and treatment of encephalitis and meningitis. According to the expert consensus on the application of diagnostic technology for viral encephalitis pathogens released in 2023, the total amount of pathogens that cause encephalitis is large, and most patients are initially diagnosed in county / township hospitals. However, grassroots hospitals lack the software and hardware conditions to identify pathogens in a timely and accurate manner. Therefore, strengthening the research and development of encephalitis pathogen detection kits with wide pathogen coverage, high sensitivity, accurate quantification, and suitable for grassroots hospitals is of great significance for accurate clinical diagnosis. Summary of the Invention

[0011] The purpose of the present invention is to address the existing problems and provide a multiplex droplet digital PCR detection kit for central nervous system infection pathogens and its application.

[0012] The present invention is achieved through the following technical solutions:

[0013] A multiplex droplet digital PCR detection kit for central nervous system infection pathogens, comprising:

[0014] Target fragment detection mixture A, including target fragment amplification primers and probes, the sequences of the target fragment amplification primers are shown in SEQ ID No. 1 to SEQ ID No. 10, and the sequences of the target fragment probes are shown in SEQ ID No. 51 to SEQ ID No. 55;

[0015] Target fragment detection mixture B, including target fragment amplification primers and probes, the sequences of the target fragment amplification primers are shown in SEQ ID No. 11 to SEQ ID No. 18, and the sequences of the target fragment probes are shown in SEQ ID No. 56 to SEQ ID No. 59;

[0016] Target fragment detection mixture C, including target fragment amplification primers and probes, the sequences of the target fragment amplification primers are shown as SEQ ID No. 19 to SEQ ID No. 26, and the sequences of the target fragment probes are shown as SEQ ID No. 60 to SEQ ID No. 63;

[0017] A target fragment detection mixture D includes target fragment amplification primers and probes, wherein the sequences of the target fragment amplification primers are shown in SEQ ID No. 27 to SEQ ID No. 34, and the sequences of the target fragment probes are shown in SEQ ID No. 64 to SEQ ID No. 67;

[0018] A target fragment detection mixture E comprises target fragment amplification primers and probes, wherein the sequences of the target fragment amplification primers are shown in SEQ ID No. 35 to SEQ ID No. 42, and the sequences of the target fragment probes are shown in SEQ ID No. 68 to SEQ ID No. 71;

[0019] A target fragment detection mixture F comprises target fragment amplification primers and probes, wherein the sequences of the target fragment amplification primers are shown in SEQ ID No. 43 to SEQ ID No. 50, and the sequences of the target fragment probes are shown in SEQ ID No. 72 to SEQ ID No. 75;

[0020] Positive quality control products A to F, including the pathogen plasmid containing the target fragment as a positive quality control product and 1×TE (pH 8.0) solution for dilution;

[0021] Negative control, including 1×TE (PH 8.0) solution as a negative control.

[0022] Preferably, the target fragment is a DNA or RNA fragment of Neisseria meningitidis (Nm), Mycobacterium tuberculosis (MTB), Listeria monocytogenes (LM), Streptococcus pneumoniae (SP), Streptococcus agalactiae (GBS), Haemophilus influenzae (Hi), Escherichia coli universal type (E.Coli) and Escherichia coli K1 type (E.ColiK1), Acinetobacter baumannii (Ab), Klebsiella pneumoniae (KPn), Staphylococcus aureus (SA), enterovirus (Evs), human double echovirus (HPeV), Japanese encephalitis virus (JEV), mumps virus (MuV), cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV6), Mycoplasma pneumoniae (MP), Cryptococcus neoformans (CN), Candida albicans (CA) and human internal standard (RPP30).

[0023] Preferably, the target fragment detection mixed solution A contains primer probes for detecting the target fragment of Escherichia coli (E.Coli), primer probes for detecting the target fragment of Escherichia coli K1 type (E.ColiK1), primer probes for detecting the target fragment of Listeria monocytogenes (LM), primer probes for detecting the target fragment of Cryptococcus neoformans (CN), and primer probes for detecting the target fragment of Streptococcus agalactiae (GBS);

[0024] The target fragment detection mixture B contains a primer probe for detecting the target fragment of Haemophilus influenzae (Hi), a primer probe for detecting the target fragment of Streptococcus pneumoniae (SP), a primer probe for detecting the target fragment of Neisseria meningitidis (Nm), and a primer probe for detecting the target fragment of human internal control (RPP30);

[0025] The target fragment detection mixed solution C contains a primer probe for detecting the target fragment of Staphylococcus aureus (SA), a primer probe for detecting the target fragment of Acinetobacter baumannii (Ab), a primer probe for detecting the target fragment of Klebsiella pneumoniae (KPn), and a primer probe for detecting the target fragment of Mycobacterium tuberculosis (MTB);

[0026] The target fragment detection mixed solution D contains a primer probe for detecting the target fragment of mumps virus (MuV), a primer probe for detecting the target fragment of human double echovirus (HPeV), a primer probe for detecting the target fragment of Japanese encephalitis virus (JEV), and a primer probe for detecting the target fragment of enterovirus (Evs);

[0027] The target fragment detection mixture E contains a primer probe for detecting the target fragment of Epstein-Barr virus (EBV), a primer probe for detecting the target fragment of herpes simplex virus type 1 (HSV1), a primer probe for detecting the target fragment of cytomegalovirus (HCMV), and a primer probe for detecting the target fragment of varicella-zoster virus (VZV);

[0028] The target fragment detection mixed solution F contains primer probes for detecting the target fragment of Candida albicans (CA), primer probes for detecting the target fragment of herpes simplex virus type 2 (HSV2), primer probes for detecting the target fragment of Mycoplasma pneumoniae (MP) and primer probes for detecting the target fragment of human herpes virus type 6 (HHV6).

[0029] Preferably, the primers in the target fragment detection mixed solution D are reverse transcription specific primers and specific amplification primers.

[0030] Preferably, the amplification reaction of the detection well containing the target fragment detection mixture is: 42°C reaction for 30 minutes, 1 cycle, 95°C reaction for 10 minutes, 1 cycle, 95°C reaction for 30 seconds, 60°C reaction for 60 seconds, 45 cycles, 98°C reaction for 10 minutes, 1 cycle. The amplification reaction is a combination of reverse transcription reaction and PCR amplification reaction. There is no need to open the cover to transfer the product, which reduces amplicon contamination. The MicroDrop-400B biochip analyzer (four channels) developed and produced by Guangdong Yongnuo Medical Technology Co., Ltd. is used for detection, and the four-channel positive and negative grouping is obvious.

[0031] Preferably, the kit is provided with 6 reaction well systems for each sample for detection, and one test can quantitatively detect Neisseria meningitidis (Nm), Mycobacterium tuberculosis (MTB), Listeria monocytogenes (LM), Streptococcus pneumoniae (SP), Streptococcus agalactiae (GBS), Haemophilus influenzae (Hi), Escherichia coli universal type (E.Coli) and Escherichia coli K1 type (E.ColiK1), Acinetobacter baumannii (Ab), Klebsiella pneumoniae (KPn), Staphylococcus aureus (SA), Enteroviruses (EVs), human echoviruses (HPeV), Japanese encephalitis virus (JEV), mumps virus (MuV), cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV6), Mycoplasma pneumoniae (MP), Cryptococcus neoformans (CN) and Candida albicans (CA), a total of 23 common pathogens of central nervous system infection.

[0032] Preferably, the reaction well system needs to be equipped with both positive quality control products and negative quality control products; the positive quality control products are derived from the positive quality control products A to F in the kit; the negative quality control products are derived from the negative quality control products in the kit.

[0033] Preferably, the B reaction well system of the kit comprises human internal control RPP30, which can control the sampling quality and nucleic acid extraction quality of the sample.

[0034] Preferably, the detection kit is characterized in that the kit can use the target fragment detection mixture A to detect one or more of Escherichia coli (E.Coli), Escherichia coli K1 type (E.ColiK1), Listeria monocytogenes (LM), Cryptococcus neoformans (CN), and Streptococcus agalactiae (GBS), for the identification and quantitative analysis of pathogens of central nervous system infection;

[0035] The kit can use the target fragment detection mixture B to detect one or more of Haemophilus influenzae (Hi), Streptococcus pneumoniae (SP), and Neisseria meningitidis (Nm), for the identification and quantitative analysis of pathogens of central nervous system infection;

[0036] The kit can use the target fragment detection mixture C to detect one or more of Staphylococcus aureus (SA), Acinetobacter baumannii (Ab), Klebsiella pneumoniae (KPn), and Mycobacterium tuberculosis (MTB), and is used for the identification and quantitative analysis of pathogens of central nervous system infection;

[0037] The kit can use the target fragment detection mixture D to detect one or more of mumps virus (MuV), human echovirus (HPeV), Japanese encephalitis virus (JEV), and enterovirus (Evs), for the identification and quantitative analysis of pathogens that infect the central nervous system;

[0038] The kit can use the target fragment detection mixture E to detect one or more of Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV1), cytomegalovirus (HCMV), and varicella-zoster virus (VZV), for the identification and quantitative analysis of pathogens that infect the central nervous system;

[0039] The kit can use the target fragment detection mixture F to detect one or more of Candida albicans (CA), herpes simplex virus type 2 (HSV2), Mycoplasma pneumoniae (MP), and human herpes virus type 6 (HHV6), and is used for the identification and quantitative analysis of pathogens that infect the central nervous system.

[0040] Compared with the prior art, the present invention has the following advantages:

[0041] (1) The kit of the present invention is based on digital PCR technology. Compared with real-time fluorescence PCR quantification methods, it can achieve accurate absolute quantification without the need for a standard curve. In addition, the amplification reaction is a combined reverse transcription reaction and PCR amplification reaction, eliminating the need for an additional step for cDNA preparation, making the operation simpler.

[0042] (2) The test kit of the present invention has a detection range covering 23 common pathogens of central nervous system infection in clinical practice, including bacteria, fungi, viruses and mycoplasmas, with more comprehensive pathogen coverage.

[0043] (3) The kit of the present invention utilizes optimized designed amplification primers and probes for the target fragment, as well as internal standard amplification primers and probes, to improve detection accuracy. The minimum detection concentration of each pathogen target can reach 5 copies / 20 μL to 10 copies / 20 μL. It is also suitable for complex sample detection and is not easily affected by PCR inhibitors.

[0044] (4) The kit of the present invention uses endogenous quality control to control the quality of samples and experimental operations, thereby avoiding false positive and false negative results. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The two-dimensional image results of the central nervous system pathogen multiplex droplet digital PCR detection kit and method of use of the present invention for detecting E. coli positive, E. coli K1 positive, LM positive, CN positive, and GBS positive samples;

[0046] Figure 2 The two-dimensional image results of the central nervous system pathogen multiplex droplet digital PCR detection kit and method of use of the present invention for detecting Hi-positive, SP-positive, Nm-positive, and RPP30-positive samples;

[0047] Figure 3 The two-dimensional image results of the central nervous system pathogen multiplex droplet digital PCR detection kit and method of use of the present invention for detecting SA-positive, Ab-positive, KPn-positive, and MTB-positive samples;

[0048] Figure 4 The two-dimensional image results of the central nervous system pathogen multiplex droplet digital PCR detection kit and method of use of the present invention for detecting MuV-positive, HPeV-positive, JEV-positive, and Evs-positive samples;

[0049] Figure 5 The two-dimensional image results of the central nervous system pathogen multiplex droplet digital PCR detection kit and method of use of the present invention for detecting EBV-positive, HSV1-positive, HCMV-positive, and VZV-positive samples;

[0050] Figure 6 The two-dimensional image results of the central nervous system pathogen multiplex droplet digital PCR detection kit and method of use of the present invention for detecting CA-positive, HSV2-positive, MP-positive, and HHV6-positive samples;

[0051] Figure 7 For the single-target system of Streptococcus agalactiae (GBS), two primer-probe combinations were designed to detect the one-dimensional results of positive samples;

[0052] Figure 8 For the Listeria monocytogenes (LM) single-target system, two primer-probe combinations were designed to detect the one-dimensional results of positive samples;

[0053] Figure 9 For the single-target system of Neisseria meningitidis (Nm), two primer-probe designs were designed and combined to detect the one-dimensional results of positive samples;

[0054] Figure 10 This is a multiplex system consisting of cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV1), varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV6) viral pathogens. The two-dimensional graph results of the five combinations are used to detect negative and positive samples respectively. DETAILED DESCRIPTION

[0055] In order to further explain the present invention, it is described below with reference to the following specific embodiments.

[0056] Example 1

[0057] How to use the kit

[0058] The method for using the multiplex droplet digital PCR detection kit for central nervous system pathogens of the present invention comprises:

[0059] (1) Freshly collected clinical specimens should be transported to the laboratory at 2-8°C within 48 hours. If they cannot be transported to the laboratory within 48 hours, they should be stored at -70°C or below. After the specimens are delivered to the laboratory, they should be tested as soon as possible. If they can be tested within 48 hours, they can be stored at 2-8°C; otherwise, they should be stored at -70°C or below.

[0060] (2) Sample processing (sample processing area)

[0061] 1) Negative and positive quality control products do not require extraction.

[0062] 2) The samples to be tested can be extracted using a commercial extraction kit according to the requirements and steps in the kit instructions.

[0063] 3) Each time an extraction is performed, at least one sample of nuclease-free water should be extracted simultaneously as an extraction control (NCS) to monitor whether there are any abnormalities such as cross-contamination during the extraction process.

[0064] (3) Reaction system preparation (reagent preparation area)

[0065] a. Positive and negative quality controls were set up for the detection reaction. The positive quality controls were implemented using positive quality control products A to F and the negative quality control was implemented using a negative quality control product. The detection reaction was accompanied by a droplet digital PCR multiplex reaction premix (4X) and a digital PCR device developed and produced by Guangdong Yongnuo Medical Technology Co., Ltd.

[0066] b. Preparation process

[0067] 1) Thaw all components and return to room temperature, vortex to mix evenly, and centrifuge briefly before use;

[0068] 2) Determine the number of reactions, N, where N = 1 negative control sample + 1 positive control sample + 1 extraction control NCS + the number of samples to be tested, n + 1. Each sample requires testing using 6 wells, namely Well A, Well B, Well C, Well D, Well E, and Well F:

[0069] 3) Take the appropriate amounts of Droplet Digital PCR Multiplex Reaction Master Mix (4X), CNS Detection Mix A, CNS Detection Mix B, CNS Detection Mix C, CNS Detection Mix E, endonuclease, and nuclease-free water, mix thoroughly to form reaction systems for wells A, B, C, and E, and aliquot into reaction tubes at 10 μL / tube for later use.

[0070] 4) Prepare a D-well reaction system using the appropriate amount of Droplet Digital PCR Multiplex Premix (4X), CNS Detection Mix D, reverse transcriptase, endonuclease, and nuclease-free water. Dispense 10 μL / tube into reaction tubes for later use.

[0071] 5) Take the appropriate amounts of Droplet Digital PCR Multiplex Premix (4X), CNS Detection Mix F, endonuclease, and CNS F-well Additive, mix thoroughly to form an F-well reaction system, and aliquot into reaction tubes at 10 μL / tube for later use.

[0072] 6) This embodiment is used in conjunction with the MicroDrop-400B droplet digital PCR system developed and produced by Guangdong Yongnuo Medical Technology Co., Ltd.

[0073] (4) Sample addition (sample processing area)

[0074] 1) Take 10 μL of CNS positive quality control, CNS negative quality control, extracted sample nucleic acid and extraction control NCS, and add them to the reaction tubes containing 10 μL of the reaction system in each well.

[0075] 2) Vortex and mix the above 20 μL mixture, centrifuge for 15 seconds, and let it stand at room temperature for 5 minutes before use.

[0076] (5) Chip loading (sample processing area)

[0077] 1) Place the microdroplet generation chip in the chip holder. Add 50 μL of microdroplet generation oil to the oil phase well, 20 μL of the PCR reaction aqueous phase (pre-sampled) to the sample well, and 5 μL of sealant to the sample well. After the oil and aqueous phases have been added, cover the microdroplet generation chip with the sealing gasket and place the microdroplet generation chip in a MicroDrop-400A droplet generator for microdroplet generation.

[0078] 2) After the microdroplets are generated, carefully transfer them to a 96-well PCR reaction plate (an eight-row pipette is recommended for transfer).

[0079] 3) After the microdroplets are transferred to a 96-well PCR reaction plate, cover the plate with pierceable heat-sealing film and place it on a preheated heat sealer for sealing.

[0080] (6) PCR amplification (amplification detection area)

[0081] Perform PCR amplification in the sealed 96-well PCR reaction plate. The amplification program is shown in Table 1. After the settings are completed (Note: Set the heated lid temperature to 105°C), run the reaction program.

[0082] Table 1 PCR amplification reaction program

[0083]

[0084]

[0085] (7) Digital PCR detection and result analysis

[0086] 1) The detailed experimental steps were carried out according to the instructions of the digital PCR instrument.

[0087] 2) Click "Settings." In the "Sample Definition" section, assign an experiment, name, and sample type to each sample, including the negative and positive controls. Select "A Absolute Quantitation" for "Experiment" and "Unknown Sample" for "Type." Set channels 1, 2, 3, and 4 to FAM, VIC, CY5, and CY5.5, respectively. Click "OK" after completing the settings.

[0088] 3) Click "Run" and the instrument will automatically run and display the progress. After the run is completed, a data file will be generated.

[0089] 4) Click "Analyze" to open and analyze the data. The experimental data for the selected well (sample) will be displayed, and the concentration results for each sample target will be obtained. You can also view a two-dimensional graph of the droplet fluorescence values.

[0090] Example 2

[0091] The present invention investigates Neisseria meningitidis (Nm), Mycobacterium tuberculosis (MTB), Listeria monocytogenes (LM), Streptococcus pneumoniae (SP), Streptococcus agalactiae (GBS), Haemophilus influenzae (Hi), Escherichia coli universal type (E.Coli) and Escherichia coli K1 type (E.Coli K1), Acinetobacter baumannii (Ab), Klebsiella pneumoniae (KPn), Staphylococcus aureus (SA), enterovirus (Evs), human double echovirus (HPeV), Japanese encephalitis virus (JEV), mumps virus (MuV), cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV6), Mycoplasma pneumoniae (MP), Cryptococcus neoformans (CN) and Candida albicans (CA), a total of 24 groups of pathogen detection target genes and human genome internal reference genes. Aiming at the conserved sequence positions of the above pathogens and internal standards, the present invention designed multiple primer and probe combinations respectively, which were first screened by a single target system to obtain the design with the optimal signal-to-noise ratio and copy number.

[0092] a. Screening plan:

[0093] 1) Using Streptococcus agalactiae (GBS), Listeria monocytogenes (LM), and Neisseria meningitidis (Nm) as examples, a different gene locus was selected for each pathogen, and a total of four primers and two probes were designed to form two combinations. The primer and probe sequences are shown in Table 2:

[0094] Table 2 Primer probe design for Streptococcus agalactiae (GBS), Listeria monocytogenes (LM) and Neisseria meningitidis (Nm)

[0095]

[0096] 2) Using the system preparation principles, droplet preparation, amplification, and detection methods described in Example 1, single-target primer-probe systems for Streptococcus agalactiae (GBS), Listeria monocytogenes (LM), and Neisseria meningitidis (Nm) were prepared, and two concentrations of positive samples for Streptococcus agalactiae (GBS), Listeria monocytogenes (LM), and Neisseria meningitidis (Nm) were detected, respectively.

[0097] 3) Calculate the actual detected copy number, negative droplet average fluorescence value, and positive droplet average fluorescence value, and calculate the relative concentration deviation and signal-to-noise ratio. The combination with a relative deviation between the measured and theoretical concentrations within ±20% is selected as the preferred primer-probe design. If the copy number detection results of two primer-probe designs are both within the relative deviation range and the difference is not significant, select the combination with the higher signal-to-noise ratio.

[0098] b.Test results

[0099] 1) Copy number and relative deviation test results: As shown in Table 3, for Streptococcus agalactiae (GBS) single-target primer-probe system combination 2 (GBS-F2R2P2), the relative deviation between the measured and theoretical concentrations for GBS samples 1 and 2 was within ±20%, while the relative deviation for combination 1 (GBS-F1R1P1) was outside this range. Therefore, combination 2 (GBS-F2R2P2) is preferred for the Streptococcus agalactiae (GBS) single-target primer-probe system. Similarly, as shown in Tables 4 and 5, combination 2 (LM-F2R2P2) is preferred for the single-target primer-probe system for Listeria monocytogenes (LM); and combination 2 (Nm-F2R2P2) is preferred for the single-target primer-probe system for Neisseria meningitidis (Nm).

[0100] Table 3 Results of positive sample concentrations detected by the two primer combinations of the single-target system for Streptococcus agalactiae (GBS)

[0101]

[0102] Table 4 Concentration results of positive samples detected by two primer combinations of Listeria monocytogenes (LM) single target system

[0103]

[0104] Table 5 Concentration results of positive samples detected by two primer combinations using the single-target system for Neisseria meningitidis (Nm)

[0105]

[0106] 2) Signal-to-noise ratio test results: Attached Figure 7 As shown in Table 6, the signal-to-noise ratios of the two single-target primer-probe design combinations of Streptococcus agalactiae (GBS) single-target primer-probe systems are consistent; Figure 8 As shown in Table 6, the signal-to-noise ratio of the single-target primer-probe design combination 1 (LM-F1R1P1) for Listeria monocytogenes (LM) is relatively higher; Figure 9 As shown in Table 6, the signal-to-noise ratio of Neisseria meningitidis (Nm) single-target primer-probe design combination 2 (Nm-F2R2P2) is relatively higher.

[0107] Table 6 Signal-to-noise ratio results of positive samples detected by single-target system and two primer combinations for Streptococcus agalactiae (GBS), Listeria monocytogenes (LM) and Neisseria meningitidis (Nm)

[0108]

[0109] c. Conclusion: Based on the relative copy number deviation and fluorescence signal-to-noise ratio indicators, the single-target primer-probe system combination 2 (GBS-F2R2P2) for Streptococcus agalactiae (GBS) showed accurate quantitative results and high signal differentiation; the single-target primer-probe system combination 2 (LM-F2R2P2) for Listeria monocytogenes (LM) showed accurate quantitative results and met the required signal differentiation; and the single-target primer-probe system combination 2 (Nm-F2R2P2) for Neisseria meningitidis (Nm) showed accurate quantitative results and high signal differentiation. Similarly, after screening with single-target primer probes, the primer and probe information selected by the kit described in this application is shown in the sequence listing in Table 18.

[0110] Example 3

[0111] Pathogen multiplex primer probe screening

[0112] The present invention screened 24 sets of pathogen detection target genes and one set of human genomic internal standard genes using a single-target system, resulting in a design with the optimal signal-to-noise ratio and copy number. Subsequently, multiplexed primer-probe sequence analysis and target cross-matching testing were performed. The 24 pathogen primer-probe designs and one internal standard primer-probe design were distributed across six reaction wells, ultimately yielding a combination with minimal primer-to-primer interference and high amplification efficiency.

[0113] a. Screening plan:

[0114] 1) Based on the principle of prioritizing the placement of similar pathogens in the same well, using six DNA viruses (HCMV), herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV6) as examples, primer multiplex design software analysis revealed that primers for HSV1 and HSV2 readily form primer dimers, necessitating that HSV1 and HSV2 should not be placed in the same reaction well. Based on this premise, for each of the five pathogen targets (HCMV, HSV1, VZV, EBV, and HHV6), four pathogens were assigned to a multiplex well, creating a total of five cross-matching combinations, as shown in Table 7.

[0115] Table 7 Combinations of five pathogens with multiple systems

[0116]

[0117]

[0118] 2) Using the system preparation principles, droplet preparation, amplification, and detection methods described in Example 1, reaction systems of combinations 1 to 5 were prepared, respectively, and mixed positive samples of cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV1), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV6) and nuclease-free water were detected.

[0119] 3) Calculate the actual concentration of each pathogen target in each combination, compare it to the theoretical concentration, calculate the relative deviation, and observe the positive and negative droplet clustering in the two-dimensional plot. For each pathogen target in the combination, the number of positive droplets should be less than 3 when testing nuclease-free water. For each pathogen target in the combination, the relative deviation between the measured concentration and the theoretical concentration should be within ±20%. The positive and negative droplet clustering for each pathogen target in the combination should be clear. Combinations that meet these three criteria are considered preferred.

[0120] b. Test results: As shown in Table 8, when combinations 1 to 5 were used to test nuclease-free water, the number of positive droplets for each target was 0, which met the requirement that the number of positive droplets should be less than 3 when testing nuclease-free water. From the analysis of the relative deviation between the measured concentration and the theoretical concentration, combinations 1, 2, and 3 met the requirements; the relative deviation of the measured concentration of the cytomegalovirus (HCMV) target in the VIC channel of combination 4 exceeded the range of ±20%, which did not meet the requirements; the relative deviation of the measured concentration of the herpes simplex virus type 1 (HSV1) target in the FAM channel and the cytomegalovirus (HCMV) target in the VIC channel of combination 5 exceeded the range of ±20%, which did not meet the requirements. Attachment Figure 10 As shown, the positive and negative droplet clustering of each target in combination 1 is clear and meets the requirements; the positive and negative droplet clustering of the CY5 channel and CY5.5 channel targets in combination 2, combination 4 and combination 5 are not clear and do not meet the requirements; the positive and negative droplet clustering of the CY5.5 channel target in combination 3 is not clear and does not meet the requirements.

[0121] Table 8 Results of five pathogen multiplex system combinations for detecting nuclease-free water and positive samples

[0122]

[0123] c. Conclusion: The four pathogen targets shown in Combination 1, Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV1), cytomegalovirus (HCMV), and varicella-zoster virus (VZV), can be combined into a multiplex system. Similarly, after screening with multiplex pathogen primer and probe combinations, the primer and probe combinations selected for the kit described in this application are as shown in the instructions.

[0124] Example 4

[0125] Kit minimum detection limit (LoD) and quantification limit (LoQ) test

[0126] a. The detection limit (LoD) and the limit of quantification (LoQ) of the reference substance were detected using the kit described in this application and the method described in Example 1. The specific detection method is as follows:

[0127] 1) Select a positive reference sample calibrated by digital PCR (concentration of 200 copies / 20 μL) and use 1×TE (pH 8.0) solution as a diluent to dilute to a theoretical concentration of 20 copies / 20 μL, 10 copies / 20 μL, and 5 copies / 20 μL, a total of three concentration gradients. Use the kit described in this application to perform 20 replicates of each copy number template.

[0128] 2) Count the number of positive results based on the positive judgment value for each target and internal standard, and calculate the detection rate using the formula. Count the number of accurate results based on the deviation criterion between the measured copy number and the theoretical copy number for each target and internal standard (a deviation of less than or equal to ±0.5 log is considered an accurate result), and calculate the accuracy rate using the formula. The pathogen level with a positive detection rate of 95% or more is considered the minimum detection limit. The limit of quantification should be greater than or equal to the detection limit, and the lowest pathogen level with more than 95% of quantitative results meeting the reagent accuracy requirement (copy number logarithm deviation ±0.5 log) is considered the quantification limit.

[0129] b.Test results:

[0130] 1) Minimum Detection Limit (LoD) Test Results: As shown in Tables 9 to 14, the minimum detection concentrations of 11 pathogens, including Staphylococcus aureus (SA), Acinetobacter baumannii (Ab), Klebsiella pneumoniae (KPn), Mycobacterium tuberculosis (MTB), mumps virus (MuV), Japanese encephalitis virus (JEV), enterovirus (Evs), herpes simplex virus type 1 (HSV1), varicella-zoster virus (VZV), Candida albicans (CA), and human herpesvirus type 6 (HHV6), were all 5 copies / 20 μL, and the minimum detection concentrations of the remaining pathogens were all 10 copies / 20 μL. In summary, the minimum detection limit of the kit described in this application is 5 copies / 20 μL to 10 copies / 20 μL.

[0131] 2) Limit of Quantitation (LoQ) Test Results: As shown in Tables 9 to 14, the LoQ for all pathogens is 10 copies / 20 μL. In summary, the LoQ of the kit described in this application is 10 copies / 20 μL.

[0132] Table 9 Results of the minimum detection limit (LoD) and quantification limit (LoQ) test for well A

[0133]

[0134]

[0135] Table 10 Results of the minimum detection limit (LoD) and quantification limit (LoQ) test for reaction well B

[0136]

[0137] Table 11 Results of the minimum detection limit (LoD) and quantification limit (LoQ) test for reaction well C

[0138]

[0139] Table 12 Results of the minimum detection limit (LoD) and quantification limit (LoQ) test for reaction well D

[0140]

[0141] Table 13 Results of the minimum detection limit (LoD) and quantification limit (LoQ) test for reaction well E

[0142]

[0143] Table 14 Results of the minimum detection limit (LoD) and quantification limit (LoQ) test for reaction well F

[0144]

[0145] c. Conclusion: The minimum detection limit (LOD) of the kit described in this application is 5 copies / 20 μL to 10 copies / 20 μL, and the limit of quantification (LOQ) is 10 copies / 20 μL.

[0146] Example 5

[0147] Comparison between kit and bacterial culture method

[0148] (1) Selected bacterial and fungal pathogens within the detection range of the kit described in this application were tested as test subjects. The selected pathogens were tested in parallel using the kit detection protocol described in this application and the method of use described in Example 1 as the experimental group, and the bacterial culture protocol as the control group to compare the sensitivity differences between the kit detection method described in this application and the culture method commonly used in clinical testing.

[0149] (2) Test results:

[0150] Table 15 Minimum detection concentration results of the kit and the bacterial culture method

[0151]

[0152]

[0153] (3) Conclusion: As shown in Table 15, using the bacterial culture concentration (unit: CFU / mL) as the statistical basis, Haemophilus influenzae (Hi) failed to culture twice. The third test result showed that the sensitivity of bacterial culture was slightly higher than that of the detection method of this kit, indicating that the detection of Haemophilus influenzae by bacterial culture method is unstable, with a low positive rate and a high risk of missed detection. In addition, the sensitivity of the multiplex detection kit for nucleic acid of central nervous system infection pathogens (digital PCR method) in detecting Escherichia coli E.Coli, Streptococcus pneumoniae SP and Klebsiella pneumoniae KPn is consistent with that of the bacterial culture method; the sensitivity of the multiplex detection kit for nucleic acid of central nervous system infection pathogens (digital PCR method) in detecting Listeria monocytogenes LM, Cryptococcus neoformans CN, Streptococcus agalactiae GBS, Neisseria meningitidis Nm, Staphylococcus aureus SA, Acinetobacter baumannii Ab and Candida albicans CA is higher than that of the bacterial culture method. In summary, the kit described in this application has high sensitivity and can detect low-concentration pathogens that cannot be detected by culture methods. It can serve as a technical supplement to the culture methods commonly used in clinical testing.

[0154] Example 6

[0155] Test kit test results of cerebrospinal fluid samples

[0156] (1) Using the kit described in this application and the method of use described in Example 1, a total of 49 cerebrospinal fluid samples from patients with clinical diagnosis results of meningitis, craniocerebral injury and other related symptoms were tested, and the results were compared and analyzed with the bacterial culture results of clinical examinations.

[0157] (2) Test results:

[0158] Table 16 Results of cerebrospinal fluid samples tested by the kit

[0159]

[0160] (3) Conclusion: As shown in Table 16, among the 49 cerebrospinal fluid samples, the test results of the kit described in this application were all positive for 7 positive cerebrospinal fluid samples, with a positive compliance rate of 100%; 11 positive samples were detected among 35 culture-negative samples, and 1 positive sample was detected among 7 uncultured samples. The ddPCR positive rate was 38.8% (19 / 49), which was much higher than the traditional bacterial culture positive rate of 16.7% (7 / 42). In summary, the kit described in this application has good specificity and high sensitivity, and can detect low-concentration pathogens that cannot be detected by culture methods. It can serve as a technical supplement to the culture methods commonly used in clinical testing.

[0161] Example 7

[0162] Comparison of kit and NGS method results

[0163] (1) Using the kit described in this application and the method of use described in Example 1, 119 clinical cerebrospinal fluid samples sequenced by NGS were tested and compared with the NGS results.

[0164] (2) Test results:

[0165] Table 17 Comparison of kit and NGS method results

[0166]

[0167] (3) Conclusion: As shown in Table 17, the kit described in this application was used to test the above 119 CSF samples. The NGS positive rate was 33.61% (40 / 119), and the ddPCR positive rate was 44.54% (53 / 119). This indicates that ddPCR has higher sensitivity and better accuracy than NGS in detecting CSF.

[0168] Table 18 Sequence Listing

[0169]

[0170]

[0171]

[0172] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A multiplex droplet digital PCR detection kit for central nervous system infection pathogens, characterized in that: include: Target fragment detection mixture A, including target fragment amplification primers and probes, the sequences of the target fragment amplification primers are shown in SEQ ID No. 1 to SEQ ID No. 10, and the sequences of the target fragment probes are shown in SEQ ID No. 51 to SEQ ID No. 55; Target fragment detection mixture B, including target fragment amplification primers and probes, the sequences of the target fragment amplification primers are shown in SEQ ID No. 11 to SEQ ID No. 18, and the sequences of the target fragment probes are shown in SEQ ID No. 56 to SEQ ID No. 59; Target fragment detection mixture C, including target fragment amplification primers and probes, the sequences of the target fragment amplification primers are shown as SEQ ID No. 19 to SEQ ID No. 26, and the sequences of the target fragment probes are shown as SEQ ID No. 60 to SEQ ID No. 63; A target fragment detection mixture D includes target fragment amplification primers and probes, wherein the sequences of the target fragment amplification primers are shown in SEQ ID No. 27 to SEQ ID No. 34, and the sequences of the target fragment probes are shown in SEQ ID No. 64 to SEQ ID No. 67; A target fragment detection mixture E comprises target fragment amplification primers and probes, wherein the sequences of the target fragment amplification primers are shown in SEQ ID No. 35 to SEQ ID No. 42, and the sequences of the target fragment probes are shown in SEQ ID No. 68 to SEQ ID No. 71; A target fragment detection mixture F comprises target fragment amplification primers and probes, wherein the sequences of the target fragment amplification primers are shown in SEQ ID No. 43 to SEQ ID No. 50, and the sequences of the target fragment probes are shown in SEQ ID No. 72 to SEQ ID No. 75; Positive quality control products A to F, including the pathogen plasmid containing the target fragment as a positive quality control product and 1×TE (pH 8.0) solution for dilution; Negative control, including 1×TE (PH 8.0) solution as a negative control.

2. A multiplex droplet digital PCR detection kit for central nervous system infection pathogens according to claim 1, characterized in that: The target fragments are DNA or RNA fragments of Neisseria meningitidis (Nm), Mycobacterium tuberculosis (MTB), Listeria monocytogenes (LM), Streptococcus pneumoniae (SP), Streptococcus agalactiae (GBS), Haemophilus influenzae (Hi), Escherichia coli universal type (E.Coli) and Escherichia coli K1 type (E.Coli K1), Acinetobacter baumannii (Ab), Klebsiella pneumoniae (KPn), Staphylococcus aureus (SA), enterovirus (Evs), human double echovirus (HPeV), Japanese encephalitis virus (JEV), mumps virus (MuV), cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV6), Mycoplasma pneumoniae (MP), Cryptococcus neoformans (CN), Candida albicans (CA) and human internal standard (RPP30).

3. A multiplex droplet digital PCR detection kit for central nervous system infection pathogens according to claim 2, characterized in that: The target fragment detection mixed solution A contains primer probes for detecting the target fragment of Escherichia coli (E.Coli), primer probes for detecting the target fragment of Escherichia coli K1 type (E.ColiK1), primer probes for detecting the target fragment of Listeria monocytogenes (LM), primer probes for detecting the target fragment of Cryptococcus neoformans (CN), and primer probes for detecting the target fragment of Streptococcus agalactiae (GBS); The target fragment detection mixture B contains a primer probe for detecting the target fragment of Haemophilus influenzae (Hi), a primer probe for detecting the target fragment of Streptococcus pneumoniae (SP), a primer probe for detecting the target fragment of Neisseria meningitidis (Nm), and a primer probe for detecting the target fragment of human internal control (RPP30); The target fragment detection mixed solution C contains a primer probe for detecting the target fragment of Staphylococcus aureus (SA), a primer probe for detecting the target fragment of Acinetobacter baumannii (Ab), a primer probe for detecting the target fragment of Klebsiella pneumoniae (KPn), and a primer probe for detecting the target fragment of Mycobacterium tuberculosis (MTB); The target fragment detection mixed solution D contains a primer probe for detecting the target fragment of mumps virus (MuV), a primer probe for detecting the target fragment of human double echovirus (HPeV), a primer probe for detecting the target fragment of Japanese encephalitis virus (JEV), and a primer probe for detecting the target fragment of enterovirus (Evs); The target fragment detection mixture E contains a primer probe for detecting the target fragment of Epstein-Barr virus (EBV), a primer probe for detecting the target fragment of herpes simplex virus type 1 (HSV1), a primer probe for detecting the target fragment of cytomegalovirus (HCMV), and a primer probe for detecting the target fragment of varicella-zoster virus (VZV); The target fragment detection mixed solution F contains primer probes for detecting the target fragment of Candida albicans (CA), primer probes for detecting the target fragment of herpes simplex virus type 2 (HSV2), primer probes for detecting the target fragment of Mycoplasma pneumoniae (MP) and primer probes for detecting the target fragment of human herpes virus type 6 (HHV6).

4. A multiplex droplet digital PCR detection kit for central nervous system infection pathogens according to claim 3, characterized in that: The primers in the target fragment detection mixed solution D are reverse transcription specific primers and specific amplification primers.

5. A multiplex droplet digital PCR detection kit for central nervous system infection pathogens according to claim 4, characterized in that: The amplification reaction of the detection well containing the target fragment detection mixture is: 42°C reaction for 30 minutes, 1 cycle, 95°C reaction for 10 minutes, 1 cycle, 95°C reaction for 30 seconds, 60°C reaction for 60 seconds, 45 cycles, 98°C reaction for 10 minutes, 1 cycle. The amplification reaction is a combination of reverse transcription reaction and PCR amplification reaction. There is no need to open the lid to transfer the product, which reduces amplicon contamination. The MicroDrop-400B biochip analyzer (four channels) developed and produced by Guangdong Yongnuo Medical Technology Co., Ltd. is used for detection, and the four-channel positive and negative grouping is obvious.

6. A multiplex droplet digital PCR detection kit for central nervous system infection pathogens according to claim 5, characterized in that: The kit has a total of 6 reaction well systems for each sample for detection. One test can quantitatively detect Neisseria meningitidis (Nm), Mycobacterium tuberculosis (MTB), Listeria monocytogenes (LM), Streptococcus pneumoniae (SP), Streptococcus agalactiae (GBS), Haemophilus influenzae (Hi), Escherichia coli universal type (E.Coli) and Escherichia coli K1 type (E.ColiK1), Acinetobacter baumannii (Ab), Klebsiella pneumoniae (KPn), Staphylococcus aureus (SA), Enterococcus pyogenes (ES), and Enterococcus spp. The 23 common pathogens of central nervous system infection included 23 common pathogens, including E. coli, human echovirus (HPeV), Japanese encephalitis virus (JEV), mumps virus (MuV), cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV6), Mycoplasma pneumoniae (MP), Cryptococcus neoformans (CN) and Candida albicans (CA).

7. A multiplex droplet digital PCR detection kit for central nervous system infection pathogens according to claim 6, characterized in that: The reaction well system must be equipped with both positive quality control products and negative quality control products; the positive quality control products are derived from the positive quality control products A to F in the kit; the negative quality control products are derived from the negative quality control products in the kit.

8. A multiplex droplet digital PCR detection kit for central nervous system infection pathogens according to claim 7, characterized in that: The B reaction well system of the kit includes human internal control RPP30, which can control the sampling quality and nucleic acid extraction quality of the sample.

9. A multiplex droplet digital PCR detection kit for central nervous system infection pathogens according to claim 8, characterized in that: The kit can use the target fragment detection mixture A to detect one or more of Escherichia coli (E.Coli), Escherichia coli K1 (E.ColiK1), Listeria monocytogenes (LM), Cryptococcus neoformans (CN), and Streptococcus agalactiae (GBS), and is used for the identification and quantitative analysis of pathogens of central nervous system infection; The kit can use the target fragment detection mixture B to detect one or more of Haemophilus influenzae (Hi), Streptococcus pneumoniae (SP), and Neisseria meningitidis (Nm), for the identification and quantitative analysis of pathogens of central nervous system infection; The kit can use the target fragment detection mixture C to detect one or more of Staphylococcus aureus (SA), Acinetobacter baumannii (Ab), Klebsiella pneumoniae (KPn), and Mycobacterium tuberculosis (MTB), and is used for the identification and quantitative analysis of pathogens of central nervous system infection; The kit can use the target fragment detection mixture D to detect one or more of mumps virus (MuV), human echovirus (HPeV), Japanese encephalitis virus (JEV), and enterovirus (Evs), for the identification and quantitative analysis of pathogens that infect the central nervous system; The kit can use the target fragment detection mixture E to detect one or more of Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV1), cytomegalovirus (HCMV), and varicella-zoster virus (VZV), for the identification and quantitative analysis of pathogens that infect the central nervous system; The kit can use the target fragment detection mixture F to detect one or more of Candida albicans (CA), herpes simplex virus type 2 (HSV2), Mycoplasma pneumoniae (MP), and human herpes virus type 6 (HHV6), and is used for the identification and quantitative analysis of pathogens that infect the central nervous system.

10. Use of the multiplex droplet digital PCR detection kit for central nervous system infection pathogens according to any one of claims 1 to 9 in detecting central nervous system infection pathogens.

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