Multiplex gene detection system and kit for viral and bacterial infection of central nervous system

Abstract

The present invention relates to a multiplex gene detection system for viral and bacterial infection of the central nervous system, comprising forward and reverse primers for detecting enterovirus, human double echovirus, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus pneumoniae, varicella zoster virus, mumps virus, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, Neisseria meningitidis, Epstein-Barr virus, Streptococcus agalactiae, Haemophilus influenzae, human herpes virus type 6, Escherichia coli K1 and Japanese encephalitis virus, and the detection sample is cerebrospinal fluid. The multiplex gene detection product for viral and bacterial infection of the central nervous system of the present invention has high throughput, low cost, high sensitivity and good specificity, and is used for synchronous identification and semi-quantitative analysis of multiple viral and bacterial infections of the central nervous system.

Description

Technical Field

[0001] The invention relates to a multiple gene detection product and a detection system used by the product, and belongs to the field of biotechnology. Background Art

[0002] Encephalitis and meningitis are infectious diseases caused by infection with various pathogens such as bacteria, fungi, and viruses, and mainly cause damage to the central nervous system.

[0003] The currently commonly used pathogen detection methods, such as cerebrospinal fluid culture and Gram staining, have low positive rates, long culture cycles (at least 24-48 hours), and their sensitivity is affected by the use of antibiotics. In addition, in patients with encephalitis and meningitis, the negative rate of cerebrospinal fluid Gram staining exceeds 90%, which cannot fully meet the clinical requirements for detection speed and accuracy. Virus detection has also always been a shortcoming of domestic microbial detection.

[0004] At present, the detection of pathogens of central nervous system (CNS) infection mainly relies on cerebrospinal fluid culture, smear staining, specific antibodies and PCR, but all of them have certain limitations: (1) CSF culture and smear staining: low positive rate, possible reasons include the large amount of bacteria required for culture, but the limited amount and content of cerebrospinal fluid samples; clinical empirical medication inhibits bacterial growth; external factors such as the specimen collection process limit bacterial survival, etc. (2) Specific antibodies: IgM antibody detection is often used as a basis for early viral infection, but in the early stage of infection, the lack of virus-specific IgM antibodies or very low titers will lead to negative results. The sensitivity and timeliness of the detection are low, and only qualitative analysis can be performed, not quantitative analysis. (3) PCR: Due to the limitation of fluorescence channels and the interaction between primers, most current PCR methods can only detect limited viral targets. For example, FilmArray can detect 14 pathogens, loop-mediated isothermal amplification (LAMP) and real-time fluorescence quantitative PCR (RT-PCR) can only detect two targets, HSV-1 and HSV-2.

[0005] Therefore, it is of great clinical significance to develop a multiplex gene detection kit product with high throughput, low cost, high sensitivity and good specificity for the identification and quantitative analysis of pathogens of central nervous system infection. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a multiple gene detection product for viral and bacterial infection of the central nervous system with high throughput, low cost, high sensitivity and good specificity for simultaneous identification and semi-quantitative analysis of multiple central nervous system infection pathogens.

[0007] The present invention proposes a technical solution to solve the above technical problems: a multiple gene detection system for viral and bacterial infection of the central nervous system, including forward and reverse primers for detecting enterovirus, human double echovirus, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus pneumoniae, varicella-zoster virus, mumps virus, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, Neisseria meningitidis, Epstein-Barr virus, Streptococcus agalactiae, Haemophilus influenzae, human herpes virus type 6, Escherichia coli K1 and Japanese encephalitis virus, and the detection sample is cerebrospinal fluid.

[0008] In the aforementioned multiple gene detection system for viral and bacterial infection of the central nervous system, the nucleotide sequence of the forward primer for enterovirus is shown in SEQ ID No.3, and the nucleotide sequence of the reverse primer for enterovirus is shown in SEQ ID No.4;

[0009] The nucleotide sequence of the forward primer for human double echovirus is shown in SEQ ID No.5, and the nucleotide sequence of the reverse primer for human double echovirus is shown in SEQ ID No.6;

[0010] The nucleotide sequence of the forward primer for Mycobacterium tuberculosis is shown in SEQ ID No.7, and the nucleotide sequence of the reverse primer for Mycobacterium tuberculosis is shown in SEQ ID No.8;

[0011] The nucleotide sequence of the forward primer for Listeria monocytogenes is shown in SEQ ID No.9, and the nucleotide sequence of the reverse primer for Listeria monocytogenes is shown in SEQ ID No.10;

[0012] The nucleotide sequence of the forward primer for Streptococcus pneumoniae is shown in SEQ ID No. 11, and the nucleotide sequence of the reverse primer for Streptococcus pneumoniae is shown in SEQ ID No. 12;

[0013] The nucleotide sequence of the forward primer for varicella-zoster virus is shown in SEQ ID No. 13, and the nucleotide sequence of the reverse primer for varicella-zoster virus is shown in SEQ ID No. 14; the nucleotide sequence of the forward primer for mumps virus is shown in SEQ ID No. 15, and the nucleotide sequence of the reverse primer for mumps virus is shown in SEQ ID No. 16;

[0014] The nucleotide sequence of the forward primer for cytomegalovirus is shown in SEQ ID No. 17, and the nucleotide sequence of the reverse primer for cytomegalovirus is shown in SEQ ID No. 18;

[0015] The nucleotide sequence of the forward primer for herpes simplex virus type 1 is shown in SEQ ID No. 19, and the nucleotide sequence of the reverse primer for herpes simplex virus type 1 is shown in SEQ ID No. 20;

[0016] The nucleotide sequence of the forward primer for herpes simplex virus type 2 is shown in SEQ ID No. 21, and the nucleotide sequence of the reverse primer for herpes simplex virus type 2 is shown in SEQ ID No. 22;

[0017] The nucleotide sequence of the forward primer for Neisseria meningitidis is shown in SEQ ID No. 23, and the nucleotide sequence of the reverse primer for Neisseria meningitidis is shown in SEQ ID No. 24;

[0018] The nucleotide sequence of the forward primer for Epstein-Barr virus is shown in SEQ ID No. 25, and the nucleotide sequence of the reverse primer for Epstein-Barr virus is shown in SEQ ID No. 26;

[0019] The nucleotide sequence of the forward primer for Streptococcus agalactiae is shown in SEQ ID No. 27, and the nucleotide sequence of the reverse primer for Streptococcus agalactiae is shown in SEQ ID No. 28;

[0020] The nucleotide sequence of the forward primer for Haemophilus influenzae is shown in SEQ ID No. 29, and the nucleotide sequence of the reverse primer for Haemophilus influenzae is shown in SEQ ID No. 30;

[0021] The nucleotide sequence of the forward primer for human herpesvirus type 6 is shown in SEQ ID No. 31, and the nucleotide sequence of the reverse primer for human herpesvirus type 6 is shown in SEQ ID No. 32;

[0022] The nucleotide sequence of the forward primer for E. coli K1 is shown in SEQ ID No. 33, and the nucleotide sequence of the reverse primer for E. coli K1 is shown in SEQ ID No. 34;

[0023] The nucleotide sequence of the forward primer for Japanese encephalitis virus is shown in SEQ ID No.39, and the nucleotide sequence of the reverse primer for Japanese encephalitis virus is shown in SEQ ID No.40.

[0024] The above-mentioned multiple gene detection system for viral and bacterial infection of the central nervous system also includes forward and reverse primers for detecting human RNA internal reference, forward and reverse primers for detecting human DNA internal reference and forward and reverse primers for detecting system quality control internal reference; the nucleotide sequence of the forward primer for the human RNA internal reference is shown in SEQ ID No.1, and the nucleotide sequence of the reverse primer for the human RNA internal reference is shown in SEQ ID No.2; the nucleotide sequence of the forward primer for the human DNA internal reference is shown in SEQ ID No.35, and the nucleotide sequence of the reverse primer for the human DNA internal reference is shown in SEQ ID No.36; the nucleotide sequence of the forward primer for the system quality control internal reference is shown in SEQ ID No.37, and the nucleotide sequence of the reverse primer for the system quality control internal reference is shown in SEQ ID No.38.

[0025] The final concentrations of forward and reverse primers for human RNA internal reference and human DNA internal reference in the detection system were 900nM; the final concentrations of forward and reverse primers for enterovirus, mumps virus, herpes simplex virus type 1, Haemophilus influenzae, and Escherichia coli K1 in the detection system were 2400nM; the final concentrations of forward and reverse primers for human double echovirus, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus pneumoniae, herpes simplex virus type 2, Neisseria meningitidis, Epstein-Barr virus, Streptococcus agalactiae, and human herpesvirus type 6 in the detection system were 1800nM; the final concentrations of forward and reverse primers for varicella-zoster virus and cytomegalovirus in the detection system were 1200nM; the final concentrations of forward and reverse primers for Japanese encephalitis virus in the detection system were 3600nM.

[0026] The central nervous system viral and bacterial infection multiple gene detection system also includes reverse transcription PCR buffer, reverse transcription PCR enzyme and dNTP.

[0027] In the above-mentioned multiple gene detection system for viral and bacterial infection of the central nervous system, all forward primers are provided with fluorescent markers, and the fluorescent markers are CY5, CY3 or FAM.

[0028] The central nervous system viral and bacterial infection multiple gene detection system also includes ultrapure water and empty test tubes.

[0029] The above-mentioned multiple gene detection system for viral and bacterial infection of the central nervous system has the following components: 3 volumes of 5× reverse transcription PCR buffer, 0.6 volumes of dNTP, 1 volume of reverse transcription PCR enzyme, 1.5 volumes of primer mixture, 7.5 volumes of template, and 1.4 volumes of pure water.

[0030] A technical solution proposed by the present invention to solve the above technical problems is: a multiple gene test kit for viral and bacterial infection of the central nervous system including the above detection system.

[0031] Neisseria meningitidis (NM) is an encapsulated, aerobic, Gram-negative bacterium best known for the meningitis it causes. NM is primarily transmitted through respiratory and throat secretions. Bacteria carried in the blood, mouth, nose, sexual organs, and excrement of infected people can be transmitted to others through food, air, water, blood, sexual intercourse, etc. Most patients infected with Neisseria meningitidis present with acute symptoms. Early symptoms include fever, headache, nausea or vomiting, rash, stiff neck, leg pain, mental confusion, photophobia, and abnormal skin color.

[0032] Haemophilus influenzae (HI) is a non-motile Gram-negative bacillus. The bacterium is widely present in the upper respiratory tract of normal people. The disease is mainly transmitted by respiratory secretions through air droplets and close contact. Infection in the throat can lead to asymptomatic carriage in the throat, which can last for several months. A small number of infected people develop invasive diseases. The initial clinical manifestations include changes in crying, nausea, vomiting, fever, headache, photophobia, meningeal irritation, irritability, anorexia, and convulsions.

[0033] Mycobacterium tuberculosis (TB) is the causative agent of tuberculous meningitis. It is a small, aerobic, inactive bacterium belonging to the genus Bacillus. The bacterium is mainly transmitted through droplets, multiplying in alveolar macrophages and spreading to other tissues outside the lungs through the blood circulation. The early clinical manifestations are not obvious, making it difficult to distinguish TB from meningitis caused by other reasons. Once the disease worsens, neurological symptoms appear, such as cough, convulsions, increased intracranial pressure, and mild hemiplegia.

[0034] Streptococcus pneumonia (SP) is a spherical Gram-positive bacterium that is the most common cause of community-acquired pneumonia, meningitis, and bacteremia in children and adults, and the most common cause of acute otitis media in children. The nasopharynx is the main colonization site of SP, and transmission from the nasopharynx to the lower respiratory tract or other places may cause invasive diseases. Its clinical manifestations include upper respiratory tract infection, otitis media, sinusitis, conjunctivitis, bacteremia, pneumonia, empyema, meningitis, endocarditis, septic arthritis, cellulitis, etc.

[0035] Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is an aerobic Gram-positive streptococcus that normally resides in the lower respiratory tract, vagina and rectum. It is a conditional pathogen that colonizes the nasopharynx and rectum of newborns for a long time and can cause neonatal sepsis, pneumonia, meningitis, and even death. Symptoms of neonatal meningitis include respiratory distress, suffocation, lethargy, fever or hypothermia, jaundice, vomiting, diarrhea, skin symptoms include petechiae, abscesses and sclerosis, and neurological symptoms also include excitability, changes in consciousness, poor voice tone, tremors, and facial muscle twitching.

[0036] Escherichia coli K1 (E.coliK1) is a representative strain of Escherichia coli that causes neonatal sepsis and meningitis. It is the leading cause of bacterial meningitis in premature infants and the second leading cause of bacterial meningitis in full-term infants. Neonatal meningitis caused by this strain has a higher morbidity and mortality rate. Most E. coli meningitis is the result of hematogenous transmission, entering the brain through the blood circulation and causing disease. The initial clinical manifestations include unstable body temperature, irritability, lethargy, feeding difficulties, hypotension, convulsions, and suffocation.

[0037] Listeria monocytogenes (LM) is a facultative anaerobic bacterium and the pathogen of listeriosis. It is mainly transmitted through food and is one of the most deadly foodborne pathogens. Invasive infection with Listeria monocytogenes causes listeriosis, including sepsis, meningitis (or meningoencephalitis), encephalitis, corneal ulcers, pneumonia, and intrauterine infection or cervical infection in pregnant women. Non-invasive infection manifests as febrile gastroenteritis.

[0038] Enterovirus (EV), also known as enterovirus, is a type IV RNA virus that mainly parasitizes in the intestine. EV includes 67 serotypes, including poliovirus (PV1-3), Coxsackievirus (CAV Al-A22, A24), Coxsackievirus (CBV BI-B6), echovirus (Enteric cytopathic human orphan virus, ECHO 1-9, 11-27, 29-33) and new enterovirus (EV68-71). EV is mainly transmitted through fecal-oral or respiratory droplets, and can also be infected by contact with the patient's skin and mucous membrane vesicle fluid. Its clinical manifestations are diverse, ranging from asymptomatic, respiratory tract infection to myocarditis, chronic cardiovascular disease, poliomyelitis, paralysis, meningitis or meningoencephalitis, neonatal disease, diabetes, post-viral fatigue syndrome, chest muscle pain, herpetic angina, hand, foot and mouth disease, eye disease, gastrointestinal disease, etc. In encephalitis, the types of enterovirus infected mainly refer to three types of picornaviruses, namely poliovirus, coxsackievirus and echovirus.

[0039] Mumps virus (Muv), also known as mumps virus, is an enveloped RNA virus. MuV is highly contagious among people and can be transmitted through direct contact, respiratory droplets or infected fomites. MuV is highly neurotropic and can enter the cerebrospinal fluid through the choroid plexus. The virus can also penetrate into the brain parenchyma and infect other brain parenchymal cells such as pyramidal cells and ependymal epithelial cells in the cerebral cortex and hippocampal tissue, causing meningoencephalitis. 4% of patients infected with MuV will experience deafness. Other neurological complications include cerebellar ataxia, transverse myelitis, poliomyelitis, etc.

[0040] Herpes virus is a class of DNA viruses with an envelope, of which more than 120 species are known. It is named because the representative species, herpes simplex virus, can cause serpiginous herpes. According to its physical and chemical properties, it is divided into four subfamilies: alpha herpes virus (such as herpes simplex virus, varicella-zoster virus), beta herpes virus (such as cytomegalovirus), gamma herpes virus (such as Epstein-Barr virus) and unclassified herpes virus. There are eight known viruses in this family that can cause human diseases, collectively referred to as human herpes virus. This kit detects six types of herpes virus, including herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (HCMV) and human herpes virus type 6 (HHV-6). Herpes virus mainly invades tissues of ectoderm origin, including skin, mucous membranes and nerve tissues. The infection sites and diseases caused are diverse, and there is a tendency for latent infection. Typical clinical symptoms of HSV encephalitis include fever, confusion, disorientation, behavioral disorders, language disorders, epilepsy or status epilepticus, and focal neurological deficits. The initial symptom of VZV infection is chickenpox, which can cause herpes zoster, dermatomal distribution, pain, and rash with aging or immunosuppression. Chronic pain, cranial nerve palsy, herpes zoster paralytic dementia, vascular lesions, meningoencephalitis, and a variety of eye diseases are also common after herpes zoster.

[0041] Japanese encephalitis virus (JEV), also known as Japanese encephalitis virus, is spherical and its nucleic acid is single-stranded RNA. Mosquitoes are the medium of transmission of Japanese encephalitis virus. When a person is bitten by a mosquito carrying the virus, the virus enters the human body, proliferates in phagocytic cells such as vascular endothelial cells, lymph nodes, liver, spleen, etc., and reaches the brain through the blood circulation to cause inflammation. The main symptoms are high fever, headache, vomiting, lethargy, convulsions, etc. Severe cases may have a high fever all over the body, convulsions, cerebral edema, respiratory or circulatory failure and die, and some patients have sequelae.

[0042] Humanparechovirus (HPeV) is a single-stranded positive-sense RNA virus belonging to the family Picornaviridae and the genus Parechovirus. Eight genotypes have been discovered so far. HPeV is mainly transmitted through the fecal-oral and respiratory routes. The clinical symptoms caused by HPeV infection are similar to those of human enterovirus, and may cause relatively mild gastrointestinal and respiratory symptoms. In addition, fatal symptoms such as encephalitis, myocarditis, flaccid paralysis, and neonatal sepsis have also been reported.

[0043] The present invention has positive effects:

[0044] (1) The central nervous system viral and bacterial infection multiple gene kit of the present invention adopts a fully automatic multiplex PCR detection method, which has the characteristics of simple operation, rapid detection, wider coverage of pathogens, higher detection sensitivity and specificity, etc. It can quickly detect 17 common encephalitis and meningitis pathogens in cerebrospinal fluid samples within 4 hours, including 10 viruses (enterovirus, human double echovirus, Japanese encephalitis virus, mumps virus, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, human herpes virus type 6), 7 bacteria (Neisseria meningitidis, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, Escherichia coli K1).

[0045] (2) The central nervous system viral bacterial infection multiple gene kit of the present invention uses the specific primers of 17 genes with FAM fluorescently labeled 5′ end primers to optimize the reaction system, realize the systematic detection of 17 target gene sites in one PCR system, overcome the deviation caused by unequal amplification in traditional PCR, and can accurately and sensitively quantify the expression of a group of target genes, saving costs and improving efficiency. In addition to detecting the above 17 gene sites at one time, the central nervous system viral bacterial infection multiple gene kit of the present invention can also simultaneously detect 2 human internal reference gene sites Hum RNA and Hum DNA and 1 system internal reference IC.

[0046] (3) The capillary electrophoresis fragment analysis technology used in the fragment analysis of the central nervous system viral and bacterial infection multiple gene kit of the present invention is different from the traditional gel electrophoresis analysis mode, making the PCR result analysis more intuitive, concise, reliable, easy to identify and judge, and more conducive to standardized operation.

[0047] (4) The central nervous system viral and bacterial infection multiple gene kit of the present invention is easy to operate, has low detection cost, high accuracy, good repeatability, and the use conditions can be modeled, which is convenient for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The capillary electrophoresis patterns of the kit of the embodiment of the present invention for detecting standard strains and strains corresponding to various pathogens respectively;

[0049] Figure 2 It is a capillary electrophoresis pattern of the test kit of the embodiment of the present invention after detecting the standard strains and strains corresponding to various pathogens mixed with negative cerebrospinal fluid;

[0050] Figure 3 It is a capillary electrophoresis spectrum of common infectious pathogens detected by the kit of the embodiment of the present invention after being mixed in pairs and then mixed with negative cerebrospinal fluid;

[0051] Figure 4 It is a standard curve diagram of the concentration of various pathogens detected by the kit of the embodiment of the present invention and the detection peak height. DETAILED DESCRIPTION

[0052] The present invention is described in detail below by way of examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned content of the present invention. In the following examples, unless otherwise indicated, the reagents used are all analytically pure and all the reagents used can be obtained from commercial channels. The experimental methods for which specific conditions are not indicated in the text are usually in accordance with conventional conditions such as the conditions described in the book "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al. and published by Science Press in 2002, or in accordance with the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the recorded contents can be applied to the present invention.

[0053] Example

[0054] 1. Composition of the kit

[0055] The central nervous system virus and bacterial infection multiple gene detection kit of this embodiment comprises: reverse transcription PCR buffer (RT-PCR Buffer), reverse transcription PCR enzyme (RT-PCR Enzyme), dNTP, primers pool, ultrapure water (ddH 2 O) and an empty test tube.

[0056] Primer design principles: Download the gene sequences of 17 target pathogens from Genebank, use Vector NTI software for multiple sequence alignment, select highly conserved, single-copy specific sequences to design primers, and use FAM fluorescent dye to label the 5′ end of the primers. Use Oligo7 to design primers, and perform sequence alignment at the National Center for Biotechnology Information (NCBI) to verify the specificity of each primer.

[0057] In addition to the basic principles of PCR primer design, the main design principles of multiple primers include: ① The length of the amplified product fragment of all primers is limited to between 100-400 bp, and the length difference of the amplified product of different primers is greater than 3 bp; ② Try to avoid the formation of dimer structure between different primers; ③ Avoid non-specific amplification of each pair of primers and any two unpaired primers. Primer synthesis was completed by Shanghai Bioengineering Co., Ltd. Reverse transcription PCR buffer (RT-PCR Buffer), reverse transcription PCR enzyme (RT-PCR Enzyme) and dNTP were all provided by QIAGEN. The sequence characteristics of each primer are shown in Table 1.

[0058] Table 1 Primer sequence characteristics

[0059]

[0060]

[0061] 2. How to use the kit Synthetic construct

[0062] The central nervous system viral and bacterial infection multiple gene detection kit of this embodiment was used to detect the expression levels of 17 pathogen target genes closely related to the selection of identification and semi-quantitative detection schemes in cerebrospinal fluid specimens of 700 patients with suspected central nervous system infection. The specific detection steps are as follows:

[0063] 1. Collect cerebrospinal fluid samples and extract the pathogen genome.

[0064] The cerebrospinal fluid sample was thoroughly shaken and mixed, and 300 μL was pipetted into the bacterial genome rapid extraction kit, and the pathogen genome was extracted using SmartLab Assist. After the concentration was measured by UV spectrophotometer, it was stored at 4°C or -20°C for future use.

[0065] 2. Perform multiplex PCR reaction using cDNA as template.

[0066] The cDNA obtained in the previous step was used as a template, and the forward and reverse primers labeled with FAM were used to amplify the PCR product. The preparation of the reaction solution in each tube is shown in Table 2.

[0067] Table 2 Reaction solution composition

[0068] Reaction liquid composition volume 5×RT-PCR Buffer 3μL dNTP 0.6μL RT-PCR Enzyme 1μL Primer pool 1.5μL <![CDATA[ddH 2 The]]> 1.4μL template 7.5μL Total volume 15μL

[0069] The prepared system was run on a PCR instrument. The reaction conditions are shown in Table 3.

[0070] Table 3 Reaction conditions

[0071]

[0072] 3. Use 3500Dx genetic analyzer to perform capillary electrophoresis, detect FAM fluorescence signals, and perform data analysis.

[0073] Add 9 μL of highly deionized formamide (HiDi) and 1 μL of multiplex PCR product to each well of a 96-well sample plate.

[0074] According to the operating manual of the 3500Dx Genetic Analyzer, install the capillaries and gel. Place the sample plate into the machine and run the separation program. Execute the default 3500Dx analysis method and finally save the data.

[0075] GeneMapper ID-X software was used to further analyze the results of capillary electrophoresis. The PCR product fragments of each gene were of different sizes. Figure 1 As shown, the abscissa represents the fragment length, and the ordinate represents the peak height.

[0076] 4. CNS-HMGS is specific to all target detection targets.

[0077] Specific fluorescent primers for the specific identification genes of 17 target pathogens were designed and optimized. CNS-HMGS was used to detect the standard strains and strains corresponding to the 17 pathogens. The results showed that all targets produced specific peaks at the target position, that is, at the position of ±1.5bp of the amplified product fragment length. Figure 1 shown.

[0078] 5. CNS-HMGS can simultaneously detect 20 target sites in a single multiplex PCR reaction.

[0079] The plasmids of 17 pathogens and three internal reference genes, Human DNA, Human RNA and IC, were all inoculated at the same concentration of 3×10 4 The CNS-HMGS system was able to simultaneously amplify 17 pathogens and 3 reference genes, and the peak values ​​of each target were higher than the critical value of 300 rfu.

[0080] 6. CNS-HMGS has high sensitivity in detecting 17 pathogen targets.

[0081] The standard strains and toxins of 17 pathogens were mixed with negative cerebrospinal fluid, and the cerebrospinal fluid was diluted in series to simulate positive samples to obtain 1×10 6 ,1×10 5 ,1×10 4 ,1×10 3 and 1×10 2The nucleic acid of 100 copies / mL was detected by CNS-HMGS, and each gradient was repeated 20 times. The criterion for the minimum detection limit was the concentration corresponding to the positive detection rate ≥ 90% in 20 repeated tests, and the positive detection rate of the next lower concentration of the gradient was < 10%. The results showed that the minimum detection limit of each pathogen could reach 1×10 3 copies / mL( Figure 2 ), CNS-HMGS has a high sensitivity.

[0082] 7. CNS-HMGS can accurately detect multiple infections in cerebrospinal fluid.

[0083] There are cases of multiple central nervous system infections in clinical practice. It is very important to accurately detect multiple infection pathogens in cerebrospinal fluid based on CNS-HMGS. Five common infection pathogens (M. tuberculosis, 131bp; C. neoformans, 262bp; EV, 118bp; CMV, 180bp; EBV, 228bp) were used as nucleic acid templates. They were mixed in pairs with negative cerebrospinal fluid. CNS-HMGS was used to detect and compare the peak positions and signal intensities of the pairwise mixtures and single targets. The results are shown in Figure 2. Figure 3 As shown, the position and signal intensity of the mixed target peak are consistent with those of the single target, so CNS-HMGS is capable of detecting multiple infectious pathogens.

[0084] 8. CNS-HMGS has semi-quantitative analysis performance.

[0085] ddPCR was used to quantify the concentrations of standard strains and strains of 17 pathogens, ranging from 1×10 6 The number of copies / mL was gradually diluted to 1×10 2 The results showed that the detection peak area of ​​CNS-HMGS increased with the increase of pathogen concentration, and the correlation coefficient R between pathogen concentration and detection peak height was 2 All>0.98 Figure 4 As shown, it is proved that CNS-HMGS has semi-quantitative detection performance.

[0086] 9. Comparison of the clinical application performance of the CNS-HMGS system.

[0087] The CNS-HMGS system was used to detect 700 cerebrospinal fluid samples of suspected CNS infection. The results showed that the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of CNS-HMGS were all high (≥0.90), as shown in Table 4.

[0088] Table 4 CNS-HMGS clinical application performance

[0089]

[0090] Obviously, the above embodiments are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. However, these obvious changes or modifications derived from the spirit of the present invention are still within the scope of protection of the present invention.

Claims

1. A multiplex gene detection system for viral and bacterial infection of the central nervous system, characterized in that: Including forward and reverse primers for detecting enterovirus, human double echovirus, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus pneumoniae, varicella zoster virus, mumps virus, cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2, Neisseria meningitidis, Epstein-Barr virus, Streptococcus agalactiae, Haemophilus influenzae, human herpes virus type 6, Escherichia coli K1 and Japanese encephalitis virus, and the test sample is cerebrospinal fluid; The nucleotide sequence of the forward primer for enterovirus is shown in SEQ ID No.3, and the nucleotide sequence of the reverse primer for enterovirus is shown in SEQ ID No.4; The nucleotide sequence of the forward primer for human double echovirus is shown in SEQ ID No.5, and the nucleotide sequence of the reverse primer for human double echovirus is shown in SEQ ID No.6; The nucleotide sequence of the forward primer for Mycobacterium tuberculosis is shown in SEQ ID No.7, and the nucleotide sequence of the reverse primer for Mycobacterium tuberculosis is shown in SEQ ID No.8; The nucleotide sequence of the forward primer for Listeria monocytogenes is shown in SEQ ID No.9, and the nucleotide sequence of the reverse primer for Listeria monocytogenes is shown in SEQ ID No.10; The nucleotide sequence of the forward primer for Streptococcus pneumoniae is shown in SEQ ID No. 11, and the nucleotide sequence of the reverse primer for Streptococcus pneumoniae is shown in SEQ ID No. 12; The nucleotide sequence of the forward primer for varicella-zoster virus is shown in SEQ ID No. 13, and the nucleotide sequence of the reverse primer for varicella-zoster virus is shown in SEQ ID No. 14; the nucleotide sequence of the forward primer for mumps virus is shown in SEQ ID No. 15, and the nucleotide sequence of the reverse primer for mumps virus is shown in SEQ ID No. 16; The nucleotide sequence of the forward primer for cytomegalovirus is shown in SEQ ID No. 17, and the nucleotide sequence of the reverse primer for cytomegalovirus is shown in SEQ ID No. 18; The nucleotide sequence of the forward primer for herpes simplex virus type 1 is shown in SEQ ID No. 19, and the nucleotide sequence of the reverse primer for herpes simplex virus type 1 is shown in SEQ ID No. 20; The nucleotide sequence of the forward primer for herpes simplex virus type 2 is shown in SEQ ID No. 21, and the nucleotide sequence of the reverse primer for herpes simplex virus type 2 is shown in SEQ ID No. 22; The nucleotide sequence of the forward primer for Neisseria meningitidis is shown in SEQ ID No. 23, and the nucleotide sequence of the reverse primer for Neisseria meningitidis is shown in SEQ ID No. 24; The nucleotide sequence of the forward primer for Epstein-Barr virus is shown in SEQ ID No. 25, and the nucleotide sequence of the reverse primer for Epstein-Barr virus is shown in SEQ ID No. 26; The nucleotide sequence of the forward primer for Streptococcus agalactiae is shown in SEQ ID No. 27, and the nucleotide sequence of the reverse primer for Streptococcus agalactiae is shown in SEQ ID No. 28; The nucleotide sequence of the forward primer for Haemophilus influenzae is shown in SEQ ID No. 29, and the nucleotide sequence of the reverse primer for Haemophilus influenzae is shown in SEQ ID No. 30; The nucleotide sequence of the forward primer for human herpesvirus type 6 is shown in SEQ ID No. 31, and the nucleotide sequence of the reverse primer for human herpesvirus type 6 is shown in SEQ ID No. 32; The nucleotide sequence of the forward primer for E. coli K1 is shown in SEQ ID No. 33, and the nucleotide sequence of the reverse primer for E. coli K1 is shown in SEQ ID No. 34; The nucleotide sequence of the forward primer for Japanese encephalitis virus is shown in SEQ ID No.39, and the nucleotide sequence of the reverse primer for Japanese encephalitis virus is shown in SEQ ID No.40; The multiple gene detection system for viral and bacterial infection of the central nervous system also includes forward and reverse primers for detecting human RNA internal reference, forward and reverse primers for detecting human DNA internal reference and forward and reverse primers for detecting system quality control internal reference; the nucleotide sequence of the forward primer for the human RNA internal reference is shown in SEQ ID No.1, and the nucleotide sequence of the reverse primer for the human RNA internal reference is shown in SEQ ID No.2; the nucleotide sequence of the forward primer for the human DNA internal reference is shown in SEQ ID No.35, and the nucleotide sequence of the reverse primer for the human DNA internal reference is shown in SEQ ID No.36; the nucleotide sequence of the forward primer for the system quality control internal reference is shown in SEQ ID No.37, and the nucleotide sequence of the reverse primer for the system quality control internal reference is shown in SEQ ID No.38; The final concentration of forward and reverse primers for human RNA internal reference, human DNA internal reference and system quality control internal reference in the detection system is 900nM; the final concentration of forward and reverse primers for enterovirus, mumps virus, herpes simplex virus type 1, Haemophilus influenzae and Escherichia coli K1 in the detection system is 2400nM; The final concentrations of forward and reverse primers for human double echovirus, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus pneumoniae, herpes simplex virus type 2, Neisseria meningitidis, Epstein-Barr virus, Streptococcus agalactiae and human herpes virus type 6 in the detection system were all 1800 nM; the final concentrations of forward and reverse primers for varicella zoster virus and cytomegalovirus in the detection system were all 1200 nM; the final concentrations of forward and reverse primers for Japanese encephalitis virus in the detection system were all 3600 nM; The multiplex gene detection system for viral and bacterial infection of the central nervous system also includes reverse transcription PCR buffer, reverse transcription PCR enzyme and dNTP. The components used in the system during the reaction are 3 μL of 5× reverse transcription PCR buffer, 0.6 μL of dNTP, 1 μL of reverse transcription PCR enzyme, 1.5 μL of primer mixture, 7.5 μL of template, and 1.4 μL of pure water.

2. The multiplex gene detection system for viral and bacterial infection of the central nervous system according to claim 1, characterized in that: All forward primers are provided with a fluorescent label, and the fluorescent label is CY5 or CY3 or FAM.

3. The multiplex gene detection system for viral and bacterial infection of the central nervous system according to claim 2, characterized in that: Ultrapure water and empty test tubes are also included.

4. A multiple gene test kit for viral and bacterial infection of the central nervous system comprising the detection system as claimed in claim 1.

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