CN116855634A - Nucleic acid detection kit for integrally and rapidly detecting central nervous system infectious disease pathogen - Google Patents

Nucleic acid detection kit for integrally and rapidly detecting central nervous system infectious disease pathogen Download PDF

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CN116855634A
CN116855634A CN202310613612.7A CN202310613612A CN116855634A CN 116855634 A CN116855634 A CN 116855634A CN 202310613612 A CN202310613612 A CN 202310613612A CN 116855634 A CN116855634 A CN 116855634A
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primer
probe
kit
upstream
downstream
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苗佳音
陈星宇
蔡晓沂
阳清伟
曾建琪
彭锋
陈丽文
张纯真
陈易谦
董佳萌
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Zhongshan Hospital Xiamen University
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Abstract

The invention discloses a primer and a probe for integrally and rapidly detecting central nervous system infectious pathogens, which comprise specific primers and probes for 8 pathogens, and are shown as SEQ ID NO. 1-30. The invention also discloses a nucleic acid detection kit for integrally and rapidly detecting central nervous system infectious pathogens, which comprises the specific primers and probes for 8 pathogens. The invention carries out bioinformatics analysis through respective genomes of 8 bacterial pathogens, screens out respective conserved gene sequences, designs primer probes aiming at 8 pathogens, has high sensitivity, sensibility, specificity and precision, is suitable for three-stage hospitals or primary hospitals, can independently finish lumbar puncture operation, can be used for primary screening of clinical diseases suspected of central nervous system infection, and can rapidly guide treatment.

Description

Nucleic acid detection kit for integrally and rapidly detecting central nervous system infectious disease pathogen
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a nucleic acid detection kit for integrally and rapidly detecting central nervous system infectious disease pathogens.
Background
The nerve syphilis is known to be variable in symptoms and signs, so that the laboratory diagnosis is particularly important, but the most classical cerebrospinal fluid venereal disease research laboratory test (VDRL) is relatively low in sensitivity, and meanwhile, the problems of relatively complex operation and the like exist. Whereas treponema pallidum particle agglutination Test (TPPA) can only be used as an auxiliary diagnosis of neurosyphilis and cannot be used for definitive diagnosis. Central nervous system infections with which they are of differential diagnostic interest are among the most serious infectious diseases, including meningitis, encephalitis, and cerebrospinal meningitis. In recent years, the incidence of the diseases is on the rise, and the disability rate and the mortality rate are increased year by year. Pathogens causing central nervous system infection are many, including bacteria, fungi, viruses, tubercle bacillus, treponema pallidum, and the like. The bacterial meningitis is the most dangerous, but the positive rate of the bacterial culture of the cerebrospinal fluid is obviously reduced due to the unreasonable use of antibiotics, and the conventional cerebrospinal fluid culture usually needs at least 7-10 days, so that the early diagnosis and recognition of the bacterial meningitis are seriously affected. The American society for Infectious Diseases (IDSA) indicates that the use of sensitive antibiotics as early as possible reduces unnecessary examinations, effectively improves patient survival and shortens hospitalization time, and is of exceptional prognostic significance. Bacterial infections are similar to early symptoms and signs of intracranial infections caused by viruses, tubercles and other pathogens, and traditional laboratory detection indexes such as cerebrospinal fluid white blood cell count (white blood cell count, WBC), total Protein (TP), glucose (Glu), procalcitonin (PCT) and the like are difficult to accurately identify pathogens causing intracranial infections, although cerebrospinal fluid bacterial culture has been regarded as a gold standard for diagnosing bacterial intracranial infections, often resulting in difficult diagnosis and even misdiagnosis due to long time consumption and low positive rate. The clinical manifestation of the novel cryptococcus meningitis and the tubercular meningitis are very similar to the biochemical manifestation of cerebrospinal fluid, the positive rate of the novel cryptococcus and the tubercular mycobacterium in the cerebrospinal fluid is low (< 10%), and the sensitivity of the novel cryptococcus antigen detection, the ink staining and the cerebrospinal fluid to find the acid-fast bacillus is poor, so that the early clinical misdiagnosis rate is up to more than 60%, and therefore, the differential diagnosis of the novel cryptococcus meningitis and the tubercular mycobacterium is always a difficult problem in the intracranial infection field.
In summary, the clinical manifestations of various central nervous system infection diseases are similar, and diagnosis of the diseases often requires searching and even culturing cerebrospinal fluid pathogens, and the methods have low positive rate, long bacterial and fungal culture period, are unfavorable for early diagnosis, lead to delayed illness and cannot be used for rapid diagnosis of infection.
In recent years, along with the development of accurate medical treatment, the cerebrospinal fluid gene detection technology has the advantages of less detection time consumption, high sensitivity, no interference by antibacterial or bacteriostatic substances in a sample and the like, but has the defects of complex primer design, high experimental condition requirement, high reagent price, high false positive rate after pollution and the like, and particularly has low detection rate on mycobacterium tuberculosis and fungi, is easy to cause false negative, and cannot be widely applied to clinic at present.
Thus, there is an urgent need to find a practical laboratory method to rapidly diagnose central nervous system infections.
Disclosure of Invention
Aiming at the technical problems, the invention provides a primer, a probe and a nucleic acid detection kit for integrally and rapidly detecting central nervous system infectious disease pathogens. The invention selects 8 most common pathogens of central nervous system infection for detection, and can accurately identify each pathogen with micro infection.
The invention is realized by the following technical scheme:
a primer and probe for integrally and rapidly detecting central nervous system infectious disease pathogens comprises specific primers and probes for 8 pathogens, wherein the specific sequences of the primers and probes are as follows:
(1) Herpes simplex virus type 1:
an upstream primer: 5'-GAGACGCTGATGAAGCGCGAACTGA-3';
a downstream primer: 5'-ATAGTGCCACGCCCACCACGTTCGAGCT-3';
and (3) probe: FAM-5'-TGACGAGGCCGCAGCTCACCAAG-3' -TAMRA;
(2) Herpes simplex virus type 2:
an upstream primer: 5'-CCCAAGCTCCCGCTA AGG-3';
a downstream primer: 5'-CGCCTTGGCAGCA CAACT-3';
and (3) probe: FAM-5'-CCTGCTCT AGATATCCT-3' -TAMRA;
(3) EB virus:
an upstream primer: 5'-GCCATTTTTCCACCCTGTAG-3'
A downstream primer: 5-ACCATCTGGGCCACCTT-3'
And (3) probe: fam-5'-TGCCGATTATTTTGAATACCTCCAA-3' -eclipse
(4) Varicella zoster virus:
an upstream primer: 5'-CGA ACA CGT TCC CCA TCA A-3'
A downstream primer: 5'-CCC GGC TTT GTT AGT TTT GG-3'
And (3) probe: FAM-5'-TCC AGG TTT TAG TTG ATA CCA-3' -BkFQ
(5) Staphylococcus aureus:
nuc F-upstream: 5'-CACCTGAAACAAAGCATCCTAAA-3'
nuc F-downstream: 5'-CGCTAAGCCACGTCCATATT-3'
And (3) probe: texas-Red-5'-TGGTCCTGAAGCAAGTGCATTTACGA-3' -BHQ1
Upstream of mecA: 5'-CCC AAT TTT GAT CCA TTT GTT-3'
Downstream of mecA: 5'-GGC CAA TAC AGG AAC AGC ATA-3'
And (3) probe: mecA P HEX-5'-CAT TCT TTG GGA CGA TGC CTA TCT CA-3' -BHQ1
(6) Novel cryptococcus:
cryptococcus cyt b-upstream: 5'-TTCTAGCAGCTCTAGCTCTAG-3'
Cryptococcus cyt b-downstream: 5'-GCATTTGAGCTAATACCTTCAGG-3'
And (3) probe: FAM-5'-TACATATGCTAACACTTCACACACA-3' -BHQ1
(7) Treponema pallidum:
upstream of TP-F1: 5'-GAGGTATTGGGCGAAAAGGTT-3'
Downstream of TP-R1: 5'-CGCTTGGGTCAGTCTCGTACTC-3'
And (3) probe: FAM-5'-AAGCAGGAGACCGAAGACAGC-3' -eclipse
(8) Mycobacterium tuberculosis:
primer 1-upstream: 5'-CCGAAGAATCCGCTGAGCT-3'
Primer 1-downstream: 5'-AGAAAGCCGACGCGGTCT-3'
Probe 1: FAM-5'-CAGCGCACAACGCCGAATTGCGAAGGCGCTG-3' -BHQ primer 2-upstream: 5'-ATGCACTAGCCGAGACGATCA-3'
Primer 2-downstream: 5'-GCCAACTCGACATCCTCGA-3'
Probe 2:
FAM-5′-CAGCGCGAGCTGATCAAACCCGGCAAGCGCTG-3′-BHQ
the nucleotide sequences of the primer and the probe are shown as SEQ ID NO. 1-30.
A nucleic acid detection kit for integrally and rapidly detecting central nervous system infectious disease pathogens comprises the primer and the probe.
Further, reagents for nucleic acid detection are also included.
Further, the reagents for nucleic acid detection include buffer mixture, hot start taq enzyme, UNG enzyme, dUTPS.
Compared with the prior art, the invention has the following advantages:
1. the advanced DNA extraction method can extract the DNA component of pathogen in the clinic collected sample to the maximum extent, greatly improves the DNA extraction effect and obviously improves the detection sensitivity of the product.
2. The key technology of the product is that the design of the molecular beacon fluorescent probe and the specific primer is realized by adopting methods such as software assistance, experimental screening and the like, meanwhile, the probe and the primer sequences are determined by considering various factors such as the mutual influence of 8 pathogenic microorganism conserved DNA sequences, the probe and the primer sequences are the specific sequences of target strains, and experiments prove that various parameters (length, annealing temperature and delta G, tm) of the probe and the primer are very suitable.
3. Has high sensitivity, specificity and precision, and the sensitivity can reach 5.0X10 when the subject group is used for preparing and accurately quantifying the reference substance for detection 2 The copies/mL is negative in all detection, the yin-yang coincidence rate of the sensitive reference is 100%, and the CV value of the precision test is less than 10%.
4. Pollution prevention: unlike electrophoresis method, the kit has no need of post-treatment of PCR, complete closed-tube amplification and detection, and adopts dUTP-UNG system to prevent PCR amplified product from being polluted effectively, avoid false positive result and raise the reliability of clinical diagnosis basis.
5. The operation is simple and the time consumption is short. The kit performs amplification and detection on a specific fluorescent PCR detector, does not need electrophoresis or hybridization operation, can complete the PCR process within 1.5-2 hours, and is beneficial to improving the reporting speed of experimental results.
6. The result is objective and reliable, no artificial judgment is needed, and the instrument automatically collects and analyzes the data.
7. The kit can rapidly distinguish several common pathogens at one time, wins precious time for clinical diagnosis and treatment, is suitable for three-stage hospitals or primary hospitals to independently complete lumbar puncture, and can rapidly guide treatment and primary screening of clinical diseases suspected of central nervous system infection.
Drawings
FIG. 1 is a schematic diagram of a double-ring probe and its reaction principle.
Fig. 2 is a general technical scheme adopted by the invention.
Fig. 3 is a flow chart of a patient visit to a doctor for a suspected central nervous system infection.
Fig. 4 is a flow chart of a patient visit to a doctor for a suspected central nervous system infection.
FIG. 5 is a fluorescent PCR experimental route.
Detailed Description
For a better understanding of the present invention, reference will now be made in detail to the following examples and accompanying drawings, which are included to provide a further understanding of the invention, and it is to be understood by those skilled in the art that the following examples are not intended to limit the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Main raw material source and preparation method
The main raw materials in the kit are ordered from internationally known molecular biology reagent company, so that the kit is reliable in source, safe and stable in quality, and can be used as a stable raw material source (as follows) of the kit.
Chemical raw material
Tris: analytically pure, sigma company products or qualified suppliers, 99.7% infrared qualified, pH (5% water) 10.3-10.9, water content 0.3%, melting point 167-171 ℃, qualified absorption system, and highest impurity content.
MgCl 2 : analytically pure, sigma products or products from suppliers with qualified quality, the content is not less than 99%, the aqueous solution is qualified in reaction, and the highest content of impurities is qualified. (MgCl) 2 Is easy to absorb moisture, and is stored under a dryer after a new bottle is started).
KCl: analytically pure, beijing chemical reagent company products or qualified suppliers' products
EDTA: the analytical purity, sigma company or qualified supplier is white crystalline powder, which is dissolved in water, the solution is acidic and insoluble in alcohol, the content is not less than 99.5%, the aqueous solution is qualified in reaction, the complexing force test is qualified, and the highest content of impurities is qualified.
HCl: analytically pure, beijing chemical reagent plant product or a qualified vendor product.
Purified water: the Lebai barreled purified water was purchased and then treated by a Milli-Q Biocel water purifier from Millipore company to have a resistivity of 16 to 18M omega.
Primer and probe
Primers and probes applied in the development and production processes of the product are synthesized by Sigma and Biosearch company, and are qualified through quality inspection (including mass spectrum identification) of Sigma; and (5) detecting the product according to the corresponding quality standard of the company, and using the product after the product is qualified.
dNTPs
dATP, dCTP, dGTP, dUTP is purchased from Shanghai Hongyao or Shanghai Bioengineering, or other qualified suppliers, and is qualified by factory inspection; and (5) detecting the product according to the corresponding quality standard of the company, and using the product after the product is qualified.
Taq enzyme
In the process of development and production, the applied Taq enzyme is purchased from Dalian TaKaRa company or other qualified suppliers, and is used after being detected to be qualified according to the corresponding quality standard of the company.
Basic reagent preparation
10 Xconcentrated cleaning solution A
Preparing 0.2N NaOH and split charging 5 mL/pipe;
10X concentrated cleaning solution B
Preparing 10 xTE buffer (pH 8.0), and sub-packaging with 10 mL/tube;
DNA extracting solution
DNA extract was prepared at Triton X-100% concentration, NP-401% concentration and n-octanoic acid 0.04M concentration, and was packaged in 5 mL/tube after thoroughly mixing.
Extracting solid material
Two glass beads with the diameters of 0.5mm and 1.0mm are taken, and the weight ratio is about: the extracted solids were prepared in a ratio of 0.5mm:1.0 mm=9:1, and were dispensed at about 0.15g per tube.
Preparation of dNTPS
Name of the name dATP dCTP dGTP dUTP Ultrapure water
Volume of 100μL 100μL 100μL 150μL 550μL
The required reagents were removed from the refrigerator/cabinet and equilibrated to room temperature. All the reagents are added according to the proportion and then are mixed uniformly by a magnetic stirrer.
Dilution of primers and probes
The synthesized primers and probes were removed and centrifuged at 8000r/min for 3min in a high-speed bench refrigerated centrifuge. And taking out the primer and the probe to be detected, calculating and adding ultrapure water to dilute the primer and the probe to 100 mu M, uniformly mixing on a vortex mixer, taking out, and centrifuging in a palm type centrifuge.
Preparation method and traceability condition of quality control product:
internal reference
The composition is 10 3 cobies/mL plasmid containing internal reference gene; the high concentration plasmid containing the internal reference gene is precisely quantified by a spectrophotometer and diluted to 10 times by 10 times gradient with 1 xTE 3 The copies/mL was used as an internal reference, and 1 mL/tube was dispensed.
Positive control
The composition is 10 6 copies/mL～10 7 The copies/mL contains the plasmid of the target fragment; the high concentration plasmid containing the target fragment is precisely quantified by an ultraviolet spectrophotometer and is diluted to 10 times by 10 times of gradient with 1 xTE prepared in advance 6 copies/mL～10 7 The copies/mL was used as a positive control and 1 mL/tube was dispensed.
Negative control
The composition was 1 Xwashing liquid B, and the prepared 10 Xwashing liquid B was diluted 10 times with purified water to prepare 1 Xwashing liquid B as a negative control, and was dispensed 1 mL/tube.
Example 1 primer, probe, nucleic acid detection kit
According to the literature and clinical experience, four pathogenic viruses with higher detection positive rate in cerebrospinal fluid are herpes simplex virus type I (HSV-1), herpes simplex virus type II (HSV-2), EB virus and varicella zoster virus. Therefore, the rapid detection kit for central nervous system infection designed by researchers mainly detects 8 pathogens including HSV-1, HSV-2, EB virus, varicella zoster virus, bacteria, fungi, treponema pallidum and mycobacterium tuberculosis. The 8 pathogens are detected by adopting a real-time fluorescence PCR technology.
1. Implementation of the test method, key technology and taking action a) real-time fluorescence PCR technology
The kit adopts a real-time fluorescence quantitative PCR technology, namely a method for adding a fluorescent group into a PCR reaction system, monitoring the whole PCR process in real time by utilizing accumulation of fluorescent signals and finally quantitatively analyzing a template through a standard curve. With the advent of fluorescent PCR technology and related PCR apparatus, the previous method of quantifying genes using the terminal method was thoroughly changed. The real-time PCR quantification technique has the following advantages over the traditional method:
a. the experimental time is shortened, electrophoresis gel running identification after PCR is not needed, and quantitative work is completed by a PCR instrument;
b. because the data acquisition and analysis are completed by the instrument and the result of electrophoresis is not observed by naked eyes, the sensitivity and repeatability of detection are greatly improved;
c. since the cover is not required to be opened after PCR, the occurrence of contamination can be prevented very well.
The test procedure included two parts: preparing a sample to be detected and detecting by PCR.
b) Taqman probe mechanism
We refer to the corresponding pathogenic cryptic plasmid as target sequence, design primer, use nucleic acid amplification technology to proceed specific geneAnd (3) performing row detection, designing Taqman probes and specific primers required by real-time PCR, and adopting a schematic diagram as shown in figure 1. Specific primers are respectively designed aiming at the corresponding virus conserved sequences, the sample to be detected is detected, and only the sample with the corresponding pathogenic DNA can be smoothly amplified to obtain a double-stranded DNA product, the double-stranded DNA product can be combined with the Taqman probe to emit a fluorescent signal so as to be detected, so that whether the sample contains the corresponding pathogenic DNA can be accurately indicated. The content of the kit in the sample is as low as 5 multiplied by 10 2 The nucleic acid DNA of the gene copy/mL can be detected. The nucleic acid amplification method has high sensitivity, strong specificity and good repeatability.
In the aspect of product analysis, the Taqman probe real-time tracking analysis technology is adopted, so that the detection method is easy to automate. The probe is a fluorescent-labeled oligonucleotide probe whose fluorescent properties are related to the amplification of the sequence of interest. It is designed to pair with the sequence between the upstream primer and the downstream primer of the target sequence. The fluorescent group is connected to the 5 '-end of the probe and is called a reporter group, and the quencher with the absorption spectrum coincident with the emission spectrum of the reporter group is marked on the 3' -end of the probe and becomes a quencher group. In the free state of the probe, fluorescence excited by the fluorescent group is absorbed by the quenching group through resonance energy transfer, which is represented by quenching of fluorescence. However, in the process of nucleic acid amplification, during the extension reaction of the amplified product, the amplified product is combined with the probe, so that the Taqman probe is subjected to enzyme digestion degradation by the 5'-3' exonuclease activity of Taq enzyme, and the fluorescent emission group and the fluorescent quenching group are separated, so that a fluorescent signal can be received by a fluorescent monitoring system, namely, one fluorescent molecule is formed every time one DNA chain is amplified, and the accumulation of the fluorescent signal and the formation of a PCR product are completely synchronous. Studies have shown that the Ct value of each template has a linear relationship with the logarithm of the starting copy number of the template, the more the starting copy number, the smaller the Ct value.
c) Principle of internal reference
The kit is provided with an internal reference gene, namely an Arabidopsis gene. It has no homologous genes in both the pathogenic genome and the human genome, and thus can be used as an internal reference to detect whether PCR inhibitors are present in each PCR reaction, thereby ensuring the credibility of the PCR results. When the internal reference result is positive, the PCR reaction system is indicated and the operation is normal; therefore, when the target gene is negative, it is important that the internal reference result is positive. However, when the result of the target gene is positive, the amplification curve of the internal reference is delayed from that of the target gene, or when the result of the internal reference is negative, it is normal. However, when both the target gene and the internal reference gene were negative, the test was considered ineffective and repeated.
d) Principle of positive and negative control
Positive controls were run simultaneously for each test. Positive control results are positive, which indicates that the detection system for the target gene is normal; and when the result is negative, the experiment is invalid and needs to be repeated.
To demonstrate the presence of contamination, negative controls were also run simultaneously for each test. The negative control result is negative, which indicates that the test has no pollution; if the result is positive, the experiment is invalid and needs to be repeated.
The whole technical route of the invention is shown in fig. 2, fig. 3 and fig. 4 are respectively a flow chart of treatment of a patient suspected of central nervous system infection, and a fluorescent PCR experimental route is shown in fig. 5.
Example 2 test of Performance of the kit
1. Detection sensitivity: the lower limit of detection of the positive quality control product detected by the kit is verified
The detection sensitivity of 8 pathogens of HSV-1, HSV-2, EB virus, varicella zoster virus, staphylococcus aureus, cryptococcus, treponema pallidum and mycobacterium tuberculosis are shown in tables 1 to 16.
Table 1 determination result table of sensitivity of kit for detecting HSV-1 (ABI 7500)
Table 2 determination result table of sensitivity of the kit for detecting HSV-1 (STRATAGENE Mx3000 p)
Table 1, table 2 shows that on the ABI7500 fluorescence PCR instrument and STRATAGENE Mx3000p fluorescence PCR instrument, 5.0X10 2 Each batch of kit can be detected by copies/mL samples, 5.0X10 1 The samples of copies/mL, each batch of reagent shows different instruments, and the total detection rate is less than 100%, so we set the minimum detection amount of the kit HSV-1 to be 5.0X10 2 copies/mL。
Table 3 determination result table of sensitivity of kit for detecting HSV-2 (ABI 7500)
Table 4 determination result table of sensitivity of kit for detecting HSV-2 (STRATAGENE Mx3000 p)
Table 3, table 4 shows that on the ABI7500 fluorescent PCR apparatus and STRATAGENE Mx3000p fluorescent PCR apparatus, 5.0X10 2 Each batch of kit can be detected by copies/mL samples, 5.0X10 1 The samples of copies/mL, each batch of reagent shows different instruments, and the total detection rate is less than 100%, so we set the minimum detection amount of HSV-2 of the kit to be 5.0X10 2 copies/mL。
TABLE 5 determination of sensitivity of the kit to detect Staphylococcus aureus (ABI 7500)
Table 6 determination of sensitivity of the kit to Staphylococcus aureus (STRATAGENE Mx3000 p)
Table 5, table 6 shows that, on an ABI7500 fluorescent PCR apparatus and a STRATAGENE Mx3000p fluorescent PCR apparatus, 10 3 Each batch of kit can be detected by the copies/mL sample, 10 2 The samples with copies/mL, each batch of reagents is different on different instruments, and the total detection rate is less than 50%, so that the minimum detection amount of the kit is 10 3 copies/mL。
TABLE 7 minimum detection of Mycobacterium tuberculosis by kit (ABI 7500)
Table 8 test results of the test for the lowest detection amount of Mycobacterium tuberculosis (STRATAGENE Mx3000 p)
Table 7 and Table 8 show that on an ABI7500 fluorescent PCR apparatus and a STRATAGENE Mx3000p fluorescent PCR apparatus, 10 1 Each batch of the kit can detect 100 bacteria/ml samples, each batch of the reagents are different in different instruments, and the total detection rate is 50%, so that the minimum detection amount of the kit is 10 1 Bacteria/ml.
Table 9 shows the results of the detection of cryptococcus sensitivity by the kit (ABI 7500)
Table 10 determination result table of sensitivity of Cryptococcus detection by kit (STRATAGENE Mx3000 p)
Table 9, table 10 test results show, in ABI7500 fluorescent PCR instrument and STRATAGENE Mx3000p fluorescent PCR instrument, 5.0X10 2 Each batch of kit can be detected by copies/mL samples, 5.0X10 1 The samples of the copies/mL, each batch of reagents are different on different instruments, and the total detection rate is less than 100%, so that the minimum detection amount of the cryptococcus in the kit is 5.0X10 2 copies/mL。
Table 11 determination result table of sensitivity of kit for detecting treponema pallidum (ABI 7500)
Table 12 determination result table of sensitivity of kit for detecting treponema pallidum (STRATAGENE Mx3000 p)
Table 11, table 12 shows that on the ABI7500 fluorescent PCR apparatus and STRATAGENE Mx3000p fluorescent PCR apparatus, 5.0X10 2 Each batch of kit can be detected by copies/mL samples, 5.0X10 1 The samples of copies/mL, each batch of reagent shows different instruments, and the total detection rate is less than 100%, so we set the minimum detection amount of treponema pallidum in the kit to be 5.0X10 2 copies/mL。
Table 13 determination of sensitivity of the kit to Staphylococcus aureus (ABI 7500)
Table 14 determination of sensitivity of the kit to Staphylococcus aureus (STRATAGENE Mx3000 p)
Table 13, table 14 shows that on an ABI7500 fluorescent PCR apparatus and a STRATAGENE Mx3000p fluorescent PCR apparatus, 10 3 Each batch of kit can be detected by the copies/mL sample, 10 2 The samples with copies/mL, each batch of reagents is different on different instruments, and the total detection rate is less than 50%, so that the minimum detection amount of the kit is 10 3 copies/mL。
Table 15 test kit Mycobacterium tuberculosis minimum detection measurement results (ABI 7500)
Table 16 test results of the test for the lowest detection amount of Mycobacterium tuberculosis (STRATAGENE Mx3000 p)
Table 15, table 16 show that on the ABI7500 fluorescence PCR instrument and STRATAGENE Mx3000p fluorescence PCR instrument, 10 1 Each batch of the kit can detect 100 bacteria/ml samples, each batch of the reagents are different in different instruments, and the total detection rate is 50%, so that the minimum detection amount of the kit is 10 1 Bacteria/ml.
2. Detection specificity: and verifying whether the kit only amplifies pathogens related to the kit and has no cross reaction with other pathogens.
The detection specificity of 8 pathogens of HSV-1, HSV-2, EB virus, varicella zoster virus, staphylococcus aureus, cryptococcus, treponema pallidum and mycobacterium tuberculosis are shown in tables 17 to 32.
Table 17 determination results of the kit for detecting HSV-1 specificity (ABI 7500)
Table 18 determination of HSV-1 specificity by kit (STRATAGENE Mx3000 p)
According to the test results in Table 17 and Table 18, all negative reference products of three batches of reagents for detecting HSV-1 are negative, which shows that the reagents have good specificity.
Table 19 determination of HSV-2 specificity by kit (ABI 7500)
Table 20 determination results of the kit for detecting HSV-2 specificity (STRATAGENE Mx3000 p)
According to the test results in Table 19 and Table 20, all negative references of three batches of reagents for detecting HSV-2 were negative, indicating that the reagents have good specificity and do not cross react with other pathogens.
Table 21 determination of the specificity of the kit for Staphylococcus aureus (ABI 7500)
Table 22 determination of the specificity of the kit for Staphylococcus aureus (STRATAGENE Mx3000 p)
According to the test results in Table 21 and Table 22, all negative reference samples of three batches of reagents for detecting Staphylococcus aureus were negative, indicating that the reagents have good specificity and do not cross react with other pathogens.
Table 23 detection results (ABI 7500) of the coincidence rate of the Mycobacterium tuberculosis negative reference in the kit
Table 24 test results (STRATAGENE Mx3000 p) of the coincidence rate of the Mycobacterium tuberculosis negative reference in the kit
According to the experimental results of tables 23 and 24, the results of three batches of reagents for detecting 16 negative reference products of bacillus tuberculosis in Chinese medicine biological products are all negative, which shows that the reagents have good specificity.
Table 25 determination of Cryptococcus specificity by kit (ABI 7500)
Table 26 determination of Cryptococcus specificity by kit (STRATAGENE Mx3000 p)
According to the test results in Table 25 and Table 26, all negative reference samples of the three batches of reagents for detecting Cryptococcus saphenous are negative, which shows that the reagents have good specificity and do not cross react with other pathogens.
Table 27 determination results of the kit for detecting treponema pallidum specificity (ABI 7500)
Table 28 determination results of the kit for detecting treponema pallidum specificity (STRATAGENE Mx3000 p)
According to the test results in Table 27 and Table 28, all negative reference samples of the treponema pallidum detected by the three batches of reagents were all negative, indicating that the reagents had good specificity and did not cross react with other pathogens.
Table 29 determination of the specificity of Staphylococcus aureus (ABI 7500)
Table 30 determination of the specificity of the kit for Staphylococcus aureus (STRATAGENE Mx3000 p)
According to the test results in Table 29 and Table 30, all negative reference samples of three batches of reagents for detecting Staphylococcus aureus were negative, indicating that the reagents had good specificity and did not cross react with other pathogens.
Table 31 detection results (ABI 7500) of the coincidence rate of the Mycobacterium tuberculosis negative reference in the kit
Table 32 test kit Mycobacterium tuberculosis negative reference coincidence rate determination result (STRATAGENE Mx3000 p)
According to the experimental results of tables 31 and 32, the results of three batches of reagents for detecting 16 negative reference products of bacillus tuberculosis in Chinese medicine biological products are all negative, which shows that the reagents have good specificity.
3. Detection accuracy: clinical specimens are used as detection objects to verify the accuracy of the kit for repeated detection of the clinical specimens, and the kit has good repeatability and anti-interference capability.
The results of the detection accuracy of 8 pathogens of HSV-1, HSV-2, EB virus, varicella zoster virus, staphylococcus aureus, cryptococcus, treponema pallidum and Mycobacterium tuberculosis are shown in tables 33 to 48.
Table 33 determination result of HSV-1 accuracy of kit (ABI 7500)
Table 34 determination result of HSV-1 accuracy test (STRATAGENE Mx3000 p)
According to the test results of Table 33 and Table 34, all positive accuracy references of three batches of reagent detection HSV-1 are positive, and all negative accuracy references are negative, so that the kit has good accuracy for clinical sample detection.
Table 35 determination result of HSV-2 accuracy (ABI 7500)
Table 36 determination of HSV-2 accuracy (STRATAGENE Mx3000 p)
According to the test results of the table 35 and the table 36, all positive accuracy references of three batches of reagents for detecting HSV-2 are positive, and all negative accuracy references are negative, so that the kit has good accuracy for detecting clinical samples.
Table 37 determination of accuracy of staphylococcus aureus (ABI 7500)
Table 38 determination of the accuracy of the test kit (STRATAGENE Mx3000 p)
According to the test results in Table 37 and Table 38, all positive accuracy references of three batches of reagents for detecting staphylococcus aureus are positive, and all negative accuracy references are negative, so that the reagent has good accuracy.
Table 39 reagent kit Mycobacterium tuberculosis positive reference coincidence rate determination result (ABI 7500)
Table 40 reagent kit Mycobacterium tuberculosis positive reference coincidence rate determination result (STRATAGENE Mx3000 p)
According to the results of tables 39 and 40, the results of three batches of kits for detecting 14 positive reference products of tubercle bacillus in Chinese medicine biological products detection are all positive, which shows that the kit has good sensitivity.
Table 41 determination of accuracy of Cryptococcus test kit (ABI 7500)
Table 42 determination of accuracy of Cryptococcus detection kit (STRATAGENE Mx3000 p)
According to the test results of table 41 and table 42, all positive accuracy references of the three batches of the reagents for detecting cryptococcus are positive, and all negative accuracy references are negative, so that the kit has good accuracy for detecting clinical samples.
Table 43 determination result of the accuracy of the reagent kit for detecting treponema pallidum (ABI 7500)
Table 44 determination result of the accuracy of the reagent kit for detecting treponema pallidum (STRATAGENE Mx3000 p)
According to the test results of table 43 and table 44, all positive accuracy references of three batches of reagent detection treponema pallidum are positive, and all negative accuracy references are negative, so that the kit has good accuracy for clinical sample detection.
Table 45 determination of accuracy of staphylococcus aureus (ABI 7500)
Table 46 determination of the accuracy of the test kit (STRATAGENE Mx3000 p)
According to the test results in Table 45 and Table 46, all positive accuracy references of three batches of reagents for detecting staphylococcus aureus are positive, and all negative accuracy references are negative, so that the reagent has good accuracy.
Table 47 reagent kit Mycobacterium tuberculosis positive reference coincidence rate determination result (ABI 7500)
Table 48 reagent kit Mycobacterium tuberculosis positive reference coincidence rate determination result (STRATAGENE Mx3000 p)
According to the results of tables 47 and 48, the results of three batches of kits for detecting 14 positive reference products of tubercle bacillus in Chinese medicine biological products detection are all positive, which shows that the kit has good sensitivity.
4. Detection precision: verifying the accuracy of the kit detection
The results of the detection precision of 8 pathogens of HSV-1, HSV-2, EB virus, varicella zoster virus, bacteria, fungi, treponema pallidum and Mycobacterium tuberculosis are shown in tables 49 to 64.
Table 49 results of 5 times repeated measurement of HSV-1 with three lot number reagents (ABI 7500)
Table 50 results of 5 times repeated measurement of HSV-1 with three lot number reagents (STRATAGENE Mx3000 p)
Table 49 and Table 50 show that three batches of kits for detecting HSV-1 have good precision, and CV values are less than 10%.
Table 51 results of 5 replicates of HSV-2 assays (ABI 7500) on samples with three lot reagents
Table 52 results of 5 repeated determinations of HSV-2 (STRATAGENE Mx3000 p) for three lots of reagents for the sample
Table 51 and Table 52 show that three batches of kits for detecting HSV-2 have good precision, and CV values are less than 10%.
Table 53-1: for 1.0X10 6 Table of test results for samples positive for staphylococci (ABI 7500)
Table 53-2 vs. 1.0X10 4 Table of test results for samples positive for staphylococci (ABI 7500)
Table 54-1 vs. 1.0X10 6 Copies/mL staphylococcus aureus positive sample determination result table (STRATAGENE Mx3000 p)
Table 54-2 vs. 1.0X10 4 Copies/mL staphylococcus aureus positive sample determination result table (STRATAGENE Mx3000 p)
Table 53 and Table 54 show that the three kits have good precision in detecting staphylococcus aureus, and CV values are less than 10%.
Table 55 10 3 The results of 10 tube assays (ABI 7500) were repeated for individual bacteria/ml sensitivity standards
Table 55 shows that the three kits have good accuracy in detecting the mycobacterium tuberculosis.
Table 56 results of 5 repeated measurements of herpes zoster virus on samples with three lots of reagents (STRATAGENE Mx3000 p)
Table 55 and Table 56 show that the three kits have good accuracy in detecting the herpes zoster virus, and CV value is less than 10%.
Table 57 results of 5 replicates of Cryptococcus for samples with three lots of reagents (ABI 7500)
Table 58 results of 5 replicates of Cryptococcus for samples with three lots of reagents (STRATAGENE Mx3000 p)
Table 57 and Table 58 show that the three kits detect cryptococcus with good precision and CV less than 10%.
Table 59 results of repeated 5 times determination of treponema pallidum on samples with three lot number reagents (ABI 7500)
Table 59 and Table 60 show that the three batches of the kit for detecting treponema pallidum have good precision, and CV value is less than 10%.
Table 61-1: for 1.0X10 6 Table of test results for samples positive for staphylococci (ABI 7500)
Table 61-2 vs. 1.0X10 4 Table of test results for samples positive for staphylococci (ABI 7500)
Table 62-1 vs. 1.0X10 6 Copies/mL staphylococcus aureus positive sample determination result table (STRATAGENE Mx3000 p)
Table 62-2 vs. 1.0X10 4 Copies/mL staphylococcus aureus positive sample determination result table (STRATAGENE Mx3000 p)
Table 61 and Table 62 show that the three kits are accurate for detecting staphylococcus aureus, and the CV value is less than 10%.
Table 63 10 3 The results of 10 tube assays (ABI 7500) were repeated for individual bacteria/ml sensitivity standards
The results in Table 63 show that the three kits have good accuracy in detecting the Mycobacterium tuberculosis.
EXAMPLE 3 clinical examination
In this study, cerebrospinal fluid routine examination included: cerebrospinal fluid routine, biochemical, cytologic, bacterial culture, fungal culture, ink staining, cryptococcus antigen detection, and acid fast finding bacillus. A total of 421 patients perfected the above test, with a total of 281 patients diagnosed with intracranial infection or neurosyphilis by routine examination of cerebrospinal fluid, of which 98 viral meningitis or meningoecto, 130 neurosyphilis, 18 tuberculous meningitis, 20 suppurative meningitis, 15 cryptococcoid meningitis. The kit is used for checking 290 patients diagnosed with intracranial infection or neurosyphilis, wherein 101 patients with viral meningitis or meningoepitis, 132 patients with neurosyphilis, 19 patients with tuberculous meningitis, 21 patients with suppurative meningitis and 17 patients with cryptococcosis meningitis. The patient with positive detection and negative clinical routine detection of the kit is added with gene detection (Table one). The specificity of the kit is 100% by combining the gene detection result (Table II), the sensitivity is not inferior to the conventional cerebrospinal fluid examination (Table III), the time period is short, and the time period from collecting the specimen to obtaining the data is not more than 3 hours (Table IV).
The clinical common detection method is compared with the positive rate of the diagnosis of intracranial infection by the kit
Disease species Clinical common detection method/number of positive cases Positive rate of the kit P value
Viral meningitis or meningitis CSF conventional biochemical sum or Gene detection/98 101 p>0.05
Neurosyphilis CSF TPPA+CLIA/130 132 p>0.05
Tubercular meningitis CSF tubercle bacillus/18 19 p>0.05
Suppurative meningitis CSF bacterial culture/20 21 p>0.05
Cryptococcosis meningitis CSF ink staining+cryptococcus antigen+fungal culture/15 17 p>0.05
The kit is used for detecting the gene of a patient positive in detection and negative in clinical routine detection.
The specificity of the conventional clinical detection method for the surface two times is compared with that of the diagnosis of intracranial infection by the kit
The general clinical detection method of the exterior three is compared with the diagnosis of the sensitivity of intracranial infection by the kit
Disease species Clinical common detection method Sensitivity of the kit P value
Viral meningitis or meningitis CSF conventional biochemical and/or genetic assays 103.06％ p>0.05
Neurosyphilis CSF TPPA+CLIA 101.54％ p>0.05
Tubercular meningitis CSF tubercle bacillus 105.56％ p>0.05
Suppurative meningitis CSF bacterial culture 105.00％ p>0.05
Cryptococcosis meningitis CSF ink staining+cryptococcus antigen+fungus culture 113.33％ p>0.05
The kit is positive and patients with negative clinical routine detection are added for gene detection.
Time-limited comparison between the four clinical common detection methods and the diagnosis of intracranial infection by the kit
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not intended to limit the scope of the invention, and that equivalent modifications and variations of the invention in light of the spirit of the invention will be covered by the claims of the present invention.

Claims (4)

1. A primer and a probe for integrally and rapidly detecting central nervous system infectious disease pathogens are characterized in that: the kit comprises specific primers and probes for 8 pathogens, wherein the specific sequences of the primers and the probes are as follows:
(1) Herpes simplex virus type 1:
an upstream primer: 5'-GAGACGCTGATGAAGCGCGAACTGA-3';
a downstream primer: 5'-ATAGTGCCACGCCCACCACGTTCGAGCT-3';
and (3) probe: FAM-5'-TGACGAGGCCGCAGCTCACCAAG-3' -TAMRA;
(2) Herpes simplex virus type 2:
an upstream primer: 5'-CCCAAGCTCCCGCTA AGG-3';
a downstream primer: 5'-CGCCTTGGCAGCA CAACT-3';
and (3) probe: FAM-5'-CCTGCTCT AGATATCCT-3' -TAMRA;
(3) EB virus:
an upstream primer: 5'-GCCATTTTTCCACCCTGTAG-3'
A downstream primer: 5-ACCATCTGGGCCACCTT-3'
And (3) probe: fam-5'-TGCCGATTATTTTGAATACCTCCAA-3' -eclipse
(4) Varicella zoster virus:
an upstream primer: 5'-CGA ACA CGT TCC CCA TCA A-3'
A downstream primer: 5'-CCC GGC TTT GTT AGT TTT GG-3'
And (3) probe: FAM-5'-TCC AGG TTT TAG TTG ATA CCA-3' -BkFQ
(5) Staphylococcus aureus:
nuc F-upstream: 5'-CACCTGAAACAAAGCATCCTAAA-3'
nuc F-downstream: 5'-CGCTAAGCCACGTCCATATT-3'
And (3) probe: texas-Red-5'-TGGTCCTGAAGCAAGTGCATTTACGA-3' -BHQ1
Upstream of mecA: 5'-CCC AAT TTT GAT CCA TTT GTT-3'
Downstream of mecA: 5'-GGC CAA TAC AGG AAC AGC ATA-3'
And (3) probe: mecA P HEX-5'-CAT TCT TTG GGA CGA TGC CTA TCT CA-3' -BHQ1
(6) Novel cryptococcus:
cryptococcus cyt b-upstream: 5'-TTCTAGCAGCTCTAGCTCTAG-3'
Cryptococcus cyt b-downstream: 5'-GCATTTGAGCTAATACCTTCAGG-3'
And (3) probe: FAM-5'-TACATATGCTAACACTTCACACACA-3' -BHQ1
(7) Treponema pallidum:
upstream of TP-F1: 5'-GAGGTATTGGGCGAAAAGGTT-3'
Downstream of TP-R1: 5'-CGCTTGGGTCAGTCTCGTACTC-3'
And (3) probe: FAM-5'-AAGCAGGAGACCGAAGACAGC-3' -eclipse
(8) Mycobacterium tuberculosis:
primer 1-upstream: 5'-CCGAAGAATCCGCTGAGCT-3'
Primer 1-downstream: 5'-AGAAAGCCGACGCGGTCT-3'
Probe 1: FAM-5'-CAGCGCACAACGCCGAATTGCGAAGGCGCTG-3' -BHQ
Primer 2-upstream: 5'-ATGCACTAGCCGAGACGATCA-3'
Primer 2-downstream: 5'-GCCAACTCGACATCCTCGA-3'
Probe 2:
FAM-5′-CAGCGCGAGCTGATCAAACCCGGCAAGCGCTG-3′-BHQ 。
2. a nucleic acid detection kit for integrally and rapidly detecting central nervous system infectious disease pathogens is characterized in that: comprising the primer and probe of claim 1.
3. The nucleic acid detection kit of claim 2, wherein: reagents for nucleic acid detection are also included.
4. The nucleic acid detection kit according to claim 3, wherein: the reagent for detecting nucleic acid comprises buffer mixed solution, hot start taq enzyme, UNG enzyme and dUTPS.
CN202310613612.7A 2023-05-29 2023-05-29 Nucleic acid detection kit for integrally and rapidly detecting central nervous system infectious disease pathogen Pending CN116855634A (en)

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