CN111440859A - Biomarker for human respiratory syncytial virus infection and application - Google Patents
Biomarker for human respiratory syncytial virus infection and application Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
Abstract
The invention discloses a biomarker PF4 gene for evaluating the severity of RSV infection, an expression product thereof and application thereof. The corresponding primer group, the probe and the protein can be used for preparing a kit, and have excellent sensitivity and specificity when being used for evaluating the RSV infection severity degree from nasopharyngeal aspirate and plasma samples.
Description
Technical Field
The present invention relates to biomarkers of respiratory syncytial virus infection and their use and methods for assessing the severity of disease symptoms caused by the virus.
Background
Respiratory Syncytial Virus (RSV) belongs to the genus Pneumovirus of the subfamily Pneumovirinae of the family Paramyxoviridae and is an enveloped single-stranded negative-strand RNA Virus. RSV is transmitted via the respiratory tract and is the most important pathogen causing acute lower respiratory tract infections in young children1Serious lower respiratory tract diseases, including bronchiolitis and pneumonia, can result. Research showsShows that the RSV detection rate in the capillary bronchitis case can reach 43 percent and the RSV detection rate in the pneumonia case is 25 percent in infants treated in hospitalization due to respiratory diseases2。
Children are the major susceptible population to RSV, and by the age of 2 years more than 95% of children have been infected with RSV. RSV is more likely to cause lower respiratory tract infections than other common respiratory tract viruses, such as influenza and parainfluenza viruses3Acute lower respiratory tract infection is one of the leading causes of death of children worldwide4. One meta-analysis showed that children under 6.6-19.9 ten thousand 5 years of age died of RSV infection and associated complications worldwide in 2005, with 99% occurring in developing countries4. In addition, researches report that 23.4 ten thousand children died from RSV infection in 2010 worldwide under 5 years old and 25.35 ten thousand died in all age groups5. In addition to children, RSV also frequently infects the elderly and people with low immunity.
There are two main categories of current clinical diagnostic methods for RSV infection: virus antigen detection and virus nucleic acid detection. The detection method aiming at the virus antigen, such as the direct immunofluorescence technology, can detect the RSV antigen from a respiratory tract sample through a specific fluorescent antibody, has strong subjectivity, is easy to cause false positive, needs special equipment and professional technicians, wastes time and labor7. Enzyme immunoassay is also the most commonly used method for diagnosing viral antigens in laboratories, but the sensitivity and specificity vary widely. In addition, immunochromatography is favored for detection of RSV viral antigens because of its short reaction time, no need for special equipment and technicians, but lower sensitivity6. The detection method aiming at the RSV virus nucleic acid mainly comprises nucleic acid hybridization, polymerase chain reaction and reverse transcription polymerase chain reaction technology. The detection technology aiming at the RSV nucleic acid, particularly the polymerase chain reaction technology, has the advantages of rapidness, sensitivity and specificity, but also has the defects of easy pollution influence and high false positive rate.
The invention provides a biomarker related to RSV infection and a corresponding auxiliary diagnosis kit thereof, has the advantages of rapidity, specificity and sensitivity, can powerfully promote early diagnosis, predictive treatment and the like of RSV infection, and has important clinical application value.
Disclosure of Invention
The invention aims to provide a biomarker related to the pathogenic development of the respiratory syncytial virus infection, and the biomarker can be used for evaluating the severity of the respiratory syncytial virus infection.
According to one aspect of the present invention, the present application provides a novel biomarker for assessing the severity of respiratory syncytial virus infection, the Platelet Factor 4(PF4) gene and its expression products.
According to another aspect of the present invention, there is provided a kit for assessing the severity of respiratory syncytial virus infection, wherein the kit comprises a product for detecting the platelet factor 4(PF4) gene or a functional equivalent thereof or the expression level thereof.
In a preferred embodiment, the product for detecting the platelet factor 4(PF4) gene or functional equivalent thereof or expression level thereof is a probe that specifically recognizes the gene or a primer that specifically amplifies the gene.
According to another aspect of the present invention, there is provided a kit for assessing the severity of respiratory syncytial virus infection, wherein the kit comprises a product for detecting the PF4 protein or a functional equivalent thereof or the expression level thereof.
In a preferred embodiment, the product for detecting the PF4 protein or the functional equivalent thereof is a reagent for detecting the PF4 protein in human serum in the E L ISA method.
In a more preferred embodiment, the product for the detection of PF4 protein or a functional equivalent thereof is an antibody to PF4 protein in human serum.
According to another aspect of the present invention, there is provided the use of the PF4 gene or a functional equivalent thereof for the manufacture of a medicament or kit for assessing the severity of respiratory syncytial virus infection.
In a preferred embodiment, the agent is a product for detecting the PF4 gene or a functional equivalent thereof or the expression level thereof.
In a more preferred embodiment, the kit comprises a product for detecting the PF4 gene or a functional equivalent thereof or the expression level thereof.
According to another aspect of the present invention, the present application provides the use of PF4 protein or a functional equivalent thereof for the preparation of a medicament or kit for assessing the severity of respiratory syncytial virus infection.
In a preferred embodiment, the agent is a product for detecting the level of PF4 protein or a functional equivalent thereof or expression thereof.
In a more preferred embodiment, the kit comprises a product for the detection of the PF4 protein or a functional equivalent thereof.
According to another aspect of the present invention, the PF4 gene or PF4 protein or functional equivalent thereof is used as a detection target or standard in the above reagent or kit.
According to another aspect of the present invention, the present application provides a use of a product for detecting the PF4 gene or its functional equivalent or its expression level for the preparation of a medicament or kit for assessing the severity of respiratory syncytial virus infection.
In a preferred embodiment, the product is a probe that specifically recognizes the gene or a primer that specifically amplifies the gene.
According to another aspect of the present invention, a product for detecting the expression level of PF4 gene comprises: and (3) detecting the expression level of the PF4 gene by using the technologies such as RT-PCR, real-time fluorescence quantitative PCR, in-situ hybridization, a chip or a high-throughput sequencing platform and the like to evaluate the severity of the respiratory syncytial virus infection.
According to another aspect of the present invention, a product for detecting the expression level of PF4 gene comprises: a probe that specifically recognizes the gene or a primer that specifically amplifies the gene.
In a preferred embodiment, the probes or primers may be obtained based on the PF4 gene sequence using means conventional in the art.
According to another aspect of the present invention, the present application provides the use of a product for detecting the PF4 protein or its functional equivalent or its expression level for the preparation of a medicament or kit for assessing the severity of respiratory syncytial virus infection.
In a preferred embodiment, the product is a reagent for detecting PF4 protein in human serum by the ISA method of E L.
In a more preferred embodiment, the product is an antibody for the detection of PF4 protein in human serum.
According to another aspect of the present invention, there is provided a method for assessing the severity of respiratory syncytial virus infection, comprising detecting the level of PF4 gene, or a functional equivalent thereof, or expression thereof, in a sample from a subject, thereby determining the severity of infection by respiratory syncytial virus in the subject.
In another preferred embodiment, the detection is carried out using the above-mentioned product for detecting the PF4 gene or a functional equivalent thereof or the expression level thereof.
According to another aspect of the present invention, there is provided a method for assessing the severity of respiratory syncytial virus infection, comprising detecting the level of PF4 protein or a functional equivalent thereof or expression thereof in a sample from a subject, thereby determining the severity of respiratory syncytial virus infection in the subject.
In another preferred embodiment, the detection is performed using the product described above for detecting the level of PF4 protein or a functional equivalent thereof or expression thereof.
According to another aspect of the invention, the above-mentioned detection is carried out in a sample from the subject.
In a preferred embodiment, the sample is a bodily fluid of the subject.
In a more preferred embodiment, the sample is plasma and/or nasopharyngeal aspirate from the subject.
The invention has the advantages and beneficial effects that:
the invention discovers a new biomarker related to the severity of human respiratory syncytial virus infection for the first time, wherein the biomarker is PF4 gene and an expression product thereof; by detecting the expression level of the genes or the expression level of the proteins in the blood of the subject, the evaluation of the severity of the respiratory syncytial virus infection can be realized, so that the early diagnosis can be rapidly, specifically and sensitively carried out for intervention treatment.
Drawings
Figure 1 shows the PF4 levels in plasma of RSV infected, pneumonia infants infected with other respiratory viruses, and normal control children.
Figure 2 shows PF4 levels in nasopharyngeal aspirates from infants with RSV and other respiratory virus infections in pneumonia.
Figure 3 shows PF4 levels in nasopharyngeal aspirates of RSV infected children with pneumonia in different clinical scoring groups.
Description of sequence listing
SEQ ID NO. 1 shows the nucleotide sequence of the human platelet factor 4 gene;
SEQ ID NO 2 shows the amino acid sequence of the human platelet factor 4 protein.
Detailed Description
The invention provides a new and effective biomarker for evaluating the severity of RSV infection through extensive and intensive research. The invention discovers that PF4 gene and protein are significantly and specifically upregulated in RSV infected patients for the first time and are associated with disease severity, and the PF4 gene and protein can be used as detection indexes for clinical diagnosis of RSV infection severity.
PF4 gene and product thereof
The PF4 gene is located on human chromosome 4 and in the context of the present invention, the "PF 4 gene" includes polynucleotides of the human PF4 gene as well as any functional equivalent of the human PF4 gene, wherein said PF4 gene functional equivalent has at least about 60% homology, preferably at least about 70%, more preferably at least about 80%, preferably at least about 90%, and more preferably at least about 95, 96, 97, 98 or 99% homology to the PF4 gene. In a specific embodiment of the present invention, the nucleotide sequence of a representative PF4 gene is set forth in SEQ ID NO 1.
One skilled in the art will recognize that the present invention is not limited to quantifying gene expression for any particular variant of the target gene of the present invention. Two sequences are "substantially homologous" (or substantially similar) if, when the nucleic acid or fragment thereof is optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, preferably at least about 70%, more preferably at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases.
The mature HUMAN PF4 protein contains 70 amino acids, has a monomer molecular weight of 7.8kDa, and has an accession number P02776(http:// www.uniprot.org/uniprot/P02776, P L F4_ HUMAN, glutamic acid at position 32-serine at position 101) in the UniProtKB database, and the protein sequence of HUMAN PF4 is shown in SEQ ID NO: 2.
One skilled in the art will recognize that the present invention is not limited to quantifying any particular variant of the target gene product (protein) of the present invention. "PF 4 protein" includes PF4 protein as well as any functional equivalent of PF4 protein. The functional equivalents comprise a conservative variant protein of PF4 protein, or an active fragment or an active derivative thereof, an allelic variant, a natural mutant, an induced mutant, a protein encoded by DNA capable of hybridizing with the DNA of PF4 under high or low stringency conditions.
In general, it is known that modification of one or more amino acids in a protein does not affect the function of the protein. One skilled in the art will recognize that individual amino acid changes or small percentage amino acids or individual additions, deletions, insertions, substitutions to an amino acid sequence are conservative modifications, wherein a change in a protein results in a protein with a similar function. Conservative substitution tables providing functionally similar amino acids are well known in the art.
Biomarkers
A "biomarker," also referred to as a "molecular marker," or a "gene marker," is any gene or protein whose expression level in a tissue or cell is altered compared to the expression level of a normal or healthy cell or tissue. Any method available in the art for detecting biomarker expression is encompassed herein. Expression of the biomarkers of the invention can be detected at the nucleic acid level (e.g., RNA transcript) or at the protein level. By "detecting expression" is intended to determine the amount or presence of an expression product of an RNA transcript or a biomarker gene thereof. Thus, "detecting expression" encompasses instances where the biomarker is determined to be not expressed, not to be detected expressed, expressed at a low level, expressed at a normal level, or overexpressed. To determine overexpression, the body sample being tested can be compared with a corresponding body sample from a healthy person. That is, the "normal" level of expression is the level of expression of the biomarker. This sample may be presented in a normalized form. In some embodiments, biomarker overexpression is determined by comparing a body sample to a corresponding body sample from a healthy person.
Detection method
The present invention can be detected using a variety of nucleic acid and protein techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing, nucleic acid amplification, nucleic acid hybridization and protein immunization.
Illustrative, non-limiting examples of nucleic acid sequencing techniques described herein include, but are not limited to, chain terminator sequencing (Sanger sequencing) and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.
Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing) and third generation single molecule sequencing, a high throughput sequencing technique that is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary principles of reversible termination chemical reactions.
The nucleic acid amplification techniques described herein are selected from the group consisting of Polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), ligase chain reaction (L CR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA), wherein PCR entails reverse transcription of RNA to DNA prior to amplification (RT-PCR), TMA, and NASBA direct amplification of RNA.
Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.
Nucleic acid hybridization techniques in the present invention include, but are not limited to, In Situ Hybridization (ISH), microarray, and southern or northern blotting. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously. Southern and northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a matrix gel, and then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Detecting the hybridization probes bound to the filter. A variation of this procedure is reverse northern blotting, in which the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from the tissue and labeled.
Protein immunoassays include sandwich immunoassays, such as sandwich E L ISA, in which detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker, Radioimmunoassays (RIA), direct, indirect or contrast enzyme-linked immunosorbent assays (E L ISA), Enzyme Immunoassays (EIA), Fluorescence Immunoassays (FIA), western blots, immunoprecipitations, and immunoassays based on any particle (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots).
The immunization method according to the present invention may be based, for example, on any of the above methods.
Statistical method
In the present invention, the experiment was repeated at least 3 times, the data were expressed as mean ± standard deviation, and the statistical analysis was performed using Graphpad prism5.0 statistical software, and the difference between the two was considered to be statistically significant when P <0.05 using t-test.
The following examples are not intended to be exhaustive of the specific experimental procedures, as the methods or methods described in the molecular cloning handbook (Sambrook, et al. New York: Cold Spring Harbor L laboratory Press,1989) are followed by procedures provided by the manufacturer of the product.
Example 1 determination of PF4 concentration in plasma of RSV infected patients and healthy persons
1.1 case Collection
Pneumonia patients treated in the pediatric hospital of the beijing children hospital affiliated to the university of capital medicine during 2007 to 2015 (547 cases) and routine physical examination children in the pediatric growth and development clinic (control group, 45 cases). Viruses screened for respiratory pathogens in children with pneumonia include Respiratory Syncytial Virus (RSV), Adenovirus (ADV), Enterovirus (EV), human coronavirus (HCoVs), human bocavirus (HBoV), Human Metapneumovirus (HMPV), influenza virus (IFV), parainfluenza virus (PIV).
1.2 clinical sample Collection and handling
Collecting peripheral blood of children patients, and placing the children patients in an EDTA anticoagulation tube. Plasma was separated according to conventional methods.
1.3 determination of PF4 concentration in plasma samples
The content of PF4 protein in the plasma of patients is detected by a commercial human PF 4E L ISA kit (Abcam company product, ab 189573). The specific operation is as follows:
1) the kit is balanced to room temperature, and meanwhile, required solution and standard substance are prepared according to the kit specification;
2) adding 50 μ l of standard substance and sample diluent NS of 2.5ng/ml, 1.25ng/ml, 0.63ng/ml, 0.31ng/ml, 0.16ng/ml, 0.08ng/ml and 0.04ng/ml into the reaction well in turn to obtain standard curve and blank control;
3) adding the sample into reaction holes after properly diluting, wherein each hole is 50 mu l;
4) add 50. mu.l of Antibody Cocktail (Antibody Cocktail) per well;
5) sealing the reaction wells with sealing plate gummed paper, and incubating for 40 minutes at room temperature using a micro-oscillator (400 rpm);
6) adding 350 mul of washing buffer PT into each hole, and washing for 3 times;
7) add 100. mu.l TMB substrate per well;
8) incubating the cells in a micro-shaker (400rpm) for 4 minutes at room temperature in the dark;
9) add 100. mu.l stop solution per well;
10) incubating the cells in a micro-oscillator (400rpm) for 1 min at room temperature in the dark;
11) OD was measured at 450nm with a microplate reader, and the concentration of PF4 in the sample was calculated from the standard curve.
1.4 statistical analysis of results
Statistical analysis of the PF4 content in the plasma of the RSV group, other respiratory virus infected group and normal control group showed that the plasma PF4 content (1376 + -781.3 ng/ml) of the RSV infected group was significantly higher than that of the other respiratory virus infected group (1127 + -861.1 ng/ml) and normal control group (609.9 + -502.5 ng/ml) in the RSV infected group, and the difference was statistically significant (p < 0.0001).
It can be seen that a significant increase in PF4 protein levels in the plasma of RSV infected patients compared to the other groups can be used as an indication of RSV infection.
Example 2 determination of PF4 concentration in nasopharyngeal aspirates from patients infected with RSV and other respiratory viruses
2.1 case Collection
Pneumonia patients treated in the pediatric hospital of the beijing children hospital affiliated to the university of capital medicine during 2007 to 2015 (547 cases). Viruses screened for respiratory pathogens in children with pneumonia include Respiratory Syncytial Virus (RSV), Adenovirus (ADV), Enterovirus (EV), human coronavirus (HCoVs), human bocavirus (HBoV), Human Metapneumovirus (HMPV), influenza virus (IFV), parainfluenza virus (PIV).
2.2 clinical sample Collection and handling
The patient is subjected to bronchoscopy by collecting 1-2ml of nasopharyngeal aspirate and storing at-80 ℃ for later use.
2.3 determination of PF4 concentration in samples
The content of PF4 protein in nasopharyngeal aspirate of patients was measured by commercially available kit PF 4E L ISA (Abcam corporation.) the detailed procedure was as follows:
1) the kit is balanced to room temperature, and meanwhile, required solution and standard substance are prepared according to the kit specification;
2) adding 50 μ l of standard substance and sample diluent NS of 2.5ng/ml, 1.25ng/ml, 0.63ng/ml, 0.31ng/ml, 0.16ng/ml, 0.08ng/ml and 0.04ng/ml into the reaction well in turn to obtain standard curve and blank control;
3) adding the sample into reaction holes after properly diluting, wherein each hole is 50 mu l;
4) add 50. mu.l of antibody mix per well;
5) sealing the reaction wells with sealing plate gummed paper, and incubating for 40 minutes at room temperature using a micro-oscillator (400 rpm);
6) adding 350 mul of washing buffer PT into each hole, and washing for 3 times;
7) add 100. mu.l TMB substrate per well;
8) incubating the cells in a micro-shaker (400rpm) for 4 minutes at room temperature in the dark;
9) add 100. mu.l stop solution per well;
10) incubating the cells in a micro-oscillator (400rpm) for 1 min at room temperature in the dark;
11) OD was measured at 450nm with a microplate reader, and the concentration of PF4 in the sample was calculated from the standard curve.
2.4 statistical analysis of results
The result of statistical analysis of the content of PF4 in nasopharyngeal aspirates of patients in RSV group and other respiratory virus infected groups is shown in FIG. 2, the content of PF4 (1085 + -1389 pg/ml) in nasopharyngeal aspirates of RSV infected groups is significantly higher than that of other respiratory virus infected groups (679 + -1216 pg/ml), and the difference is statistically significant (p < 0.01).
It can be seen that a significant increase in PF4 protein content in nasopharyngeal aspirates in RSV infected patients compared to the other groups can be used as an indicator of RSV infection.
Example 3 correlation of PF4 levels in nasopharyngeal aspirates from RSV-infected patients with disease severity
3.1 case Collection and clinical Scoring of disease severity
Pneumonia patients treated in the pediatric hospital of the children's hospital beijing, affiliated to the university of capital medicine during 2007 to 2015 (547 cases), Respiratory Syncytial Virus (RSV) infected patients were confirmed by respiratory pathogen screening (158 cases). Assessment of disease severity in RSV infected patients according to a clinical scoring system8。
3.2 clinical sample Collection and handling
The patient is subjected to bronchoscopy by collecting 1-2ml of nasopharyngeal aspirate and storing at-80 ℃ for later use.
3.3 determination of PF4 concentration in samples
The content of PF4 protein in nasopharyngeal aspirate of patients was measured by commercially available kit PF 4E L ISA (Abcam corporation.) the detailed procedure was as follows:
1) the kit is balanced to room temperature, and meanwhile, required solution and standard substance are prepared according to the kit specification;
2) adding 50 μ l of standard substance and sample diluent NS of 2.5ng/ml, 1.25ng/ml, 0.63ng/ml, 0.31ng/ml, 0.16ng/ml, 0.08ng/ml and 0.04ng/ml into the reaction well in turn to obtain standard curve and blank control;
3) adding the sample into reaction holes after properly diluting, wherein each hole is 50 mu l;
4) add 50. mu.l of antibody mix per well;
5) sealing the reaction wells with sealing plate gummed paper, and incubating for 40 minutes at room temperature using a micro-oscillator (400 rpm);
6) adding 350 mul of washing buffer PT into each hole, and washing for 3 times;
7) add 100. mu.l TMB substrate per well;
8) incubating the cells in a micro-shaker (400rpm) for 4 minutes at room temperature in the dark;
9) add 100. mu.l stop solution per well;
10) incubating the cells in a micro-oscillator (400rpm) for 1 min at room temperature in the dark;
11) OD was measured at 450nm with a microplate reader, and the concentration of PF4 in the sample was calculated from the standard curve.
3.4 correlation analysis of PF4 levels in samples with clinical scores for disease severity
RSV patients can be divided into two groups according to disease severity score: the scores were 2-7 groups and 10-14 groups (the higher the score, the more severe the disease severity, see reference 8 for details). As shown in fig. 3, the statistical results showed that the concentration of nasopharyngeal aspirate PF4 (median 528pg/ml) was significantly higher in the 10-14 groups than in the 2-7 groups (median 167 pg/ml; P0.003).
Thus, the corresponding increase in PF4 protein content in nasopharyngeal aspirates from RSV infected patients with increased disease severity can be used to indicate disease severity of RSV infection.
The above embodiments are exemplary embodiments and should not be construed as limiting the invention. Those skilled in the art can recognize that any modification, equivalent replacement, improvement or the like which comes within the spirit and principle of the present invention is included in the scope of the present invention.
Reference to the literature
1.Hall C B,Weinberg G A,Iwane M K,et al.The burden of respiratorysyncytial virus infection in young children[J].N Engl J Med,2009,360(6):588-98；
2.Kim H W,Arrobio J O,Brandt C D,et al.Epidemiology of respiratorysyncytial virus infection in Washington,D.C.I.Importance of the virus indifferent respiratory tract disease syndromes and temporal distribution ofinfection[J].Am J Epidemiol,1973,98(3):216-225；
3.Fisher R G,Gruber W C,Edwards K M,et al.Twenty years of outpatientrespiratory syncytial virus infection:a framework for vaccine efficacy trials[J].Pediatrics,1997,99(2):E7；
4.Nair H,Nokes D J,Gessner B D,et al.Global burden of acute lowerrespiratory infections due to respiratory syncytial virus in young children:asystematic review and meta-analysis[J].Lancet,2010,375(9725):1545-1555；
5.Lozano R,Naghavi M,Foreman K,et al.Global and regional mortalityfrom 235causes of death for 20age groups in 1990and 2010:a systematicanalysis for the Global Burden of Disease Study 2010[J].Lancet,2012,380(9859):2095-128；
6.Selvarangan R,Abel D,Hamilton M.Comparison of BD Directigen EZ RSVand Binax NOW RSV tests for rapid detection of respiratory syncytial virusfrom nasopharyngeal aspirates in a pediatric population.[J]Diagn MicrobiolInfect Dis.2008 Oct；62(2):157-61；
7.Jonathan N.Diagnostic utility of BINAX NOW RSV--an evaluation ofthe diagnostic performance of BINAX NOW RSV in comparison with cell cultureand direct immunofluorescence.[J]Ann Clin Microbiol Antimicrob.2006 Jun 6；5:13；
8.Larranaga CL,Ampuero SL,Luchsinger VF,et al.Impaired immuneresponse in severe human lower tract respiratory infection by respiratorysyncytial virus.Pediatr Infect Dis J 2009；28:867-873.
Sequence listing
<120> biomarker for human respiratory syncytial virus infection and application
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>1217
<212>DNA
<213> human (Homo sapiens)
<400>1
attggccaca gagacccagc ccgagtttcc catcgcactg agcactgaga tcctgctgga 60
agctctgccg cagcatgagc tccgcagccg ggttctgcgc ctcacgcccc gggctgctgt 120
tcctggggtt gctgctcctg ccacttgtgg tcgccttcgc cagcggtgag agcagaagcc 180
aggctgtgag ggctggcagc ggcgaggggg agtccgggaa gccctggggc tggggaggaa 240
tcctctagga tcatgatcac agccacactt aacggagagc ctgctgagtg tctggccaca 300
gtgccaggcg ctgcacctgc acctcccacc tggttagaac aacttctgtc tgggggaggt 360
gtgatttatg gtaaggaaaa gaaggctgaa ggtagtggaa aaagtcccta aagtacctct 420
ggctactcat ggagtcacca ctccctcccc tcctcctctc ttaccctctc catttccccc 480
tcagctgaag ctgaagaaga tggggacctg cagtgcctgt gtgtgaagac cacctcccag 540
gtccgtccca ggcacatcac cagcctggag gtgatcaagg ccggacccca ctgccccact 600
gcccaactga tgtgagtcct tgcactgcat ctgtcagtgc tcccgctccg tgcctcctct 660
gcccaaccct cccccttcta atgccatttg caaacccaag gactgaaagt cacatctctt 720
ctcttttccc tgccagagcc acgctgaaga atggaaggaa aatttgcttg gacctgcaag 780
ccccgctgta caagaaaata attaagaaac ttttggagag ttagctacta gctgcctacg 840
tgtgtgcatt tgctatatag catacttctt ttttccagtt tcaatctaac tgtgaaagaa 900
cttctgatat ttgtgttatc cttatgattt taaataaaca aaataaatca agttgtagta 960
tagtcaaaat acttcttaat aatagtgcaa aaattgtgtt gacacataac aatttcatgg 1020
aagaaaaaaa ttccggtatt ttaagcaaaa agtattttga aggaaggtgt gaatactggt 1080
tatgcttggt gttacatgtt ggctgataca tattcatgca tttacatgat tgcagtactt 1140
tatagctaca tatttacctt gaccattatt attacctttg ccaataaata tcagtaacac 1200
agatggcttt taaaaaa 1217
<210>2
<211>70
<212>PRT
<213> human (Homo sapiens)
<400>2
Glu Ala Glu Glu Asp Gly Asp Leu Gln Cys Leu Cys Val Lys Thr Thr
1 5 10 15
Ser Gln Val Arg Pro Arg His Ile Thr Ser Leu Glu Val Ile Lys Ala
20 25 30
Gly Pro His Cys Pro Thr Ala Gln Leu Ile Ala Thr Leu Lys Asn Gly
35 40 45
Arg Lys Ile Cys Leu Asp Leu Gln Ala Pro Leu Tyr Lys Lys Ile Ile
50 55 60
Lys Lys Leu Leu Glu Ser
65 70
Claims (10)
1. A kit for assessing the severity of respiratory syncytial virus infection, wherein the kit comprises a product for detecting the level of platelet factor 4(PF4) gene or a functional equivalent thereof or expression thereof; preferably, the product for detecting the platelet factor 4(PF4) gene or its functional equivalent or its expression level is a probe that specifically recognizes the gene or a primer that specifically amplifies the gene.
2. A kit for evaluating the severity of respiratory syncytial virus infection, wherein the kit comprises a product for detecting PF4 protein or a functional equivalent thereof or an expression level thereof, preferably, the product for detecting PF4 protein or a functional equivalent thereof is a reagent for detecting PF4 protein in human serum by the E L ISA method, and more preferably, the product for detecting PF4 protein or a functional equivalent thereof is an antibody for detecting PF4 protein in human serum.
Use of the PF4 gene or a functional equivalent thereof for the manufacture of a medicament or kit for assessing the severity of respiratory syncytial virus infection; preferably, wherein the agent is a product for detecting the level of expression of the PF4 gene or a functional equivalent thereof; preferably wherein the kit comprises a product for detecting the level of PF4 gene or a functional equivalent thereof or expression thereof.
Use of the PF4 protein or a functional equivalent thereof for the manufacture of a medicament or kit for assessing the severity of respiratory syncytial virus infection; preferably wherein the agent is a product for detecting the level of PF4 protein or a functional equivalent thereof or expression thereof; preferably wherein the kit comprises a product for the detection of PF4 protein or a functional equivalent thereof.
5. Use of a product for detecting the PF4 gene or a functional equivalent thereof or the expression level thereof for the preparation of a medicament or a kit for evaluating the severity of respiratory syncytial virus infection.
6. Use of a product for detecting the PF4 protein or functional equivalent thereof or the expression level thereof for the preparation of a medicament or kit for assessing the severity of respiratory syncytial virus infection.
7. The use of claim 5, wherein the product is a probe that specifically recognizes the gene or a primer that specifically amplifies the gene.
8. The use of claim 6, wherein the product is a reagent for detecting PF4 protein in human serum by E L ISA method, preferably the product is an antibody for detecting PF4 protein in human serum.
9. A method of assessing the severity of respiratory syncytial virus infection comprising detecting the level of PF4 gene, or a functional equivalent thereof, or expression thereof, in a sample from a subject, thereby determining the severity of infection by the subject with respiratory syncytial virus.
10. A method of assessing the severity of respiratory syncytial virus infection comprising detecting the level of PF4 protein or a functional equivalent thereof or expression thereof in a sample from a subject, thereby determining the severity of respiratory syncytial virus infection in the subject.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010192972.0A CN111440859A (en) | 2020-03-18 | 2020-03-18 | Biomarker for human respiratory syncytial virus infection and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010192972.0A CN111440859A (en) | 2020-03-18 | 2020-03-18 | Biomarker for human respiratory syncytial virus infection and application |
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| CN111440859A true CN111440859A (en) | 2020-07-24 |
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| CN202010192972.0A Pending CN111440859A (en) | 2020-03-18 | 2020-03-18 | Biomarker for human respiratory syncytial virus infection and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112225805A (en) * | 2020-10-20 | 2021-01-15 | 长春百克生物科技股份公司 | Nanobodies and uses thereof |
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| CN105622743A (en) * | 2016-02-22 | 2016-06-01 | 西安交通大学 | Autistic serum polypeptide marker PF4-A and application thereof |
| CN108060224A (en) * | 2018-01-08 | 2018-05-22 | 聂绍平 | A kind of gene and its expression product for merging cardiac rupture screening for acute myocardial infarction AMI |
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2020
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| CN105622743A (en) * | 2016-02-22 | 2016-06-01 | 西安交通大学 | Autistic serum polypeptide marker PF4-A and application thereof |
| CN108060224A (en) * | 2018-01-08 | 2018-05-22 | 聂绍平 | A kind of gene and its expression product for merging cardiac rupture screening for acute myocardial infarction AMI |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112225805A (en) * | 2020-10-20 | 2021-01-15 | 长春百克生物科技股份公司 | Nanobodies and uses thereof |
| CN112225805B (en) * | 2020-10-20 | 2023-03-21 | 长春百克生物科技股份公司 | Nanobodies and uses thereof |
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