CN117969829A - Kit for rapidly detecting H1N1 influenza virus based on FRET fluorescent probe and latex turbidimetry technology - Google Patents
Kit for rapidly detecting H1N1 influenza virus based on FRET fluorescent probe and latex turbidimetry technology Download PDFInfo
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The invention relates to an innovative diagnostic kit for H1N1 influenza virus, which adopts the cooperative use of a FRET fluorescent probe and a latex turbidimetry technology. The kit remarkably improves the detection accuracy and speed by combining the high specificity and sensitivity of FRET and the rapid visual detection capability of latex turbidimetry. According to the invention, through analyzing the gene sequence of H1N1, a special fluorescent probe is designed, and a monoclonal antibody with a good effect is modified, so that an antibody conjugate with good specificity is obtained, and the modified antibody is coated on latex microspheres to prepare a latex turbidimetric kit for in-vitro rapid detection. The method can further improve the accuracy and the speed of detection, is suitable for most biochemical analyzers, and is very suitable for wide application.
Description
Technical Field
The invention relates to the field of detection, in particular to a novel improved in-vitro detection kit based on the combination of a fluorescent probe technology and a latex turbidimetric technology, which is used for detecting specific influenza viruses.
Background
Introduction of the characteristics of influenza virus H1N1, limitations of existing detection methods, and fundamental principles of FRET technology and latex turbidimetry.
Influenza a virus (H1N 1), commonly referred to as influenza a, is a subtype of influenza virus that can cause infection in humans, pigs and other animals. The most well known H1N1 virus outbreaks are the 2009 global pandemic, when new H1N1 strains have led to a global health crisis. Symptoms of a flow are similar to other types of influenza, including fever, cough, sore throat, body pain, headache, chills, and fatigue. Serious cases may lead to pneumonia, respiratory failure, and death. The nail stream may be spread by droplets, and when an infected person coughs, sneezes or talks, the virus may be spread to nearby people. Preventive measures include vaccination, hand washing, avoiding contact with the patient, and maintaining proper hygiene habits in public places. Since alphaviruses can vary, monitoring and studying their spread and evolution patterns is critical to public health. Outbreaks of H1N1 influenza viruses, such as the 2009 pandemic, have highlighted the urgent need for a rapid, accurate diagnostic method. Although various detection means exist at present, the problems of insufficient sensitivity, complex operation, long response time and the like often exist.
Traditional methods for detecting H1N1 influenza virus, such as the real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), have some limitations, although they are widely used in clinical diagnosis. First, these methods typically require specialized laboratory equipment and highly trained technicians, limiting their use in resource-constrained environments. Second, these techniques tend to be complex to operate and take a long time, which can be a significant hurdle in rapidly coping with influenza large flows. In addition, RT-PCR places high demands on sample quality and handling, and sample contamination or improper handling may lead to detection failure or erroneous results. ELISA, while simpler than RT-PCR, is generally less sensitive and less specific than RT-PCR, which can lead to missed detection of low viral load samples. Therefore, developing a novel detection method that is both rapid and accurate, and at the same time is easy to operate is an important requirement in the current influenza virus detection field.
Fluorescence Resonance Energy Transfer (FRET) is a technique based on energy transfer between two fluorescent molecules, and is widely used in biological and medical research. This transfer only occurs when the intermolecular distance is very close, so FRET can act as a powerful molecular scale "ruler" to study interactions between biomolecules.
In the field of in vitro diagnostics, FRET has the major advantage of its extremely high sensitivity and specificity. FRET can detect specific biomarkers, such as proteins, nucleic acids, and small molecules, making it very useful in pathogen detection, drug screening, and biomarker analysis. FRET probes can be designed to be highly specific for molecules of a particular sequence or structure, thereby providing accurate detection results.
Another significant advantage of FRET technology is that it requires relatively low sample handling requirements and can be directly detected in complex biological samples, which is particularly important for rapid diagnosis. In addition, FRET allows for real-time monitoring of molecular processes, providing a dynamic view for studying disease mechanisms and drug actions.
In practical applications, the use of FRET technology in combination, such as with nanotechnology or other high throughput screening methods, further widens the application range. For example, by binding nanoparticles, FRET technology can be used to develop highly sensitive and customizable biosensors that can be used to detect low concentrations of pathogens or disease markers.
In summary, FRET technology has great potential in the field of in vitro diagnostics, especially in applications requiring high sensitivity, high specificity and real-time detection. With the development of technology, FRET and its related technologies are expected to play a greater role in early diagnosis and real-time monitoring of diseases.
Latex nephelometry is based on the scattering and absorption properties of nanoparticles in solution. In such tests, latex particles (e.g., polystyrene microspheres) are typically used as the reaction medium. When a specific antigen reacts with latex microspheres surface-modified with the corresponding antibody, the microspheres are caused to aggregate, thereby increasing turbidity of the solution. By measuring the change in turbidity, the presence of antigen can be quantitatively analyzed. This method has historically been widely used for rapid detection of various proteins and antibodies, for example for the identification of certain disease markers in the blood.
The main advantage of latex turbidimetry is the simplicity of operation and the fast response. Latex turbidimetry does not require expensive equipment or highly specialized handling skills compared to other more complex biochemical methods. The testing process can typically be completed in a matter of minutes, and is well suited for clinical sites requiring rapid diagnostic results, such as emergency medical services or primary medical facilities. In addition, due to the simplicity of this method, it is also suitable for large-scale screening.
Although latex nephelometry is simple and quick to operate, its sensitivity and specificity are challenging in complex virus assays, such as influenza virus assays. For these viruses, it is often desirable to detect low concentrations of pathogens or specific protein components thereof. To increase the sensitivity and specificity of the detection, it can be improved by optimizing the surface modification of the latex microspheres and using finer detection techniques. For example, the accuracy of detection can be significantly improved by binding fluorescent markers or using monoclonal antibodies with higher specificity as probes.
In conclusion, the kit combines the advantages of fluorescent probes and latex turbidimetry, and develops a rapid detection kit aiming at the A-flow virus, which is not available in the market at the present.
Disclosure of Invention
Detailed description of specific compositions of the invention, including sequence information of FRET fluorescent probes and selection of latex microspheres.
For latex microspheres, the product of JSR life science is selected, and the specific model and reason for selection are based on product specifications, particle size, surface functionalization ability, and the like.
The operation steps are as follows: methods of use of the kit are described in detail, including sample preparation, mixing of fluorescent probes and latex microspheres, reaction conditions, and observation and interpretation of results.
The invention mainly aims to improve the existing rapid detection technical means of the first stream, amplifies the signal of the specific binding of the antigen and the antibody through fluorescence resonance energy transfer, and simultaneously simplifies the operation steps and time during detection by using the latex turbidimetric technology. In terms of the practical operation principle, the FRET technology and the antigen-antibody specificity technology are organically combined to achieve double detection, expand the detected strain types and amplify detection signals at the same time, and solve the defect that quantitative analysis is not easy to realize in a short-time rapid detection.
Probes are a complementary conserved region of DNA, specific pathogens and specific binding. The probe has a 5 'reporter fluorophore and a 3' quencher, and a complementary DNA sequence directed to the genomic region of the pathogen. In the actual reaction process, the quenching signal is enhanced along with the combination of the fluorescent group and the target antigen, and the more the combined antigen is, the stronger the signal is.
The kit firstly constructs a FRET-Immune Latex system for reflecting the specific binding level of antigen and antibody, so that an antibody complex in the reagent carries one part of a fluorescent group, and a sample is simply treated during detection, so that the antigen can carry the fluorescent group of the other part. The antibody complex is uniformly coated on latex microspheres, and then prepared into a latex reagent for preservation and use when detection is carried out. When the latex reagent is filled with the target antigen, the internal molecular structure of the latex reagent is changed, turbidity is improved, and a fluorescence signal is enhanced.
The probe sequence and the antibody sequence designed in the kit are as follows:
Antibody forward primer ABSeq-1F:
5’-GAAATT AATACGACTCACTATAGGGCTAATGAGTGTGCTCAAGTATTGAGTGAAAT-3’
Reverse primer ABSeq-1R: 5'-CAATGTTAAAAACACTATTAGCATAAGCAGT-3' A
Probe sequence FretSeq-1:5'-6-FAM-UUUUUC-BHQ1-3'
The primer design is based on the characteristics of human, pig and poultry H1N1, and four primers and probes of InF, SH1, RP and NH1 are selected for design reference according to the detection result of RT-PCR on clinical samples. And (3) strengthening the promoter part, modifying the conserved domain, simultaneously reserving a space for specific binding, and ensuring the normal occurrence of fluorescence resonance energy transfer while enhancing the specific binding.
The ABSeq-1F and R primers were specifically designed, and specifically bound biotin and biotin ligands were designed at their ends, respectively.
The kit is a non-disease diagnosis kit designed for clinical research and development, and mainly aims at exploring possible clinical diagnosis technologies. The invention provides SHERLOCK (SPECIFIC HIGH-SENSITIVITY ENZYMATIC REPORTER UNLOCK) assays for two other N-targeting genes crrna (5 '-GATTTAGAC TACCCCAAAAACGAAGGGGACTAAAACGCAGCAGCAAAGCAAGAGBHQ 1-3') and ssRNA reporter genes (5 '-6-fam-uuuuucc-bhq 1-3').
The invention mainly provides a novel thought for clinical research and development, and by combining gene detection and immunodetection, the invention provides a kit capable of utilizing a biochemical analyzer to carry out rapid detection, so that the detection speed and the detection quantity can be greatly improved, simultaneously, samples can be well quantitatively analyzed, and the gap of H1N1 in the field of chemiluminescence detection can be filled.
Drawings
FIG. 1 is a graph showing the relationship between the fluorescence value and the H1N1 concentration in the interval of 0-200 ng/ml.
FIG. 2 shows the relationship between the fluorescence value and the H1N1 concentration in the interval of 0-25 ng/ml.
Example 1
Sensitivity of the kit of the invention.
Preparing a reference by using an antigen, and selecting a lowest detection limit sample S1 and a type H1N1 in national reference
(Virus titer 9.8X10 5 TCID 50/L), 10, 20, 40, 80, 160, 320, 640, 1000, 2000 times diluted with 0.02 mol/L PBS buffer, and according to the detection result, the result is positive after 1000 times dilution, which proves that the sensitivity of the kit is about 9.8X10 2 TCID 50/L. 5 samples were prepared for each dilution to ensure statistical significance in duplicate experiments, and the kit results are shown in Table 1.
TABLE 1 results of sample detection at different dilution factors
Conclusion: the sample detection results of different dilution factors according to example 1 can clearly show the sensitivity of the kit, and the sample can still be correctly detected after the kit is diluted to 1000 times, and the sensitivity level of the kit is at an excellent level in the similar virus detection kit.
Example 2
The kit can be used for detecting H1N1 samples from different sources and displaying the specificity.
To test the effect of different strains, 200 nasal swab samples were selected from samples that had been tested by molecular diagnostics, and tested after treatment. Of the 200 samples selected, 42H 1N1 positive samples, 14H 1N5 positive samples, 21H 3N2 positive samples, and the rest were negative samples. Through detection results, 42H 1N1 positive samples are accurately detected, meanwhile, according to quantitative results and patient symptoms, the patient symptoms with larger values are found to be serious, a certain positive correlation is displayed, a doctor is facilitated to determine a clinical treatment scheme, and the results are shown in Table 2.
TABLE 2 detection results of different types of Strain positive samples
| Sample name | Quantity of | The detection result of the invention | Molecular diagnostic results |
| H1N1 | 42 | + | + |
| H1N5 | 14 | - | + |
| H3N2 | 21 | - | + |
Conclusion: from the statistical results in table 2, it can be seen that the detection results of the kit in 200 samples are consistent with the molecular diagnosis results, and H1N1 can be accurately distinguished from H1N1 in samples with similar structures, H1N5 and H3N2, thus exhibiting excellent specificity of the kit.
Example 3
In order to test the relation between the kit and symptoms, and whether the relation is approximately proportional to actual values in quantitative process, two concentration intervals (0-25 ng/ml and 0-225 ng/ml) are specially selected, the test is carried out in the interval of 0-200ng/ml, 50 samples are uniformly arranged in each interval of 0-25ng/ml, the detection values are counted, two statistical diagrams of fig. 1 and 2 are drawn, and the actual situation is displayed. In the figure, the abscissa represents the concentration of H1N1, the ordinate represents the fluorescence value, and the thumbnail represents the fitted curve.
The concentration interval of 0-20ng/ml was divided once every 5ng/ml, the obtained sample concentration was measured, and whether the distribution of the measurement results was uniform was counted, and the results are shown in Table 3.
TABLE 3 statistics of H1N1 sample detection values for different concentration intervals
| Sample class | H1N1 added value (ng/ml) | Detection value (ng/ml) | Coverage% | Reproducibility% |
| 1 | 0 | 0 | - | - |
| 2 | 5 | 5.27±0.34 | 105.49 | 6.9 |
| 3 | 10 | 10.18±0.21 | 102.16 | 3.5 |
| 4 | 15 | 14.92±0.42 | 94.87 | 4.61 |
| 5 | 20 | 20.35±0.46 | 112.64 | 7.22 |
Conclusion: the result shows that the kit has the advantages of the latex reagent after the double-tracing method is utilized, can linearly show the relation between the infection degree and the detection value in a certain range interval, and can provide favorable reference opinion for clinical diagnosis.
Claims (5)
1. The H1N1 influenza virus detection kit based on the FRET fluorescent probe and latex turbidimetry technology is characterized by comprising an antibody modified by the fluorescent probe and a special latex reagent, wherein the modified antibody has a sequence ABSeq-1 and the probe has a sequence FretSeq-1. The conserved region of the latex monoclonal antibody is added with a fluorescent group, and specific modification is carried out, and the type of fluorescent dye used is FITC.
2. The kit further comprises an engineered H1N1 antibody, the sequence of which contains engineered information and is mainly used for detecting antigenic site mutant of the H1N1 HA domain.
3. A fluorescent detection kit for specifically binding antigen and antibody is characterized in that fluorescent probes and modified antibody complexes are uniformly coated on the surfaces of latex particles, so that the fluorescent detection kit can specifically bind antigen and initiate fluorescent molecular energy transfer.
4. The detection kit is characterized by further comprising an excitation wavelength of about 490nm and an emission wavelength of about 520nm of the dye, and is suitable for detection of a biochemical analyzer in a wavelength range of 470-550nm, and parameter setting is performed according to instructions in the kit.
5. The kit is mainly used for detecting throat swab and nose swab samples, is not suitable for detecting blood and urine, and is required to simply process the samples according to instructions before testing.
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2023
- 2023-12-28 CN CN202311831888.9A patent/CN117969829A/en active Pending
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