CN113341141A - AlphaLISA detection kit for avian influenza virus H9 and detection method thereof - Google Patents
AlphaLISA detection kit for avian influenza virus H9 and detection method thereof Download PDFInfo
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Abstract
The invention discloses an avian influenza virus H9 type AlphaLISA detection kit and a detection method thereof, wherein the kit comprises the following substances A1) -A3) which are packaged independently: A1) a biotin-labeled avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody; A2) the receptor microsphere is coupled with an avian influenza virus H9-type envelope protein hemagglutinin monoclonal antibody; A3) streptavidin coupled donor microspheres; the nucleotide sequence for coding the avian influenza virus H9 type envelope protein hemagglutinin is shown as SEQ ID NO. 2. The detection method disclosed by the invention is simple, short in detection time, capable of realizing the purpose of rapid detection, free of expensive detection instruments and washing, low in detection cost, high in detection sensitivity, strong in specificity, free of false positive detection results, higher in detection reliability and more promising in market prospect.
Description
Technical Field
The invention relates to the field of veterinary medicine, immunology and biological product science, in particular to an avian influenza virus H9 type AlphaLISA detection kit and a detection method thereof.
Background
Avian influenza (avian influenza for short) is a systemic or respiratory infectious disease caused by influenza virus type A in the family of orthomyxoviridae, which is designated as a type A infectious disease by the International animal and disease administration (OIE) and listed in the list of animal infectious diseases in the International biological weapon convention, and is also listed as a type of animal infectious disease in China. Avian influenza can be manifested in various forms such as subclinical and mild respiratory system epidemic diseases, egg drop, acute lethal disease and the like. Outbreaks and epidemics of avian influenza caused by specific strains all over the world all cause massive death of birds and drastic reduction of production performance, resulting in huge economic losses. The occurrence of human events infected with avian influenza in hong Kong H5N1 and 1999 in 1997 and hong Kong H9N2 more prominently showed the public health significance of avian influenza. The avian influenza belongs to zoonosis, and the control of the avian influenza not only needs to isolate patients, but also needs to carry out early detection on animal epidemic situations and cut off the transmission path in the aspect of animals. The prevention and treatment of avian influenza is a long-term and difficult task, and China is the first major poultry raising country in the world, hundreds of billions of feathers are kept in a fence, and tens of thousands of samples are required to be diagnosed in a chicken farm every year. Therefore, animal husbandry and veterinary departments and large-scale poultry enterprises all use the avian influenza as a major epidemic disease to prevent and control. Therefore, early, rapid, accurate diagnosis of the disease is critical to the prevention and control and decontamination of the disease. .
At present, the main detection technologies for avian influenza virus include methods of virus isolation and identification, hemagglutination-hemagglutination inhibition (HA-HI) test, enzyme-linked immunosorbent assay (ELISA), Polymerase Chain Reaction (PCR), colloidal gold and the like. The virus is more accurate in separation, identification and diagnosis, but the time consumption is longer, the operation is complicated, and the rapid detection is not facilitated; the HA-HI test is sensitive, but is greatly influenced by external factors to weaken the practical value of the HA-HI test; the ELISA method is simple to operate, is suitable for large-scale serological investigation, has a great application prospect, needs repeated washing, has limited detection sensitivity and needs a certain amount of samples; although the PCR method can directly detect the gene of the virus and has high detection sensitivity, the detection cost is high, the pollution is easy, the technical requirement is higher, the kit is only suitable for laboratory diagnosis and is not suitable for rapid diagnosis of clinical infection, for example, the domestic invention patent 201310176317.6 discloses an influenza virus N9 type RT-PCR detection kit and a detection method, the invention provides a fluorescent quantitative RT-PCR detection kit for avian influenza virus N9 type, and the kit comprises deoxynucleoside triphosphate, MgCl2RT-PCR buffer solution, influenza virus N9 gene standard, RNase inhibitor, MMLV reverse transcriptase, DNA polymerase and the like, and the fluorescence PCR method for detecting the avian influenza virus is established, the method has the sensitivity which is 100-1000 times higher than that of the traditional PCR method (sensitive reaction is achieved under the concentration of 0.1TCID 50), but the time from sample treatment to result production needs 4-5 hours, and the method also needs expensive instruments such as a fluorescence quantitative PCR instrument and the like, and is not suitable for popularization in the basic level; the colloidal gold method has related products at home and abroad, but the detection sensitivity is not highThe quality of the product is greatly different, the quality control is not suitable, the product can only be qualitative and cannot be quantitative, and the method can only be used for auxiliary detection of epidemic disease diagnosis. Therefore, the exploration of efficient, specific and accurate detection means has very important significance for the prevention and control of the avian influenza.
The light-activated chemiluminescence immunoassay technology (AlphaLISA, amplified fluorescent reagent assay linked immunosorbent assay) is a novel homogeneous assay technology based on chemiluminescence of microbeads, and compared with the traditional ELISA technology, the technology has the characteristics of higher sensitivity, higher accuracy, higher uniformity, low background, wide dynamic assay range, no need of washing, extremely small sample demand and the like, and is very convenient for rapid screening and automation. In recent years, the technology has been widely applied in the fields of medicine and molecular biology. However, the detection of the AlphaLISA aiming at the avian influenza virus H9 type is not reported at home and abroad.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an avian influenza virus H9 type AlphaLISA detection kit and a detection method thereof, and solves the problems of long time consumption, complex operation, low detection sensitivity and the like in avian influenza virus detection.
In order to solve the technical problems, the invention adopts the following technical scheme: an AlphaLISA detection kit for avian influenza virus H9, comprising the following substances a1) -A3) packaged individually: A1) a biotin-labeled avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody; A2) the receptor microsphere is coupled with an avian influenza virus H9-type envelope protein hemagglutinin monoclonal antibody; A3) streptavidin coupled donor microspheres; the nucleotide sequence for coding the avian influenza virus H9 type envelope protein hemagglutinin is shown as SEQ ID NO. 2.
Preferably, the concentration of the receptor microsphere coupled with the anti-avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody is 10-100 mug/mL; the concentration of the biotin-labeled avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody is 0.2-2 mug/mL; the concentration of the streptavidin coupled donor microspheres is 10-100 mug/mL.
Preferably, the concentration of the receptor microsphere coupled with the monoclonal antibody of the anti-avian influenza virus H9 envelope protein hemagglutinin is 50 mug/mL; the concentration of the biotin-labeled avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody is 0.5 mug/mL; the concentration of the streptavidin coupled donor microspheres is 50 mug/mL.
The invention also provides the application of the kit in detecting the avian influenza virus H9; or the application of the kit in preparing products for detecting avian influenza virus H9.
The invention also provides a detection method using the kit, which specifically comprises the following steps:
1) adding the receptor microsphere coupled with the avian influenza virus H9 resistant capsular protein hemagglutinin monoclonal antibody and the biotin labeled avian influenza virus H9 type capsular protein hemagglutinin monoclonal antibody into each detection hole of a reaction plate, adding a sample to be detected into the detection holes, uniformly mixing, and placing in an incubator for incubation;
2) adding streptavidin-labeled donor microspheres into the detection holes obtained in the step 1), uniformly mixing, and placing in an incubator for incubation;
3) placing the reaction plate reacted in the step 2) on an AlphaScreen/Lisa detector to detect a signal value, and judging whether the sample to be detected contains the avian influenza virus H9 according to the detection signal value.
Preferably, the incubation temperature is 37 ℃, and the incubation time is 15-30 min.
Preferably, when the detection signal value of the sample to be detected is greater than 2 times of the background value, the sample to be detected contains the avian influenza virus H9.
Preferably, the receptor microsphere coupled with the avian influenza virus H9-type envelope protein hemagglutinin monoclonal antibody and the 1xAphaLISA buffer solution are diluted according to the volume ratio of 1: 50-1: 100; the biotin-labeled avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody and a 1xAphaLISA buffer solution are diluted according to the volume ratio of 1: 100-1: 200.
Preferably, the wavelength of the excitation light is 680nm and the detection wavelength of the emission light is 520-620 nmm when the AlphaScreen/Lisa detector detects.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention adopts a prokaryotic expression system to prepare avian influenza virus H9 type specific envelope protein Hemagglutinin (HA), adopts PEG1500 cell fusion technology to prepare avian influenza virus H9 type hemagglutinin monoclonal antibody, uses two strains of paired antibodies in the monoclonal antibody, 1 strain of antibody to coat acceptor microbeads, and 1 strain of antibody is labeled by biotin firstly and then forms an AlphaLISA reaction system together with donor microbeads of streptavidin, and establishes a double-antibody sandwich AlphaLISA detection method for detecting avian influenza virus H9 type by optimizing test reaction conditions of the donor microbeads, the acceptor microbeads, the monoclonal antibody, virus antigen and the like. Compared with the prior art, the detection method is simple, short in detection time, capable of achieving the purpose of rapid detection, free of expensive detection instruments and washing, low in detection cost, high in detection sensitivity, strong in specificity, free of false positive detection results, high in detection reliability and more promising in market prospect.
2. The detection method is simple and convenient to operate and rapid in detection, the positive hybridoma obtained by the detection method is good in stability, and the titer is good after subculture for 6 months; the detection shows that the method of the invention has the advantages of 3.42-5.67 percent of internal variation coefficient and 4.26-6.89 percent of inter-analysis variation coefficient, and good precision; in the detection, only the detection value of the avian influenza virus H9 is obviously higher, the result is judged to be positive, the detection values of other pathogens are lower, the detection values are negative according to the judgment standard, and the specificity is higher; after detection, the sample is placed at room temperature for 7 days, the change of signal values of all batches of reagents is small, the linear relation is good, the signal values of reference standard products are not obviously increased, and the stability is good; the sensitivity of detection is 98.4% and the specificity is 97.4% proved by a large batch of detection tests; the result shows that the established AlphaLISA detection method is slightly influenced by external environmental factors, has higher sensitivity, specificity and stability, can meet the requirements of high-flux and rapid detection, and has good popularization and application values.
Drawings
FIG. 1 is the restriction enzyme identification map of recombinant plasmid pET28a (+) -HA; m is DL-2000 Marker; 1-2 are pET28a (+) -HA double enzyme digestion products.
FIG. 2 is SDS-PAGE of HA protein expression under different induction temperature conditions; m is a protein molecular mass standard; 1.3, 5, 7 and 9 are 16 deg.C, 25 deg.C, 30 deg.C and 37 deg.C (0.5 mmol. L), respectively-1IPTG,10h) of the recombinant bacterium pET28a (+) -HA/BL21(DE3), wherein 2, 4, 6 and 8 are respectively 16 ℃, 30 ℃, 25 ℃ and 37 ℃ (0.5 mmol. L)-1IPTG,10h) and then the recombinant bacterium pET28a (+) -HA/BL21(DE3) is crushed by the cracking liquid to obtain a precipitate;
FIG. 3 is a SDS-PAGE of HA protein expression at different induction times; m is a protein molecular mass standard; 1-5 at 37 deg.C, and the concentration of IPTG is 0.5 mmol.L-1The induction time is 4h, 6h, h8 and 10h respectively.
FIG. 4 is an SDS-PAGE of HA protein expression under different IPTG induction concentrations; m is a protein molecular mass standard; 1-5 are respectively 0.4 mmol.L of IPTG at 37 DEG C-1、0.6mmol·L-1、0.8mmol·L-1、1.0mmol·L-1And 1.2 mmol. L-1And 6h after induction, the recombinant bacterium pET28a (+) -HA/BL21(DE3) is subjected to cracking liquid crushing to obtain a precipitate.
FIG. 5 is an SDS-PAGE electrophoresis of purified HA protein; m is a protein molecular mass standard; 1 is pET28a (+) -HA precipitation after ultrasonication without IPTG induction; 2 is a precipitate of a recombinant bacterium pET28a (+) -HA/BL21(DE3) after being crushed by a cracking liquid; 3 is the supernatant of the recombinant bacterium pET28a (+) -HA/BL21(DE3) after being crushed by a cracking liquid; 4-6 is HA penetration liquid; 7-9 are HA purified proteins.
FIG. 6 is a Western blot result chart of HA recombinant protein; 1 is the recombinant HA protein obtained after purification; 2 is a recombinant bacterium pET28a (+)/BL21(DE3) original bacterium liquid transformed by pET28a (+) vector.
FIG. 7 shows the partial cell supernatant antibody subtype identification.
FIG. 8 shows the purity of the monoclonal antibody identified by SDS-PAGE; m is a protein molecular mass standard; 1 is unpurified ascites, and 2-3 are purified antibodies.
FIG. 9 is Western-Blot of reaction of HA recombinant protein with HA monoclonal antibody; m is the standard mass of protein molecules; 1 is HA recombinant antigen and HA monoclonal antibody; 2, pET28a (+) empty vector and HA monoclonal antibody.
FIG. 10 is a Western-Blot of H9 standard virus and HA mab; m is a protein molecular mass standard; 1 is AIV H9 virus and HA monoclonal antibody; 2 is NDV F protein and HA monoclonal antibody.
FIG. 11 is a standard graph of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples. The experimental procedures described in the examples are not specifically described, i.e., they are carried out according to conventional molecular biological experimental procedures.
Examples
1. Screening of antigens
The avian influenza virus envelope protein hemagglutinin HAs important functions, can agglomerate red blood cells, belongs to type I glycoprotein, HAs the specificity of strains and subtypes, plays a key role in the virus adsorption and membrane penetration processes, further performs bioinformatics and immunological analysis on HA genes and encoding proteins thereof, and proves that HA is a specific antigen of avian influenza virus, and HAs good immunogenicity and strong stability. Therefore, the HA protein can be used as an antigen in a poultry influenza virus antibody light-induced chemiluminescence immunoassay kit.
2. Prokaryotic expression and detection of avian influenza virus H9 type HA protein
2.1 construction of pET28a (+) -HA recombinant vector
Referring to the nucleotide sequence of HA gene (GenBank accession number: EF070733.1) of avian influenza strain A/chicken/Gansu/2/99(H9N2) disclosed in GenBank, the gene sequence was optimized according to the codon preference of Escherichia coli without changing the amino acid sequence of HA protein, the optimized HA gene was chemically synthesized, and NcoI (base sequence CCATGG) restriction site was introduced at the 5 'end of the sequence, XhoI (base sequence CTCGAG) restriction site was introduced at the 3' end, and 6 histidine tags were added at the 5 'end and 3' end, respectively, as shown in SEQ ID NO. 2.
The synthesized HA gene is subjected to double enzyme digestion by NcoI and XhoI, after enzyme digestion products are recovered, the enzyme digestion products are inserted into pET28a (+) vectors which are also subjected to double enzyme digestion recovery by NcoI and XhoI, the ligation products are transformed into escherichia coli competent cells DH5 alpha, positive clones are screened out, double enzyme digestion identification is carried out on the positive clones by NcoI and XhoI, and recombinant plasmids with correct enzyme digestion identification are sent to Suzhou Jinzhi biology company for sequencing. The recombinant plasmid pET28a (+) -HA was identified by double restriction with NcoI and XhoI, and the obtained target fragment was consistent with the expected result, as shown in FIG. 1. And (3) sending the recombinant plasmid with correct enzyme digestion identification to Shanghai workers for sequencing to obtain a pET28a (+) -HA recombinant vector with correct sequence.
2.2 inducible expression Condition screening of HA recombinant proteins
Transforming recombinant plasmid pET28a (+) -HA into competent cells of Escherichia coli BL21(DE3), selecting a single colony, inoculating the single colony into 5mL of LB liquid culture medium containing kanamycin, and performing shaking culture at 37 ℃ until OD is achieved600The value is between 0.6 and 0.8, then IPTG is added into each culture bottle to ensure that the final concentration of the IPTG in the culture bottle is 0.5 mmol.L-1After being induced at 16 ℃, 25 ℃, 30 ℃ and 37 ℃ for 6 hours respectively, the samples are sampled, and then the samples are subjected to SDS-PAGE electrophoretic analysis to determine the optimal temperature, and the result is shown in figure 2, and the SDS-PAGE electrophoretic result shows that the induction condition is optimal when the induction temperature is 30 ℃.
Transforming recombinant plasmid pET28a (+) -HA into competent cells of Escherichia coli BL21(DE3), selecting a single colony, inoculating the single colony into 5mL of LB liquid culture medium containing kanamycin, and performing shaking culture at 37 ℃ until OD is achieved600The nm value is between 0.6 and 0.8, then IPTG is added into each culture bottle to ensure that the final concentration of the IPTG in the culture bottle is 0.5 mmol.L-1And after 2h, 4h, 6h, 8h and 10h of induction at 37 ℃, sampling, and performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis on the samples to determine the optimal induction time, wherein the result is shown in figure 3, and the SDS-PAGE electrophoresis result shows that the induction condition is optimal when the induction time is 6 h.
Transforming recombinant plasmid pET28a (+) -HA into competent cells of Escherichia coli BL21(DE3), selecting a single colony, inoculating the single colony into 5mL of LB liquid culture medium containing kanamycin, and performing shaking culture at 37 ℃ until OD is achieved600The value is between 0.6 and 0.8, then IPTG with different amounts is added into each culture bottle, so that the final concentration of IPTG in each culture bottle is 0.4,0.6、0.8、1.0、1.2mmol·L-1、0.6mmol·L-1、0.8mmol·L-1、1.0mmol·L-1And 1.2 mmol. L-1After the induction at 37 ℃ for 6h, the sample is sampled and then subjected to SDS-PAGE electrophoretic analysis to determine the concentration of the optimally induced IPTG, and the result is shown in figure 4, and the SDS-PAGE electrophoretic result shows that the final concentration of the IPTG is 0.8 mmol.L-1The induction conditions are optimal.
1.3 Mass expression and purification of HA proteins
Performing large-scale inducible expression on recombinant HA target protein according to the optimized optimal conditions, and purifying the HA target protein by adopting a His-tag nickel column. Before the sample is loaded on the column, the chromatographic column is equilibrated by an affinity equilibrium solution, then the obtained sample is loaded on the column, and a small amount of penetration liquid is collected. And (4) passing the affinity equilibrium solution through the column to remove the impure protein in the sample. Eluting the nickel column by using affinity eluent of imidazole with different concentration gradients, and collecting to obtain the purified HA recombinant protein. The result of the purification was analyzed by 10% SDS-PAGE, and as shown in FIG. 5, the objective HA protein was eluted when the imidazole concentration reached 200 mmol/L.
The purified recombinant HA protein was identified by immunoblotting and found to have a specific band around 63ku, which is consistent with the expected size of the target protein (see FIG. 6).
Preparation of monoclonal antibody of 3H 9N2 HA protein
3.1 establishment of H9N2 HA protein monoclonal antibody hybridoma cell line
The BALB/c mouse immunized by the HA protein prepared above is subjected to cell fusion by PEG-1500 on splenocytes and SP2/0 myeloma cells after 4 times of immunization, and cell fusion tests are carried out for two times in total, wherein the fusion rates of the two times are respectively 86.7% and 94.4%. The initial detection positive rates are 33.5% and 86.75% respectively, 30-hole subclones with higher positive values are selected, and 3 hybridoma cell strains which stably secrete antibodies are obtained through 2 times of subclones and repeated screening, and are named as 1F10E7, 2D10D10 and 5A10C3 respectively.
TABLE 1 cell fusion and Positive Rate
3.2 Positive hybridoma cell stability test
Subculturing the 1F10E7, 2D10D10 and 5A10C3 cell strains for more than 6 months, keeping the ELISA titer of the culture supernatant at 1:1280-2560, respectively freezing for 2, 4, 6 and 8 months, then recovering, keeping the mAbs titer of the induced mouse ascites at 1: 128000-256000, which shows that the positive hybridoma cell of the invention has good stability.
3.3 subtype identification of HA monoclonal antibodies
Monoclonal Antibody subtype identification was performed using Mouse Monoclonal Antibody Isotyping Kit (latex agglutination test) according to the Kit manual, and a portion of the supernatant of the Monoclonal cell line was aspirated, and the secreted Monoclonal Antibody subtype was identified by the Antibody subtype identification Kit, as shown in fig. 7 and table 2: the monoclonal antibody cell strains 1F10E7, 2D10D10 and 5A10C3 are all IgG 1.
TABLE 2 partial cell supernatant subtype identification results
3.4 purity identification of monoclonal antibody
Culturing 1F10E7 cells, injecting into abdominal cavity of BALB/c mouse to obtain mouse ascites type monoclonal antibody, collecting mouse ascites, purifying with ProteinA column antibody, and purifying collected lambda280nmThe eluted solution is identified by SDS-PAGE electrophoresis, the result is shown in figure 8, only has obvious specific bands at 25KD and 50KD without other miscellaneous bands, which accords with the expected result of IgG antibody band, and the concentration can reach 2.518mg/ml by BCA method concentration determination, which indicates that the purified antibody achieves electrophoretic purity.
3.5 specificity analysis of monoclonal antibodies
The specificity of the prepared HA monoclonal antibody is determined by Western-blot, the result is shown in figure 9, at about 63KD, the purified monoclonal antibody can be combined with HA recombinant protein to generate a specific band, and compared with the empty carrier protein, the band is not generated; and the result of Western-Blot detection performed by taking the H9N2 standard virus and the NDV F protein as antigens and taking the HA monoclonal antibody as a primary antibody is shown in figure 10, the HA monoclonal antibody can perform a specific reaction with the H9N2 standard virus but not with the NDV F protein, and the result shows that the purified monoclonal antibody HAs better specificity.
4. AlphaLISA detection kit for avian influenza virus H9 and detection method thereof
4.1 construction of the Standard Curve
1) AIV antigen was diluted to concentrations of 0, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL, 10000ng/mL, respectively. The dilution was 0.01mol/L, pH7.4 PBS buffer.
2) Diluting a receptor microsphere coupled with an avian influenza virus H9-type envelope protein hemagglutinin monoclonal antibody and a1 xAphaplisa buffer solution according to a volume ratio of 1: 100; the biotin-labeled avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody and 1 xAphaplisa buffer solution are diluted according to the volume ratio of 1: 200.
3) Respectively adding 25 mul of standard substance, 25 mul of receptor microsphere coupled with anti-avian influenza virus H9 type cyst membrane protein hemagglutinin monoclonal antibody and 25 mul of biotin-labeled avian influenza virus H9 type cyst membrane protein hemagglutinin monoclonal antibody into detection holes of a microporous plate, then placing the microporous plate on a micro oscillator to oscillate for 30-60 s, uniformly mixing, placing the mixture in an incubator at 37 ℃, incubating for 20min, then adding l75 mul of streptavidin donor microsphere under the dark condition, uniformly mixing, and placing the mixture at 37 ℃ for incubating for 20 min.
4) And (3) placing the microplate reacted in the step 3) on an AlphaScreen/Lisa detector to detect a signal value. Taking the logarithm of the concentration of the standard substance as an abscissa and the logarithm of the signal value counting as an ordinate, processing by a Log-Log function of a double-logarithm mathematical model to obtain a linear correlation coefficient r value of a dose-response curve of the AIV kit, and judging a dose-response linear relation according to the r value, as shown in FIG. 11. From the figure, it can be derived that the linear equation is: 17.687X +3.858, coefficient of correlation R20.9891, the self-made avian influenza virus photo-excitation chemiluminescence immunoassay reagent has good dose response.
4.2 sensitivity of detection method versus Linear Range of Standard Curve
The specific operation steps are the same as 4.1, and the signal value of the signal value minus the background is obtained by adding 2 times of standard deviation to the mean value of 20 measured values of the zero reference standard product and is substituted into a standard curve equation for calculation. The detection linear range is 1ng/mL-10ug/mL, so that the invention has wide linear range and high detection sensitivity.
4.3 precision test of detection method
The established detection method of the avian influenza virus H9 AlphaLISA is used for independently detecting samples to be detected with different low, medium and high concentrations, each sample is provided with 5 parallel holes, the detection is repeated for multiple times in the same experiment and different experiments, and the specific operation steps are the same as 4.1. The result shows that the self-made detection reagent has the intra-analysis variation coefficient of 3.42-5.67 percent and the inter-analysis variation coefficient of 4.26-6.89 percent, and has good precision.
4.4 specificity test of detection method
Four pathogens of avian influenza virus (AIV-H9), Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV) and bursal disease virus (IBDV) are selected as samples to be detected, and are subjected to AlphaLISA detection to determine the specificity of the method, the specific operation steps are the same as 4.1, and the results are shown in Table 3.
TABLE 3 results of specificity experiments
Remarking: the judgment standard is that the ratio of the signal value of the sample to be detected to the Negative (NC) is more than 2.0, and the sample can be judged to be positive.
And (3) displaying a detection result: the detection value of the method is lower than that of Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV) and bursal disease virus (IBDV), the detection value is negative according to the judgment standard, namely, no cross reaction occurs, and the method only has specific reaction on avian influenza virus H9, which shows that the method has better specificity.
4.5 evaluation of AlphaLISA detection method
And (2) taking 206 samples detected by a fluorescent quantitative PCR kit of foreign Saimerfin, determining 129 positive samples and 77 negative samples, detecting by a self-made avian influenza virus AlphaLISA reagent, comparing the detection positive rate and the detection negative rate with a standard kit, and performing quality evaluation on the established competitive AlphaLISA detection method. The results are shown in Table 4.
TABLE 4 AlphaLISA test method evaluation
The results show that: through the detection of the alphaLISA reagent of avian influenza virus H9 type by the method, 127 parts of true positive samples in 129 parts of samples are found, 75 parts of negative samples in 77 parts of negative samples are found, the detection sensitivity is 98.4%, and the specificity is 97.4%.
The above description is only exemplary of the present invention and should not be taken as limiting, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> animal epidemic prevention control center of Chongqing City of Chongqing university of Manifeng workers;
<120> AlphaLISA detection kit for avian influenza virus H9 and detection method thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 568
<212> PRT
<213> avian influenza Virus H9 (AIV-H9)
<400> 1
MGMHHHHHHE VVSLITIPLV VTVSNADKIC IGYQSTNSTE TVDTLTENNV PVTHAKELLH 60
TEHNGMLCAT SLGHPLILDT CTIEGLIYGN PSCNLLLGGR EWSYIVERPS AVNGLCYPGN 120
VENLEELRSL FSSASSYQRI QIFPDTIWNV SYSGTSKACS DSFYRSMRWL TQKNNAYPIQ 180
DAQYTNNRGK NILFMWGINH PPTDTVQTNL YTRTDTTTSV ATEDINRTFK PLIGPRPLVN 240
GLQGRIDYYW SVLKPGQTLR VRSNGNLIAP WYGHILSGES HGRILKTDLN SGNCVVQCQT 300
ERGGLNTTLP FHNVSKYAFG NCPKYVGVKS LKLAVGLRNV PARSSRGLFG AIAGFIEGGW 360
SGLVAGWYGF QHSNDQGVGM AADRDSTQKA IDKITSKVNN IVDKMNKQYE IIDHEFSEVE 420
TRLNMINNKI DDQILDIWAY NAELLVLLEN QKTLDEHDAN VNNLYNKVKR ALGSNAVEDG 480
KGCFELYHKC DDQCMETIRN GTYNRRKYKE ESRLERQKIE GVKLESEGTY KILTIYSTVA 540
SSLVIAMGFA AFLFWAMSNG SCRCNICI 568
<210> 2
<211> 1715
<212> DNA
<213> avian influenza Virus H9 (AIV-H9)
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ccatgggcat gcatcaccac catcaccacg aagttgtgag cctcatcacc attccactcg 60
tggtgacggt tagcaatgcg gacaagatct gcatcggcta tcagagcacc aacagtacgg 120
aaacggtgga caccctcacc gagaataacg tgccagttac gcacgcgaaa gaactgctcc 180
acaccgaaca caacggtatg ctctgtgcga cgagtctggg ccatccactc attctggata 240
cgtgcaccat cgaaggcctc atctacggca acccaagctg caatctgctg ctgggcggcc 300
gtgaatggag ctacatcgtg gaacgtccaa gcgcggtgaa cggtctctgt tacccgggca 360
acgtggagaa tctggaggaa ctgcgtagtc tgtttagcag tgccagcagc taccaacgca 420
ttcagatctt tccggacacg atctggaacg tgagttacag cggcacgagc aaagcgtgca 480
gcgatagctt ttaccgtagc atgcgctggc tcacgcaaaa gaacaatgcg tacccgatcc 540
aagatgcgca gtacaccaac aatcgcggca agaacattct gttcatgtgg ggcatcaatc 600
acccgccgac cgacaccgtt cagaccaatc tctacacccg caccgatacg acgacgagcg 660
ttgcgaccga ggacatcaac cgcaccttta aaccgctgat cggtccacgc ccgctggtta 720
acggtctgca aggccgcatc gattactact ggagcgtgct gaaaccgggc caaacgctgc 780
gtgttcgtag caatggcaat ctcatcgcgc cgtggtatgg tcacattctg agtggcgaga 840
gtcatggccg cattctgaaa accgatctga atagcggcaa ctgcgttgtt cagtgtcaga 900
ccgagcgcgg cggtctgaat acgacgctgc cgttccacaa cgttagtaag tacgccttcg 960
gtaactgccc gaagtacgtt ggcgttaaga gtctcaagct cgcggttggt ctgcgtaatg 1020
ttccagcccg tagtagccgc ggtctgtttg gcgccattgc gggtttcatt gaaggcggtt 1080
ggagcggtct ggttgccggt tggtacggtt tccagcacag caacgatcaa ggcgtgggca 1140
tggcggccga tcgtgatagc acccagaagg ccattgacaa gatcacgagc aaggtgaaca 1200
acatcgtgga caaaatgaac aagcagtatg aaatcattga tcacgaattc agcgaagtgg 1260
aaacccgtct gaatatgatt aataataaaa ttgacgacca gattctggat atctgggcgt 1320
acaacgccga gctgctggtt ctgctggaga accaaaagac gctggacgag cacgacgcga 1380
atgtgaacaa tctgtataac aaggtgaaac gcgcgctggg cagcaatgcg gttgaggatg 1440
gcaagggctg ctttgagctc taccacaaat gcgacgacca gtgtatggaa acgatccgca 1500
acggcaccta caatcgccgc aagtacaagg aagagagccg tctggaacgc cagaaaattg 1560
agggcgtgaa gctggaaagc gagggcacgt acaagattct gaccatctac agcacggttg 1620
cgagcagtct ggtgattgcg atgggcttcg ccgcgtttct gttctgggcc atgagcaatg 1680
gcagctgtcg ctgcaacatc tgcatctaac tcgag 1715
Claims (9)
1. An AlphaLISA detection kit for avian influenza virus H9, characterized in that the kit comprises the following substances A1) -A3) packaged individually: A1) a biotin-labeled avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody; A2) the receptor microsphere is coupled with an avian influenza virus H9-type envelope protein hemagglutinin monoclonal antibody; A3) streptavidin coupled donor microspheres; the nucleotide sequence for coding the avian influenza virus H9 type envelope protein hemagglutinin is shown as SEQ ID NO. 2.
2. The kit for detecting avian influenza virus H9 according to claim 1, wherein the concentration of the receptor microsphere coupled with the monoclonal antibody against hemagglutinin of avian influenza virus H9 is 10-100 μ g/mL; the concentration of the biotin-labeled avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody is 0.2-2 mug/mL; the concentration of the streptavidin coupled donor microspheres is 10-100 mug/mL.
3. The kit for detecting avian influenza virus H9 according to claim 1, wherein the concentration of the receptor microsphere coupled with the monoclonal antibody against hemagglutinin of avian influenza virus H9 is 50 μ g/mL; the concentration of the biotin-labeled avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody is 0.5 mug/mL; the concentration of the streptavidin coupled donor microspheres is 50 mug/mL.
4. Use of the kit of any one of claims 1 to 3 for detecting avian influenza virus H9; or, the kit of any claim 1 to 3 is applied to the preparation of products for detecting avian influenza virus H9.
5. A detection method using the kit of any one of claims 1 to 3, which is characterized by comprising the following steps:
1) adding the receptor microsphere coupled with the avian influenza virus H9 resistant capsular protein hemagglutinin monoclonal antibody and the biotin labeled avian influenza virus H9 type capsular protein hemagglutinin monoclonal antibody into each detection hole of a reaction plate, adding a sample to be detected into the detection holes, uniformly mixing, and placing in an incubator for incubation;
2) adding streptavidin-labeled donor microspheres into the detection holes obtained in the step 1), uniformly mixing, and placing in an incubator for incubation;
3) placing the reaction plate reacted in the step 2) on an AlphaScreen/Lisa detector to detect a signal value, and judging whether the sample to be detected contains the avian influenza virus H9 according to the detection signal value.
6. The detection method according to claim 5, wherein the incubation temperature is 37 ℃ and the incubation time is 15-30 min.
7. The detection method according to claim 5, wherein the sample to be detected contains avian influenza virus H9 when the detection signal value of the sample to be detected is greater than 2 times the background value.
8. The detection method according to claim 5, wherein the receptor microsphere coupled with the anti-avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody is diluted with 1 xAaphALISA buffer solution according to a volume ratio of 1: 50-1: 100; the biotin-labeled avian influenza virus H9 type envelope protein hemagglutinin monoclonal antibody and a 1xAphaLISA buffer solution are diluted according to the volume ratio of 1: 100-1: 200.
9. The detection method according to claim 5, wherein the AlphaScreen/Lisa detector detects the excitation light with a wavelength of 680nm and the emission light with a wavelength of 520-620 nm.
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| CN117887797A (en) * | 2023-12-27 | 2024-04-16 | 中国食品药品检定研究院 | Clostridium neurotoxin titer detection method |
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