CN116718771B - Influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigen detection kit - Google Patents
Influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigen detection kit Download PDFInfo
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Abstract
The invention relates to the technical field of detection kits, and provides a kit for detecting influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigens, which comprises a first detection reagent strip and a second detection reagent strip; the first detection reagent strip is an influenza A virus and influenza B virus antigen detection reagent strip; the second detection reagent strip is a respiratory syncytial virus and novel coronavirus antigen detection reagent strip. Through the technical scheme, the problem of technical blank of detecting four viruses simultaneously in the prior art is solved.
Description
Technical Field
The invention relates to the technical field of detection kits, in particular to a kit for detecting influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigens.
Background
Influenza virus is called influenza virus for short, is a pathogen for causing influenza, and the influenza is an acute respiratory infectious disease caused by influenza virus A (A), influenza virus B (B) and influenza virus C (C), and has strong infectivity, quick transmission, short latency period and high incidence rate. Currently, the infection of people mainly comprises H1N1 and H3N2 subtype in influenza A virus and Victoria and Yamagata system in influenza B virus, the clinical manifestations of the infection are mainly systemic poisoning symptoms such as hyperpyrexia, hypodynamia, headache, cough, systemic muscular soreness and the like, and respiratory tract symptoms are lighter.
Respiratory Syncytial Virus (RSV) belongs to the genus pneumovirus of the family paramyxoviridae, and has only one serotype, mainly causing lower respiratory tract infections such as bronchiolitis and pneumonia in infants under 6 months, and upper respiratory tract infections such as rhinitis, colds, and the like in older children and adults.
The novel coronavirus is a coronavirus which is never found in human body before, and the human body is infected with the coronavirus and has common signs such as respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like.
At present, the establishment of a rapid, simple and convenient kit for simultaneously detecting influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus is particularly important for timely diagnosing and treating virus infection.
Disclosure of Invention
The invention provides an influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigen detection kit, which solves the problem of the technical blank of detecting four viruses simultaneously in the prior art.
The technical scheme of the invention is as follows:
an influenza A virus, an influenza B virus, a respiratory syncytial virus and a novel coronavirus antigen detection kit comprises a first detection reagent strip and a second detection reagent strip;
the first detection reagent strip is an influenza A virus and influenza B virus antigen detection reagent strip;
the second detection reagent strip is a respiratory syncytial virus and novel coronavirus antigen detection reagent strip.
As a further aspect, the first detection reagent strip includes a first reaction pad and a first binding pad;
the first reaction pad comprises an influenza A virus antigen detection line, an influenza B virus antigen detection line and a first quality control line; the coating liquid of the influenza A virus antigen detection line and the influenza B virus antigen detection line is 50mM HEPES buffer solution, 1% Tetronic 1307 and 0.02% BSA; the first binding pad comprises a colloidal gold-labeled influenza A virus monoclonal antibody and a colloidal gold-labeled influenza B virus monoclonal antibody.
As a further aspect, the second test reagent strip includes a second reaction pad and a second binding pad;
the second reaction pad comprises a respiratory syncytial virus antigen detection line, a novel coronavirus antigen detection line and a second quality control line;
the second binding pad comprises a colloidal gold-labeled respiratory syncytial virus monoclonal antibody and a colloidal gold-labeled novel coronavirus monoclonal antibody.
As a further technical scheme, the first quality control line and the second quality control line are coated with goat anti-mouse IgG polyclonal antibodies.
The influenza A virus antigen detection line and the influenza B virus antigen detection line are respectively an A line and a B line, the respiratory syncytial virus antigen detection line and the novel coronavirus antigen detection line are respectively an R line and an S line, and the first quality control line and the second quality control line are both C lines.
As a further technical scheme, the influenza a virus monoclonal antibody is coated on the influenza a virus antigen detection line, the concentration is 0.8-1.2mg/mL, and the preferable concentration is 1.0mg/mL;
the influenza B virus monoclonal antibody is coated on the influenza B virus antigen detection line, the concentration is 1.0-1.5mg/mL, and the preferable concentration is 1.2mg/mL.
As a further technical scheme, the respiratory syncytial virus monoclonal antibody is coated on the respiratory syncytial virus antigen detection line, the concentration is 1.0-1.5mg/mL, and the preferable concentration is 1.2mg/mL;
the novel coronavirus antigen detection line is coated with novel coronavirus monoclonal antibody, the concentration is 1.2-1.8mg/mL, and the preferable concentration is 1.5mg/mL.
As a further technical scheme, the concentration of the goat anti-mouse IgG polyclonal antibody is 1.0-1.5mg/mL, and the preferable concentration is 1.2mg/mL.
As a further technical scheme, the kit further comprises a sample treatment liquid, wherein the sample treatment liquid comprises phosphate buffer solution and tween-20.
As a further technical scheme, the pH of the phosphate buffer solution (PBS solution) is 7.4, the concentration is 0.1mol/L, and the mass concentration of the Tween-20 is 0.5%.
In the invention, PBS solution with pH of 7.4 and concentration of 0.1mol/L and Tween-20 with weight percent of 0.5 are selected as components of the sample diluent, so that false positive or false negative results can be avoided when a sample is detected, and the detection accuracy is improved.
As a further technical scheme, the kit further comprises a sample pad, and the mouse IgG blocking agent is added on the sample pad.
According to the invention, the respiratory syncytial virus and the novel coronavirus are arranged on the same reagent strip, and the false positive condition is found in the test process, so that the false positive phenomenon is eliminated by adding the mouse IgG blocker into the sample pad, and the detection accuracy is improved.
The working principle and the beneficial effects of the invention are as follows:
the invention provides a kit for qualitatively detecting influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigens in a nose swab sample in vitro, which can be used for medical institutions and home self-detection. The kit comprises two reagent strips, namely an influenza A virus/influenza B virus antigen detection reagent strip and a respiratory syncytial virus/novel coronavirus antigen detection reagent strip, and can detect influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigen in a human nasal swab sample at the same time by one sample adding, so that the kit is convenient to sample, does not need blood sampling, improves the sampling efficiency, saves time and reduces exposure risk. The detection method is simple to operate, four products can be detected by one sample adding, the result can be interpreted within 10-30 min, compared with the existing detection kit, the detection result can be read within ten min at maximum, and the detection time is shortened.
Drawings
The invention will be described in further detail with reference to the drawings and the detailed description.
FIG. 1 is a schematic diagram of a kit of the present invention;
FIG. 2 shows the detection results of the kit of the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The English abbreviation of influenza A virus is called Flu A, the English abbreviation of influenza B virus is called Flu B, the English abbreviation of novel coronavirus is called 2019-nCoV, and the English abbreviation of respiratory syncytial virus is called RSV.
Example 1
As shown in fig. 1, the present embodiment provides an influenza a virus, an influenza b virus, a respiratory syncytial virus and a novel coronavirus antigen detection kit, comprising a first detection reagent strip and a second detection reagent strip;
the first detection reagent strip comprises a first reaction pad and a first binding pad, the first reaction pad comprises an influenza A virus antigen detection line (marked as A line), an influenza B virus antigen detection line (marked as B line) and a first quality control line (marked as C line), and the first binding pad comprises a colloidal gold marked influenza A virus monoclonal antibody and a colloidal gold marked influenza B virus monoclonal antibody;
the second detection reagent strip comprises a second reaction pad and a second combination pad, the second reaction pad comprises a respiratory syncytial virus antigen detection line (marked as R line), a novel coronavirus antigen detection line (marked as S line) and a second quality control line (marked as C line), and the second combination pad comprises a colloidal gold marked respiratory syncytial virus monoclonal antibody and a colloidal gold marked novel coronavirus monoclonal antibody;
the first quality control line and the second quality control line are coated with goat anti-mouse IgG polyclonal antibodies;
the kit further comprises a sample pad, wherein a mouse IgG blocking agent is added on the sample pad;
the kit also included 500. Mu.L of sample treatment fluid, which included phosphate buffer at pH 7.4 at a concentration of 0.1mol/L and 0.5wt% Tween-20.
The kit was prepared as follows:
(1) Preparing a gold-labeled antibody:
the colloidal gold-labeled Flu A monoclonal antibody, the colloidal gold-labeled Flu B monoclonal antibody, the colloidal gold-labeled 2019-nCoV monoclonal antibody and the colloidal gold-labeled RSV monoclonal antibody are prepared respectively according to the following methods:
a1, adding 12. Mu.L/mL pH-adjusting solution (2 wt% K) to the colloidal gold solution 2 CO 3 Solution), namely adding 12 mu L of pH regulating solution into each mL of colloidal gold solution, and shaking uniformly for 10min;
a2, adding the antibody into the solution according to the proportion of 15 mug antibody/mL colloidal gold, and shaking for 30min after the addition;
a3, adding a blocking agent (1 wt% BSA) according to the proportion of 10 mu L of the blocking agent/mL of colloidal gold, stirring while adding the blocking agent, and standing for 15min after adding;
and A4, centrifuging at 10000rpm for 30min at 4 ℃, discarding supernatant, and redissolving the red precipitate to 50% of the original volume by using a redissolution to obtain the marked colloidal gold.
The preparation method of the compound solution comprises the following steps: 0.50+ -0.01 g Tris, 5.00+ -0.01 g sucrose, 0.50+ -0.01 g PVP, 0.30+ -0.01 g Casein are weighed, and the process water is processed to 100mL for standby.
(2) Preparing a kit:
b1, sticking an NC film on a PVC plate, and sticking water absorbing paper on the PVC plate to press the film;
b2, setting parameters of a film scribing instrument to be 1.0 mu L/cm of a C line and 1.0 mu L/cm of an A/B/R/S line; adjusting instrument parameters and positions of C/A/B/R/S lines according to the colloidal gold coating post operation rules, placing the pasted PVC plate on a film drawing instrument for drawing lines, and drying to obtain a film plate;
wherein the C line: goat anti-mouse IgG polyclonal antibody with coating solution concentration of 1.2mg/mL;
r line: RSV monoclonal antibody with the concentration of coating liquid of 1.2mg/mL; the coating liquid is TRIS-HCl buffer solution
S line: the concentration of the coating liquid is 1.5mg/mL of COVID-19 monoclonal antibody; the coating liquid is TRIS-HCl buffer solution;
line a: flu A monoclonal antibody with the concentration of the coating liquid of 1.0mg/mL; the coating was 50mM HEPES buffer, 1wt% Tetronic 1307, 0.02wt% BSA;
line B: flu B monoclonal antibody with the concentration of the coating liquid of 1.2mg/mL; the coating was 50mM HEPES buffer, 1wt% Tetronic 1307, 0.02wt% BSA;
b3, gold-labeled antibody is prepared according to 20 mu L/cm 2 Spreading on the bonding pad, and drying;
b4, murine IgG blocker was used at 3.2. Mu.g/cm 2 Spreading on sample pad, and drying;
and B5, assembling the membrane plate, the combination pad, the sample pad, the water absorption pad and the sample treatment fluid into a finished product.
Example 2
The detection method of the kit comprises the following steps:
placing the swab after collecting the sample in a sampling container containing sample treatment fluid immediately, attaching the swab head to the bottom and side of the collection tube, and rotating the swab 10 times (at least 30 seconds);
gently squeezing the sampling container to drop 6 drops of samples into the sample adding holes of the detection card;
results were interpreted for 10-30 minutes, and results were not valid for 10 minutes before and 30 minutes after.
The several detection results encountered during detection are shown in fig. 2, and the specific analysis is as follows:
(1) Positive: two or three red bands appear in each test strip. One or two are positioned in the detection area, and the other is positioned in the quality control area. The color of the strip of the detection area can be dark or light, and the detection area is positive.
(1) Influenza a virus and influenza b virus antigen test strip
Influenza a positive: lines C and a show red bands, indicating positive influenza a virus antigens.
Influenza b positive: lines C and B show red bands, indicating positive influenza B virus antigen.
Influenza a and b positive: lines C, A and B all show red bands, indicating positive influenza A and B antigens.
(2) Respiratory syncytial virus, novel coronavirus (2019-nCoV) antigen test strip
Respiratory syncytial virus positive: the C and R lines show red bands, indicating positive respiratory syncytial virus antigens.
Novel coronavirus positive: lines C and S show red bands, indicating that the novel coronavirus antigen is positive.
Respiratory syncytial virus and novel coronavirus positive: the C line, R line and S line all show red bands, indicating that the novel coronavirus and respiratory syncytial virus antigens are positive.
(2) Negative: only line C shows a red band, indicating a negative result.
(3) Invalidation: if after testing, the C line is not visible in the window, the result is considered invalid. The occurrence of an invalid result indicates incorrect handling or a failure of the kit. It is recommended to repeat the test with a new test card.
Influence of the sample treatment fluid on the Experimental group I
Comparative example 1
The sample treatment solution was a PBS solution of 0.1mol/L as compared with example 1, and the other steps were the same as in example 1.
Comparative example 2
The sample treatment solution was 0.1mol/L PBS solution+0.50 wt% BSA as in example 1, and the other steps were the same as in example 1.
Comparative example 3
The sample treatment solution was 0.1mol/L PBS+0.30wt% Tween-20 as in example 1, and the other steps were the same as in example 1.
Comparative example 4
The sample treatment solution was 0.1mol/L PBS+0.80wt% Tween-20 as in example 1, and the other steps were the same as in example 1.
The experimental method comprises the following steps:
the swab heads were placed against the bottom and sides of the collection tube, the swab heads were rotated 10 times (at least 30 seconds) to squeeze the swab head liquid through the outer wall of the collector, and the swab was discarded and the collector dripper was capped. Swab samples were treated with the sample treatment solutions of example 1, comparative example 2, comparative example 3, and comparative example 4, respectively, and the test was performed in accordance with the detection method of example 2, and each sample was repeated 3 times.
Table 1 results of the tests of example 1, comparative example 1, and comparative example 2
Description: "+++". Representation of actual detection the result is strong positive; "++" indicates that the actual detection result is positive; "+" indicates that the actual detection result is weak positive; "-" indicates that the actual detection result is negative.
The experimental result shows that when the sample treatment solution is 0.1mol/L PBS solution for detection, a false positive result appears in the negative sample; when the sample treatment solution is PBS solution of 0.1mol/L and 0.5wt% BSA, the sensitivity of the product is reduced, and a false negative result appears; the sample treatment solution is PBS solution of 0.1mol/L and 0.5wt% Tween-20, and no false negative or false positive result appears in the detection sample. Thus, a PBS solution of 0.1mol/L and Tween-20 of 0.5wt% were selected as the sample treatment solution.
Table 2 the results of the tests of example 1, comparative example 3, comparative example 4
Description: "+++". Representation of actual detection the result is strong positive; "++" indicates that the actual detection result is positive; "+" indicates that the actual detection result is weak positive; "-" indicates that the actual detection result is negative.
The experimental result shows that when the sample treatment solution is PBS solution of 0.1mol/L and Tween-20 of 0.30wt% is used for treating the sample detection, a false positive result appears in the negative sample; when the sample treatment solution is 0.1mol/L PBS solution and 0.80wt% Tween-20, the sensitivity of the product is reduced, and a false negative result appears; the sample treatment solution is PBS solution of 0.1mol/L and 0.5wt% Tween-20, and no false negative or false positive result appears in the detection sample. Thus, a PBS solution of 0.1mol/L and Tween-20 of 0.5wt% were selected as the sample treatment solution.
Influence of blocking Agents in Experimental group II
The respiratory syncytial virus and the novel coronavirus are respectively arranged on two reagent strips, and the invention discovers that the respiratory syncytial virus and the novel coronavirus are arranged on one reagent strip in the experimental process, and the false positive condition occurs in the detection process.
In order to eliminate false positive phenomenon of the product during the combined detection of the novel coronavirus and the respiratory syncytial virus, the components of the sample pad in the product are studied.
Comparative example 5
No mouse IgG blocking agent was added to the sample pad, and the other was the same as in example 1.
The experimental method comprises the following steps:
120 samples of healthy human nasal swabs were tested using the kits of comparative example 5 and example 1, respectively, to investigate whether the test results could eliminate or reduce the development of false positive after the addition of the murine IgG blocker to the sample pad.
TABLE 3 detection results of healthy human nasal swab samples
| Numbering device | Comparative example 5 | Example 1 | Numbering device | Comparative example 5 | Example 1 | Numbering device | Comparative example 5 | Example 1 |
| N1 | + | - | N41 | - | - | N81 | - | - |
| N2 | - | - | N42 | - | - | N82 | - | - |
| N3 | + | - | N43 | - | - | N83 | + | - |
| N4 | - | - | N44 | - | - | N84 | - | - |
| N5 | + | - | N45 | - | - | N85 | - | - |
| N6 | - | - | N46 | - | - | N86 | - | - |
| N7 | - | - | N47 | - | - | N87 | - | - |
| N8 | - | - | N48 | - | - | N88 | + | - |
| N9 | - | - | N49 | - | - | N89 | - | - |
| N10 | - | - | N50 | - | - | N90 | - | - |
| N11 | - | - | N51 | + | - | N91 | - | - |
| N12 | - | - | N52 | + | - | N92 | - | - |
| N13 | - | - | N53 | - | - | N93 | - | - |
| N14 | - | - | N54 | - | - | N94 | - | - |
| N15 | - | - | N55 | - | - | N95 | - | - |
| N16 | - | - | N56 | - | - | N96 | - | - |
| N17 | - | - | N57 | + | - | N97 | - | - |
| N18 | - | - | N58 | - | - | N98 | - | - |
| N19 | - | - | N59 | - | - | N99 | + | - |
| N20 | - | - | N60 | - | - | N100 | - | - |
| N21 | + | - | N61 | + | - | N101 | - | - |
| N22 | - | - | N62 | - | - | N102 | - | - |
| N23 | - | - | N63 | - | - | N103 | - | - |
| N24 | - | - | N64 | - | - | N104 | - | - |
| N25 | - | - | N65 | - | - | N105 | - | - |
| N26 | + | - | N66 | - | - | N106 | - | - |
| N27 | - | - | N67 | + | - | N107 | - | - |
| N28 | - | - | N68 | - | - | N108 | - | - |
| N29 | - | - | N69 | - | - | N109 | - | - |
| N30 | - | - | N70 | - | - | N110 | - | - |
| N31 | - | - | N71 | - | - | N111 | - | - |
| N32 | - | - | N72 | - | - | N112 | - | - |
| N33 | - | - | N73 | - | - | N113 | + | - |
| N34 | - | - | N74 | - | - | N114 | - | - |
| N35 | - | - | N75 | - | - | N115 | - | - |
| N36 | - | - | N76 | - | - | N116 | - | - |
| N37 | - | - | N77 | - | - | N117 | - | - |
| N38 | - | - | N78 | - | - | N118 | - | - |
| N39 | - | - | N79 | + | - | N119 | - | - |
| N40 | - | - | N80 | - | - | N120 | - | - |
In comparative example 5, 15 samples showed positive results, the remaining samples were negative results, and the test results of the kit in example 1 were all negative results. After the mouse IgG blocking agent is added into the sample pad, the false positive phenomenon of the product during the combined detection of the novel coronavirus and the respiratory syncytial virus can be eliminated, so that the mouse IgG blocking agent is added into the treatment fluid of the sample pad.
Influence of coating liquids on test group III, line A and line B
Comparative example 6
In comparison with example 1, both the coating solutions for line A and line B were TRIS-HCl buffer.
The experimental method comprises the following steps:
positive and weak positive samples and negative samples in Flu A and Flu B are respectively prepared, the swab head is clung to the bottom and the side surface of the collecting pipe, the swab is rotated for 10 times (at least 30 seconds), the swab head liquid is squeezed by hands through the outer wall of the collector, the swab is discarded, and the collector dripper is covered. The test was performed according to the detection method of example 2 using the kits of example 1 and comparative example 5, respectively, and each sample was repeated 3 times.
Table 4 comparison of the results of the measurements of example 1 and comparative example 5
Description: "+++". Representation of actual detection the result is strong positive; "++" indicates that the actual detection result is positive; "+" indicates that the actual detection result is weak positive; "-" indicates that the actual detection result is negative.
As is clear from the experimental results, in comparative example 5, TRIS-HCl buffer was used as coating liquid, and influenza A virus and influenza B virus were put on the same reagent strip, and negative samples were detected, and false positive was generated, but false positive was eliminated by using the coating liquid in example 1.
Experiments of experimental examples 1 to 4 were performed using the kit obtained in example 1.
Experimental example 1, minimum detection limit
(1) Initial LOD of lowest detection limit
1 sample of virus was selected and diluted in a gradient using a nasal swab negative sample to give 1 sample per concentration gradient. For each gradient sample, 1 lot number of product assays were used, and 3 replicates were tested for each concentration.
The test results of four virus samples of Flu A inactivated virus A1 (2009H 1N 1), flu B inactivated virus B1 (B/Victoria), RSV inactivated virus R1 (respiratory syncytial virus type B), 2019-nCoV inactivated virus L1 (New coronavirus culture 1#) are shown in tables 5-8, respectively.
TABLE 5 Flu A initial LOD detection results
TABLE 6 Flu B initial LOD detection results
TABLE 7 initial LOD detection results of RSV
TABLE 8 2019-nCoV initial LOD detection results
The sample detection results of different concentration gradients can be obtained, and the initial LOD of Flu A is 2 multiplied by 10 3 TCID 50 Per mL, flu B initial LOD of 500TCID 50 Per mL, RSV initial LOD of 2.4X10 4 TCID 50 1/mL, 2019-nCoV initial LOD of 800TCID 50 /mL。
(2) Determination of minimum detection limit
3 parts of Flu A inactivated viruses A1 (2009H 1N 1), A2 (seasonal H1N 1) and A3 (type A H3N 2) were selected, and the nasal swab negative sample was used for gradient dilution, and the concentration after dilution was 4×10 3 TCID 50 /mL、2×10 3 TCID 50 /mL、1×10 3 TCID 50 /mL、500TCID 50 /mL、250TCID 50 3 replicates per mL were obtained, 3 samples per concentration gradient per virus sample. 3 samples obtained by repeating 3 times for each concentration were tested sequentially using 3 batches of the product, 20 persons were tested for each sample, and the detection results of the minimum detection limit determination of influenza a virus are shown in table 9.
TABLE 9 determination of the minimum detection limit for influenza A virus
2 parts of Flu B inactivated viruses B1 (B/Victoria) and B2 (B/Yamagata) were selected, and the samples were subjected to gradient dilution using a nasal swab negative sample at a concentration of 1X 10 3 TCID 50 /mL、500TCID 50 /mL、250TCID 50 /mL、125TCID 50 /mL、62.5TCID 50 3 replicates per mL were obtained, 3 samples per concentration gradient per virus sample. 3 samples obtained by repeating 3 times of each concentration were tested sequentially using 3 batches of products, 20 persons were tested repeatedly for each sample, and the detection results of the minimum detection limit determination of influenza b virus are shown in table 10.
TABLE 10 determination of the detection results for the lowest detection limit of influenza B virus
2 RSV inactivated viruses R1 (respiratory syncytial virus type B) and R2 (respiratory syncytial virus type A) were selected, and were subjected to gradient dilution with a nasal swab negative sample at a concentration of 4.8X10 4 TCID 50 /mL、2.4×10 4 TCID 50 /mL、1.2×10 4 TCID 50 /mL、6×10 3 TCID 50 /mL、3×10 3 TCID 50 3 replicates per mL were obtained, 3 samples per concentration gradient per virus sample. 3 samples obtained by repeating 3 times of each concentration were tested sequentially using 3 batches of the product, 20 persons were tested repeatedly for each sample, and the detection results of the minimum detection limit determination of respiratory syncytial virus are shown in table 11.
TABLE 11 determination of the minimum detection limit for respiratory syncytial virus
3 parts of 2019-nCoV inactivated virus L2 (novel coronavirus Alpha strain), L3 (novel coronavirus Beta strain), L4 (novel coronavirus Delta strain) were selected and used with nasal swabsPerforming gradient dilution on the negative sample, wherein the concentration after dilution is 1.6X10 3 TCID 50 /mL、800TCID 50 /mL、400TCID 50 /mL、200TCID 50 /mL、100TCID 50 3 replicates per mL were obtained, 3 samples per concentration gradient per virus sample. 3 samples obtained by repeating 3 times of each concentration were tested sequentially using 3 batches of products, each sample was tested repeatedly for 20 persons, and the detection results of the novel coronavirus minimum detection limit determination are shown in table 12.
TABLE 12 determination of the detection results of novel coronavirus minimum detection limit
Flu A inactivated virus was diluted to 1X 10 using nasal swab negative samples 3 TCID 50 The positive detection rate can reach more than 95% in the case of/mL, so that the ratio of 1X 10 3 TCID 50 As the minimum detection limit for Flu A,/mL. Flu B inactivated virus was diluted to 250TCID using a nasal swab negative sample 50 The positive detection rate can reach more than 95% in the process of/mL, so that the detection rate of the reagent reaches 250TCID 50 As the minimum detection limit of Flu B,/mL. RSV inactivated virus diluted to 1.2 x 10 using nasal swab negative samples 4 TCID 50 The positive detection rate can reach more than 95% in the case of/mL, so that the ratio of 1.2X10 4 TCID 50 As the minimum detection limit of RSV,/mL. 2019-nCoV inactivated virus was diluted to 200TCID using nasal swab negative samples 50 at/mL, the positive detection rate can reach more than 95%, so that 200TCID is obtained 50 the/mL is taken as the minimum detection limit of 2019-nCoV.
(3) Verification of minimum detection limit
3 parts of Flu A inactivated virus A1 (2009H 1N 1), A2 (seasonal H1N 1), A3 (influenza A H3N2 virus) were selected for dilution to a minimum detection limit of 1X 10 using nasal swab negative samples 3 TCID 50 /mL. The diluted samples were each examined for 20 persons using 3 batches of the product, and the results were observed.
TABLE 13 minimum detection limit verification test results for Flu A Virus strain
3 parts of Flu B inactivated virus B1 (B/Victoria), B2 (B/Yamagata), B3 (B/Washington) were selected and diluted to a minimum detection limit of 250TCID using nasal swab negative samples 50 /mL. The diluted samples were each examined for 20 persons using 3 batches of the product, and the results were observed.
TABLE 14 minimum detection limit verification test results for Flu B Virus strain
2 RSV inactivated viruses R1 (respiratory syncytial virus type B) and R2 (respiratory syncytial virus type A) were selected and diluted to a minimum detection limit of 1.2X10 using nasal swab negative samples 4 TCID 50 /mL. The diluted samples were each examined for 20 persons using 3 batches of the product, and the results were observed.
TABLE 15 minimum detection limit verification test results for RSV strains
3 parts of 2019-nCoV inactivated virus L5 (novel coronavirus P.1 strain), L6 (novel coronavirus Kappa strain), L7 (novel coronavirus Omicron strain) were selected and diluted to a minimum detection limit of 200TCID using a nasal swab negative sample 50 /mL. The diluted samples were each examined for 20 persons using 3 batches of the product, and the results were observed.
Table 16 2019-nCoV virus strain minimum detection limit verification test results
Flu A inactivated virus (2009H 1N1, seasonal H1N1, H3N2 influenza A virus) was diluted to the lowest detection limit (1X 10) using nasal swab negative samples 3 TCID 50 /mL), the positive detection rate can reach more than 95%; flu B inactivated virus (B/Victoria, B/Yamagata, B/Washington) was diluted to minimum detection limit (250 TCID) using nasal swab negative samples 50 /mL), the positive detection rate can reach more than 95%; RSV inactivated virus (respiratory syncytial virus type B, respiratory syncytial virus type a) was diluted to the minimum detection limit (1.2x10) using a nasal swab negative sample 4 TCID 50 /mL), the positive detection rate can reach 95% or more; 2019-nCoV inactivated virus (novel coronavirus P.1 strain, novel coronavirus Kappa strain, novel coronavirus Omicron strain) was diluted to minimum detection limit (200 TCID) using a nasal swab negative sample 50 /mL), the positive detection rate can reach more than 95%.
Therefore, the minimum detection limit concentration of Flu A in the kit of the present invention is 1X 10 3 TCID 50 /mL; the minimum detection limit concentration of Flu B is 250TCID 50 /mL; the minimum detection limit concentration of RSV was 1.2X10 4 TCID 50 /mL; the minimum detection limit concentration of 2019-nCoV is 200TCID 50 /mL。
Experimental example 2, high dose HOOK effect (HOOK)
Inactivated influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus (2019-nCoV) cultures were diluted with a nasal swab negative sample matrix, respectively. Three batches of product were drawn at random, all samples after dilution were tested 3 times per sample.
TABLE 17 Flu A HOOK test results
TABLE 18 Flu B HOOK test results
TABLE 19 RSV HOOK test results
Table 20 2019-nCoV HOOK test results
The test results prove that: as the concentration of the virus culture increased, the color depth was increased, and Flu A reached 1.26X10 6 TCID 50 concentration/mL, flu B reached 7.2X10 6 TCID 50 Concentration of RSV reaches 4.8X10 7 TCID 50 Concentration of 2019-nCoV reaches 3.8X10 6 TCID 50 At a concentration of/mL, the coloration is changed from dark to light, and no HOOK effect is observed in the reagent at the concentration.
Experimental example 3 Cross-validation
The product is verified by the common cross-reactive pathogenic microorganisms which are easy to cause the same and similar symptoms clinically in the following table, and the virus concentration is l multiplied by 10 6 At PFU/mL, other bacteria and pathogens were found to be at l X10 concentrations 6 At CFU/mL, the results indicate that the product has good specificity for the cross-reactants listed below. The kit provided by the invention has negative detection results on other pathogenic microorganisms, and has strong specificity, and no cross reaction is proved between the kit and other pathogenic microorganisms.
TABLE 21 common Cross-reacting pathogenic microorganisms
Experimental example 4, interference reaction
The product is subjected to interference verification by the highest blood concentration of clinical common therapeutic drugs in the following table under the normal dosage, and the result shows that the product has good anti-interference performance.
Clinical common therapeutic medicine for exterior syndrome 22
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (6)
1. An influenza A virus, an influenza B virus, a respiratory syncytial virus and a novel coronavirus antigen detection kit is characterized by comprising a first detection reagent strip and a second detection reagent strip;
the first detection reagent strip is an influenza A virus and influenza B virus antigen detection reagent strip;
the second detection reagent strip is a respiratory syncytial virus and novel coronavirus antigen detection reagent strip;
the first detection reagent strip comprises a first reaction pad and a first binding pad;
the first reaction pad comprises an influenza A virus antigen detection line, an influenza B virus antigen detection line and a first quality control line; the coating liquid of the influenza A virus antigen detection line and the influenza B virus antigen detection line is 50mM HEPES buffer solution, 1% Tetronic 1307 and 0.02% BSA;
the first binding pad comprises a colloidal gold-labeled influenza A virus monoclonal antibody and a colloidal gold-labeled influenza B virus monoclonal antibody;
the second detection reagent strip comprises a second reaction pad and a second binding pad;
the second reaction pad comprises a respiratory syncytial virus antigen detection line, a novel coronavirus antigen detection line and a second quality control line; the coating liquid of the respiratory syncytial virus antigen detection line and the novel coronavirus antigen detection line is TRIS-HCl buffer solution;
the second binding pad comprises a colloidal gold-labeled respiratory syncytial virus monoclonal antibody and a colloidal gold-labeled novel coronavirus monoclonal antibody;
the sample processing device also comprises a sample processing liquid, wherein the sample processing liquid comprises a phosphate buffer solution and tween-20; the mass concentration of the tween-20 is 0.5%;
also included is a sample pad with a murine IgG blocker added thereto.
2. The kit of claim 1, wherein the first and second control lines are coated with goat anti-mouse IgG polyclonal antibodies.
3. The kit according to claim 1, wherein the influenza a virus antigen detection line is coated with an influenza a virus monoclonal antibody at a concentration of 0.8-1.2mg/mL;
the influenza B virus antigen detection line is coated with an influenza B virus monoclonal antibody, and the concentration is 1.0-1.5mg/mL.
4. The kit according to claim 1, wherein the respiratory syncytial virus antigen detection line is coated with respiratory syncytial virus monoclonal antibody at a concentration of 1.0-1.5mg/mL;
the novel coronavirus antigen detection line is coated with novel coronavirus monoclonal antibody, and the concentration is 1.2-1.8mg/mL.
5. The kit of claim 2, wherein the goat anti-mouse IgG polyclonal antibody is at a concentration of 1.0-1.5mg/mL.
6. The kit according to claim 1, wherein the phosphate buffer has a pH of 7.4 and a concentration of 0.1mol/L.
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