CN115453116A - Immunochromatography joint inspection kit for simultaneously detecting six respiratory pathogens and preparation method thereof - Google Patents

Abstract

The invention discloses an immunochromatography joint inspection kit for simultaneously detecting six respiratory disease pathogens and a preparation method thereof. The kit comprises a reagent card and a fluorescent particle mixed solution, wherein the reagent card comprises a reagent strip, the reagent strip comprises a PVC base plate, an integrated pad, a nitrocellulose membrane and absorbent paper, and the integrated pad, the nitrocellulose membrane and the absorbent paper are adhered to the base plate in a lap joint mode in sequence; the nitrocellulose membrane is sequentially provided with a quality control line coated with a DNP monoclonal antibody and detection lines 1, 2, 3, 4, 5 and 6 respectively coated with influenza A, influenza B, adenovirus, syncytial virus, mycoplasma pneumoniae and parainfluenza monoclonal antibody; the fluorescent particle mixed solution is formed by mixing an influenza A antibody-microsphere compound, an influenza B antibody-microsphere compound, an adenovirus antibody-microsphere compound, a syncytial virus antibody-microsphere compound, a mycoplasma pneumoniae antibody-microsphere compound, a parainfluenza antibody-microsphere compound and a DNP-BSA-microsphere compound. The joint detection kit can simultaneously detect six respiratory pathogens of influenza A, influenza B, adenovirus, syncytial virus, mycoplasma pneumoniae and parainfluenza virus, can detect the respiratory pathogens by only collecting nasal swabs and pharyngeal swabs, realizes non-wound and high specificity, and can be used for early diagnosis of diseases.

Description

An immunochromatographic joint detection kit and its preparation for simultaneously detecting six respiratory pathogens preparation method

technical field

The invention belongs to the technical field of in vitro diagnosis, in particular to an immunochromatographic joint detection kit and a preparation method for simultaneously detecting six respiratory pathogens

Background technique

Influenza virus is a single-stranded negative-sense RNA virus belonging to the family Orthomyxoviridae. According to the genetic characteristics and antigenicity of the viral nucleoprotein and matrix protein, influenza viruses can be divided into four types: A, B, C, and D. Influenza A viruses can be divided into subtypes based on differences in the hemagglutinin (HA) and neuraminidase (NA) antigens on the viral envelope. H5N1 and H7N9 avian influenza viruses infect humans and cause serious lesions, and are called highly pathogenic avian influenza viruses. According to WHO statistics, influenza A virus and influenza B virus infection lead to as many as 200,000 hospitalizations and 30,000-50,000 deaths each year, making it the main cause of death after acquired immunodeficiency syndrome.

Parainfluenza virus (PIV) belongs to Paramyxoviridae and Respirovirus. Its biological characteristics are similar to those of paramyxoviruses, and it has typical structural features of paramyxoviruses. Divided into 4 serotypes according to heredity and antigenicity, namely: PIV-1, PIV-2, PIV-3 and PIV-4, of which PIV-1, PIV-2 and PIV-3 are the main types of human infection . HPIV-3 often breaks out in spring and summer, and usually causes lower respiratory tract infections in infants. It is the second most common pathogen after RSV that causes bronchiolitis and pneumonia in infants under 6 months old. A nearly 4-year survey from New Vaccine Surveillance Network (NVSN) showed that among children ≤ 5 years old (nearly 2,800) hospitalized for fever and acute respiratory diseases, about 190 were caused by HPIV infection, accounted for nearly 7%, of which children infected with PIV-3 accounted for 50%.

Respiratory syncytial virus (RSV) is a non-segmental, single-stranded negative-sense RNA virus belonging to the family Paramyxoviridae and the genus Pneumovirus. It is one of the main pathogens causing acute respiratory infections in infants, and its incubation period is usually For 4-6d. Due to immature immune function development, RSV infection in infants aged 2-6 months can cause severe bronchiolitis and pneumonia, and RSV infection is also one of the risk factors for children with bronchial asthma. According to statistics, 24 of every 1,000 hospitalized infants and young children in the world are caused by direct RSV infection, and 66,000-199,000 children die each year due to RSV infection, and 99% of them die under the age of 5. Studies in recent years have shown that adults with immunodeficiency are also susceptible to RSV. It is estimated that 3%-10% of adults will be infected with RSV every year and develop severe lower respiratory tract infections. Therefore, in-depth study of RSV early diagnosis technology has important and positive significance for its prevention and treatment.

Adenovirus (AdV) is a non-enveloped, double-stranded DNA virus that can be transmitted through aerosol particle inhalation, fecal contamination, or exposure to infected tissue or blood. After AdV infects the upper or lower respiratory tract, it is very likely to develop pneumonia, acute respiratory distress syndrome (ARDS), or diffuse infection. Patients with ARDS present with variable bilateral lung opacity, resembling pulmonary edema on chest x-ray or CT scan, and have a mortality rate of 27%-45%. Early and timely identification of pathogens is of great help to treatment and improvement of prognosis.

Mycoplasma pneumoniae (mycoplasma pneumoniae, MP) belongs to Mycoplasma, Mycoplasma, Mycoplasma, Mycoplasma, and is the smallest microorganism capable of self-replication and survival without living cells in vitro. The clinical manifestations of MP infection are diverse, ranging from asymptomatic to nasopharyngitis, pharyngeal tonsillitis, tracheobronchitis, bronchiolitis, and pneumonia, and severe pneumonia can occur in severe cases. MP pneumonia accounts for approximately 10%-40% of community-acquired pneumonia in children. Resulting in 10% of children admitted to the ICU. Because the clinical symptoms of MP infection vary in severity and are not typical, some patients present with extrapulmonary symptoms as the first symptom, and imaging examinations lack specificity, so MP infection is often misdiagnosed or missed.

Acute respiratory infection (ART) is one of the main causes of acute and fatal diseases worldwide. Timely and accurate diagnosis of etiology at the early stage of viral infection will help medical staff to accurately grasp the patient's condition. At present, the methods for detecting respiratory pathogens mainly include virus isolation and culture, antigen detection, serum immunology antibody detection, and nucleic acid detection. Serotype detection cannot be used for early diagnosis of diseases. At present, nucleic acid detection reagents have become an important detection method for respiratory virus infection, but nucleic acid detection generally takes at least 2 hours. The requirements of the experimental environment are high, and the operation is relatively complicated, and the operator needs to have certain operating skills.

Respiratory tract infections are seasonal, contagious, and regional. There are many types of respiratory pathogens. Most respiratory pathogen detection chromatography reagents on the market can only detect a single pathogen, or splice together several rapid detection test strips for the detection of a single pathogen. There is no immunochromatographic joint detection reagent that screens six respiratory pathogens at the same time.

Contents of the invention

In view of this, the purpose of the present invention is to provide an immunochromatographic joint detection kit and preparation method for simultaneous detection of six respiratory pathogens, which solves the problems in the above-mentioned background technology.

To achieve the above object, the technical scheme of the present invention is as follows:

In the first aspect, the present invention provides an immunochromatographic joint detection kit for simultaneous detection of six respiratory disease pathogens, including a reagent card and a fluorescent particle mixture, the reagent card includes a reagent strip, and the reagent strip includes a PVC bottom plate and an adhesive An integrated pad, nitrocellulose membrane, and absorbent paper on the base plate, the integrated pad, nitrocellulose membrane, and absorbent paper are sequentially lapped and pasted on the base plate;

Quality control line, detection line 1, detection line 2, detection line 3, detection line 4, detection line 5 and detection line 6 are successively arranged on the nitrocellulose membrane, and the detection line and the quality control line are parallel to each other, and the distance between them is 2.5cm±0.1cm;

The quality control line is coated with DNP monoclonal antibody, and the detection lines 1, 2, 3, 4, 5 and 6 are respectively coated with 1 mg/mL～2 mg/mL of influenza A monoclonal antibody, influenza B monoclonal antibody Antibodies, adenovirus monoclonal antibodies, syncytial virus monoclonal antibodies, mycoplasma pneumoniae monoclonal antibodies, and parainfluenza monoclonal antibodies;

The fluorescent particle mixture is prepared by mixing the labeled influenza A antibody-microsphere complex, influenza B antibody-microsphere complex, adrenal Virus antibody-microsphere complexes, syncytial virus antibody-microsphere complexes, mycoplasma pneumoniae antibody-microsphere complexes, parainfluenza antibody-microsphere complexes were diluted at a ratio of 1% to 2.5%, and DNP-BSA-microsphere The complex is diluted and mixed according to 0.02%, and the microspheres are time-resolved fluorescent microspheres.

Preferably, the time-resolved fluorescent test paper card is fixed on the plastic cartridge by the detection test strip, the surface of the test paper is pressed tightly with the card surface, and the card surface is respectively reserved for sample injection holes and nitrocellulose membrane parts. Observation window.

Preferably, the particle size of the time-resolved fluorescent microspheres is 300 nm.

Preferably, the width of the nitrocellulose membrane is 2.5 cm between the detection strip and the quality control strip.

Preferably, the integrated pad is spread on 21mm×100mm glass fiber with a treatment liquid containing 0.02mol/L PH=7.2～7.6 phosphate buffer, 5% sucrose and 0.8% Triton-100 surfactant and freeze-dried made.

In a second aspect, the present invention provides a method for preparing an immunochromatographic joint detection kit for simultaneously detecting six respiratory disease pathogens, comprising the following steps:

S1. Time-resolved fluorescent microsphere labeling

1. Activation: Take the suspension of time-resolved fluorescent microspheres with a solid content of 1%, add it to the EP tube, take 100-1000th of the volume of the suspension above, and add 50mg/ml N-hydroxysuccinimide solution Mix well in the microsphere suspension, then add 50 mg/ml 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution with the same volume as the N-hydroxysuccinimide solution Mix evenly in the microsphere suspension, and react at room temperature for 0.5 hours;

2. Labeling: Centrifuge at 14000r/min for 15min, discard the supernatant, add 0.02mol/L phosphate buffer solution with pH=5.0～9.0 to wash and centrifuge, then use ultrasonic to disperse evenly, press the microspheres and influenza The mass ratio of A monoclonal antibody, influenza B monoclonal antibody, adenovirus monoclonal antibody, syncytial virus monoclonal antibody, Mycoplasma pneumoniae monoclonal antibody, parainfluenza monoclonal antibody and DNP-BSA conjugate is 50:4 Add marked raw materials and react for 4 hours;

3. Blocking: Centrifuge at 14000r/min for 15min, discard the supernatant, add BSA to make the final concentration of the solution 0.50%, block for 1 hour, centrifuge the sealed microspheres at 14000r/min for 15min , remove the supernatant, add 250ul preservation solution to the centrifuged microspheres, ultrasonically disperse the microspheres evenly, and set aside at 4°C;

S2. Preparation of coated plates

Add another monoclonal antibody to influenza A, another monoclonal antibody to influenza B, another monoclonal antibody to adenovirus, another monoclonal antibody to syncytial virus, another monoclonal antibody to Mycoplasma pneumoniae, another Parainfluenza monoclonal antibody and DNP-BSA monoclonal antibody were diluted to 1mg/ml with 0.01mmol/L PBS with a pH value of 7.4, respectively, and were drawn on the nitrocellulose membrane with a film-drawing device. As the quality control line (line C); the line containing influenza A monoclonal antibody as test line 1; the line containing influenza B monoclonal antibody as test line 2; the line containing adenovirus monoclonal antibody as test line 3; the line containing syncytial virus monoclonal antibody The cloned antibody was used as detection line 4; the one containing mycoplasma pneumoniae monoclonal antibody was used as detection line 5; the one containing parainfluenza monoclonal antibody was used as detection line 6, and then dried at 37°C for 16 hours in an environment with humidity <20% to make a coating plate;

S3. Preparation of Fluorescent Particle Mixture

The labeled influenza A antibody-microsphere complex, influenza B antibody-microsphere complex, and adenovirus antibody-microsphere complex were mixed with phosphate buffer solution containing 0.02mol/L PH=7.2～7.6 and 0.5% casein , Syncytial virus antibody-microsphere complex, Mycoplasma pneumoniae antibody-microsphere complex, parainfluenza antibody-microsphere complex were diluted in a ratio of 1% to 2.5%, and DNP-BSA-microsphere complex was diluted in a ratio of 0.02% Mix into the required fluorescent particle mixture;

S4. Preparation of one-piece pads

The treatment solution containing 0.02mol/L pH=7.2-7.6 phosphate buffer solution, 5% sucrose and 0.8% Triton-100 surfactant is spread on 21mm×100mm glass fiber and freeze-dried.

S5. Preparation of reagent card

Paste the one-piece blank pad on the end close to the quality control line, 1mm on the nitrocellulose membrane, stick the absorbent paper on the end close to the detection line 6, and 1mm on the nitrocellulose membrane, and then cut into strips with a strip cutter. 4mm±0.1mm test strip, put the test strip into the plastic card case and compact it.

The beneficial effect of the present invention is that: currently, only nucleic acid detection reagents are available in the market as chromatographic reagents for more than quadruple detection of respiratory pathogens. Nucleic acid testing laboratories require high environmental requirements, take a long time, and operators need certain operational skills. Generally, only tertiary hospitals have the conditions for nucleic acid testing. Most of the respiratory pathogen detection chromatography reagents on the market can only detect a single pathogen, or several rapid detection test strips for the detection of a single pathogen are spliced together, requiring multiple loadings. Generally speaking, the more testing items there are, the more cross problems need to be solved among the items, such as the accumulation of microspheres and the cleanliness of the membrane surface. The present invention overcomes the existing problems and technical obstacles in the prior art, and provides a convenient chromatography reagent capable of simultaneously detecting six respiratory pathogens including influenza A, influenza B, adenovirus, syncytial virus, mycoplasma pneumoniae and parainfluenza virus , Only need to collect nasal swabs and throat swabs for detection, which is non-invasive. And it can meet the needs of grass-roots community hospitals and can be used for early diagnosis of diseases.

At the same time, the present invention uses the integrated pad + wet method mode, which can allow the marker and the target to fully react during the liquid phase reaction, so as to ensure that the sensitivity of the multi-joint inspection can be consistent with the single-test sample pad + binding pad mode; the integrated pad, All markers can reduce residues at different interfaces during the chromatography process, and more fluorescent particles will pass through the T line, and the reaction will be more thorough; the integrated pad + wet mode makes the membrane surface more uniform and clean.

Description of drawings

Fig. 1 is a schematic structural view of the joint detection reagent detection test strip of the present invention.

Among them: 1. Test strip; 11. PVC bottom plate; 12. Integrated pad; 13. Nitrocellulose membrane; 14. Absorbent paper; 15. Quality control line (C line); 161. Influenza A detection line (T1); 162. Influenza B detection line (T2); 163. Adenovirus detection line (T3); 164. Syncytial virus detection line (T4); 165. Mycoplasma pneumoniae detection line (T5); 166. Parainfluenza virus detection line (T6 ); 2. Fluorescent particle mixture.

Fig. 2 is a schematic structural view of the detection reagent card of the joint detection reagent of the present invention.

Among them: 3. Reagent card; 31. Plastic cartridge; 32. Sample hole; 33. Observation window

detailed description

In order to better understand the present invention, the present invention will be described in further detail below in conjunction with the examples and accompanying drawings, but those skilled in the art understand that the following examples do not limit the protection scope of the present invention. Changes and changes are all within the protection scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Influenza A/Influenza B/Adenovirus/Syncytial Virus/Parainfluenza/Mycoplasma pneumoniae antigen detection test kit

An embodiment of an immunochromatographic joint detection kit for simultaneous detection of six respiratory disease pathogens of the present invention, as shown in Figure 1, includes a test strip 1 and a fluorescent particle mixture, and the test strip 1 consists of a PVC base plate 11 and The integral pad 12, the nitrocellulose membrane 13 and the absorbent paper 14 pasted on the base plate are composed, and the integrated pad 12, the nitrocellulose membrane 13 and the absorbent paper 14 are successively overlapped and pasted on the base plate; the nitrocellulose membrane is There are quality control line (C line) 15, test line 1 (T1) 161, test line 2 (T2) 162, test line 3 (T3) 163, test line 4 (T4) 164, test line 5 (T5) 165 and detection line 6 (T6) 166, the detection line and the quality control line are parallel to each other, and the distance between them is 2.5cm ± 0.1cm; the quality control line is coated with DNP monoclonal antibody, and the detection line 1, 2, 3 , 4, 5 and 6 were coated with 1mg/mL～2mg/mL influenza A monoclonal antibody, influenza B monoclonal antibody, adenovirus monoclonal antibody, syncytial virus monoclonal antibody, Mycoplasma pneumoniae monoclonal antibody and para Influenza monoclonal antibodies;

The fluorescent particle mixture is composed of phosphate buffer containing 0.02mol/L PH=7.2-7.6, 0.5% casein to label the influenza A antibody-microsphere complex, influenza B antibody-microsphere complex, adenovirus antibody -Microsphere complexes, syncytial virus antibody-microsphere complexes, mycoplasma pneumoniae antibody-microsphere complexes, parainfluenza antibody-microsphere complexes are diluted in a ratio of 1% to 2.5%, and DNP-BSA-microsphere complexes It is prepared by diluting and mixing at a ratio of 0.02%. The microspheres are time-resolved fluorescent microspheres with a particle diameter of 300nm.

In order to shorten the chromatographic time, a 25cm wide NC membrane is selected, and 7 strips need to be coated on the 25cm wide NC membrane, so the distance between each detection strip and quality control strip is 2.5cm±0.1cm. The fluorescence detection instrument can well frame the area to ensure the position of the item band.

As shown in Figure 2, the test paper card 3 is fixed on the plastic card case 31 by the test paper strip 1, the surface of the test paper strip is pressed tightly with the card surface, and the card surface is on the parts corresponding to the integral pad 12 and the nitrocellulose membrane 13 respectively. A sample injection hole 32 and an observation window 33 are reserved.

Example 2 Preparation of Influenza A/Influenza B/Adenovirus/Syncytial Virus/Parainfluenza/Mycoplasma pneumoniae Antigen Detection Kit

1. Time-resolved fluorescent microsphere labeling steps

Activation: Fluorescent microspheres are ultrasonically dispersed with an ultrasonic cleaner and mixed with an oscillator. Take 50ul of the time-resolved fluorescent microsphere suspension with a solid content of 1%, dilute it 20 times with ultrapure water, that is, 1000ul, and add it to the EP tube. Take 1~10ul 50mg/ml N-hydroxysuccinimide (NHS) solution, add it to the microsphere suspension and mix well, then add 1~10ul 50mg/ml 1-(3-dimethylaminopropyl)-3- Ethylcarbodiimide hydrochloride (EDC) solution was mixed into the microsphere suspension, and reacted at room temperature for 0.5 hours.

Marking: centrifuge at 14000r/min for 15min. Discard the supernatant, add 0.02mol/L PH=5.0～9.0 phosphate buffer solution to wash and centrifuge, and then use ultrasonic sonication to disperse evenly, press the microspheres with influenza A monoclonal antibody, influenza B monoclonal antibody, adenovirus The mass ratio of monoclonal antibody, syncytial virus monoclonal antibody, mycoplasma pneumoniae monoclonal antibody, parainfluenza monoclonal antibody and DNP-BSA conjugate was 50:4, and the labeled raw materials were added and reacted for 4 hours.

Blocking: Centrifuge for 15 minutes with 14000r/min centrifugal condition. The supernatant was discarded, BSA was added to make the final concentration of the solution 0.50%, and blocking was carried out for 1 hour. Centrifuge the sealed microspheres at 14000r/min for 15min, remove the supernatant, add 250ul preservation solution to the centrifuged microspheres, ultrasonically disperse the microspheres evenly, and set aside at 4°C.

In order to reduce the problems of non-specific crossover between projects and microsphere aggregation, this reagent adjusts the particle size of fluorescent microsphere-labeled microspheres, microsphere activation conditions and labeling conditions to achieve the optimal state.

The particle size of the time-resolved fluorescent microspheres is preferably 300 nm.

Preferably, the final concentrations of NHS and EDC used for activation are 0.5 mg/ml and 0.05 mg/ml.

Preferably, influenza A monoclonal antibody, influenza B monoclonal antibody, adenovirus monoclonal antibody, syncytial virus monoclonal antibody, Mycoplasma pneumoniae monoclonal antibody and DNP-BSA are labeled with phosphate buffered saline at pH=7.0, and parainfluenza monoclonal Antibodies were labeled with phosphate buffer saline at pH = 9.0.

2. Preparation of coated plates

Add another monoclonal antibody to influenza A, another monoclonal antibody to influenza B, another monoclonal antibody to adenovirus, another monoclonal antibody to syncytial virus, another monoclonal antibody to Mycoplasma pneumoniae, another Parainfluenza monoclonal antibody and DNP-BSA monoclonal antibody were respectively diluted to 1 mg/ml with 0.01 mmol/L PBS (PH=7.4), and were drawn on the nitrocellulose membrane with a streaking device.

Containing DNP monoclonal antibody as quality control line (C line);

Containing influenza A monoclonal antibody as test line 1 (T1);

Containing influenza B monoclonal antibody as test line 2 (T2);

Containing adenovirus monoclonal antibody as detection line 3 (T3);

Containing syncytial virus monoclonal antibody as test line 4 (T4);

Containing Mycoplasma pneumoniae monoclonal antibody as detection line 5 (T5);

Containing monoclonal antibody against parainfluenza as detection line 6 (T6).

Then dry at 37°C for 16 hours in an environment with humidity <20% to make a coated board.

In order to shorten the chromatographic time, a 25cm wide NC membrane is selected, and 7 strips need to be coated on the 25cm wide NC membrane, so the distance between each detection strip and quality control strip is 2.5cm±0.1cm. The fluorescence detection instrument can well frame the area to ensure the position of the item band.

3. Preparation of fluorescent particle mixture

The labeled influenza A antibody-microsphere complex, influenza B antibody-microsphere complex, and adenovirus antibody-microsphere complex were mixed with phosphate buffer solution containing 0.02mol/L PH=7.2～7.6 and 0.5% casein , syncytial virus antibody-microsphere complex, mycoplasma pneumoniae antibody-microsphere complex and parainfluenza antibody-microsphere complex were diluted in a ratio of 1% to 2.5%, and the DNP-BSA-microsphere complex was diluted in a ratio of 0.02% Mix into the required fluorescent particle mixture.

4. Preparation of one-piece pad

The treatment liquid containing 0.02mol/L phosphate buffer solution with pH=7.2-7.6, 5% sucrose and 0.8% Triton-100 surfactant was spread on 21mm×100mm glass fiber and freeze-dried.

This reagent uses an integrated pad + wet mode, and the conventional sample pad + binding pad mode is changed to an integrated pad + wet mode. The integrated pad + wet mode is a mode of liquid phase reaction first and then solid phase reaction. During the liquid phase reaction, the marker and the target can be fully reacted to ensure that the sensitivity of the multiple detection can be compared with that of the single detection sample pad + The mode of the binding pad is the same; with the integrated pad, all markers can reduce the residue at different interfaces during the chromatography process, and more fluorescent particles will pass through the T line, and the reaction will be more thorough; the integrated pad + wet mode makes the The film surface is more uniform and clean.

5. Preparation of reagent card

Attach the one-piece blank pad to the end close to the quality control line, and place it on the NC membrane for 1mm. Stick the absorbent paper on the end close to the detection line 6 (T6), and put it on the NC membrane for 1mm. Then use a strip cutter to cut into 4mm±0.1mm test strips. Put the test strip into a polystyrene plastic case and compact it, then put it into a packaging bag, add a bag of desiccant, and seal it. One serving of reagents includes one reagent card plus one fluorescent particle freeze-dried reaction tube.

Example 3 Detection

Using the test card prepared in Example 1 to test the sample to be tested, nasal swabs and throat swabs can be tested.

Nasal swab collection: Insert the swab into the nasal cavity and gently rotate it and push it toward the inside of the nasal cavity until the turbinate (2.0cm-2.5cm away from the nostril) and then rotate it 3 times, then take out the swab.

Pharyngeal swab collection: Insert the swab from the oral cavity into the throat completely, centering on the reddened part of the throat wall and palatine tonsil, wipe the bilateral pharyngeal tonsils and throat wall with moderate force, avoid touching the tongue, and take out the swab.

Add 0.5ml of sample extraction solution into the plastic hose, then insert the cotton swab of the collected nasal swab or throat swab into the sample extraction solution, rotate and squeeze it 5 times, so that the sample can be dissolved in the solution as much as possible. Finally squeeze the swab tip of the swab to keep as much liquid in the tube as possible, remove and discard the swab.

Add 100ul of the dissolved sample dropwise to the fluorescent particle reaction tube, mix well, then take 70ul dropwise and add it to the sample port of the test card, let it stand for 15 minutes, then insert the test card into the dry fluorescent immunoassay The test results in the instrument.

Among them, the sample extract has the ability to lyse the virus to expose the detection site. The sample extract formula includes 0.02mol/L phosphate buffer with pH=7.4, Triton 100 with a volume fraction of 0.1% to 1.0%, volume Sodium chloride with a fraction of 0.1% to 1.0% Tween 20 and a molar mass of 0.5 to 1.0 mol/L.

Example 4 Comparison between integrated pad+wet mode and sample pad+bonding pad mode

Using the reagents in Example 1, and then using the same materials in Example 1 to prepare the sample pad + binding pad mode single detection reagent and sample pad + binding pad mode joint detection reagent, the comparison results are shown in Table 1 below:

Table 1 Comparison results of integrated pad + wet mode and sample pad + bonded pad mode

From the test results in Table 1 above, the sensitivity of the one-pad+wet mode is consistent with that of the sample pad+conjugated pad (single detection) mode, and is better than that of the sample pad+conjugated pad (joint detection mode).

Embodiment 5 performance evaluation

1. Minimum detection limit

(1) Minimum detection limit of influenza A

Influenza A/B influenza virus antigen detection reagent national reference L1\L2\L5 was diluted with sample extracts at 1:10, 1:20, 1:40, 1:80 and 1:160 respectively. The test results are shown in Table 2 Shown:

Table 2 The detection card of the present invention detects the detection result table of A/B influenza virus antigen detection reagent national reference product influenza A

serial number T1:FluA T2:FluB T3:ADV T4:RSV T5: MP T6: HPIV L1(1:10) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L1(1:20) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L1(1:40) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L1(1:80) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L1(1:160) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L2(1:10) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L2(1:20) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L2(1:40) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L2(1:80) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L2(1:160) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L5(1:10) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L5(1:20) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L5(1:40) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L5(1:80) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) L5(1:160) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-)

As can be seen from the detection results of the above table 2, the respiratory combined detection card of the present invention detects the lowest detection limit of the national reference product influenza A of the influenza A/B influenza virus antigen detection reagent, and influenza A and other items No cross.

(2) Minimum detection limit of influenza B

The national reference product L3\L4 of influenza A/B virus antigen detection reagent was diluted with sample extracts at 1:10, 1:20, 1:40, 1:80 and 1:160 respectively, and the test results are shown in Table 3 below :

Table 3 The detection card of the present invention detects the detection result table of influenza A/B type influenza virus antigen detection reagent national reference product influenza B

serial number T1:FluA T2:FluB T3:ADV T4:RSV T5: MP T6: HPIV L3(1:10) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) L3(1:20) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) L3 (1:40) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) L3(1:80) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) L3(1:160) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) L4(1:10) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) L4(1:20) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) L4 (1:40) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) L4 (1:80) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) L4 (1:160) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-)

As can be seen from the detection results of the above table 3, the respiratory combined detection card of the present invention detects the lowest detection limit of the national reference product influenza B of the influenza A/B virus antigen detection reagent, and the influenza B and other items No cross.

(3) Minimum detection limit of adenovirus

Adenovirus culture solution was diluted with sample extract to 2×10 ^{^5} VP/ml (ADV1), 2×10 ^{^4} VP/ml (ADV2), 1×10 ^{^4} VP/ml (ADV3), and the test results were as follows Table 4 shows:

Table 4 The detection result table of detection card of the present invention detecting adenovirus

serial number T1:FluA T2:FluB T3:ADV T4:RSV T5: MP T6: HPIV ADV1 feminine(-) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) ADV2 feminine(-) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) ADV3 feminine(-) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-)

It can be seen from the above test results in Table 4 that the lower limit of detection of adenovirus of the respiratory combined detection card of the present invention is 1×10 ^{^4} VP/ml, and there is no crossover between adenovirus and other items.

(4) Minimum detection limit of syncytial virus

The syncytial virus culture solution was diluted with the sample extract to 4.36×10 ^{^4} pfu/ml (RSV1), 8.72×10 ^{^3} pfu/ml (RSV2), 4.36×10 ^{^3} pfu/ml (RSV3), and the test results As shown in Table 5 below:

Table 5 Detection card of the present invention detects the detection result table of syncytial virus

serial number T1:FluA T2:FluB T3:ADV T4:RSV T5: MP T6: HPIV RSV1 feminine(-) feminine(-) feminine(-) Positive (+) feminine(-) feminine(-) RSV2 feminine(-) feminine(-) feminine(-) Positive (+) feminine(-) feminine(-) RSV3 feminine(-) feminine(-) feminine(-) Positive (+) feminine(-) feminine(-)

It can be seen from the test results in Table 5 above that the detection limit of the combined detection of syncytial virus of the present invention is 4.36×10 ^{^3} pfu/ml, and there is no crossover between syncytial virus and other items.

(5) The minimum detection limit of Mycoplasma pneumoniae

Mycoplasma pneumoniae culture solution was diluted with sample extract to 3.83×10 ^{^4} pfu/ml (MP1), 1.532×10 ^{^ 4} pfu/ml (MP2), 7.66×10 ^{^3} pfu/ml (MP3), and the test results were as follows Table 6 shows:

Table 6 The detection result table of detecting card of the present invention to detect Mycoplasma pneumoniae

serial number T1:FluA T2:FluB T3:ADV T4:RSV T5: MP T6: HPIV MP1 feminine(-) feminine(-) feminine(-) feminine(-) Positive (+) feminine(-) MP2 feminine(-) feminine(-) feminine(-) feminine(-) Positive (+) feminine(-) MP3 feminine(-) feminine(-) feminine(-) feminine(-) Positive (+) feminine(-)

It can be seen from the test results in Table 6 above that the detection limit of the combined detection of Mycoplasma pneumoniae of the present invention is 7.66×10 ^{^3} pfu/ml, and there is no crossover between Mycoplasma pneumoniae and other items.

(6) Minimum detection limit of parainfluenza virus

The parainfluenza virus culture fluid was diluted with the sample extract to 1.31×10 ^{^4} pfu/ml (HPIV1), 5.24×10 ^{^ 3} pfu/ml (HPIV), 2.62×10 ^{^3} pfu/ml (HPIV3), and the test results As shown in Table 7 below:

Table 7 The detection result table of detection card of the present invention detecting parainfluenza virus

serial number T1:FluA T2:FluB T3:ADV T4:RSV T5: MP T6: HPIV HPIV1 feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) Positive (+) HPIV2 feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) Positive (+) HPIV3 feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) Positive (+)

It can be seen from the test results in Table 7 above that the detection limit of the combined detection card of the present invention for parainfluenza virus is 2.62×10 ^{^3} pfu/ml, and there is no crossover between parainfluenza virus and other items.

Finally, the detection sensitivity results of the kit of the present invention are determined in Table 8:

The detection sensitivity result table of table 8 kit of the present invention

2. Cross-reaction experiment

cross T1:FluA T2:FluB T3:ADV T4:RSV T5: MP T6: HPIV H1N1 Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) H3N2 Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) H5N1 Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) H7N9 Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) B stream V feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) B stream Y feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) feminine(-) Adenovirus disease feminine(-) feminine(-) Positive (+) feminine(-) feminine(-) feminine(-) syncytial virus feminine(-) feminine(-) feminine(-) Positive (+) feminine(-) feminine(-) Mycoplasma pneumoniae feminine(-) feminine(-) feminine(-) feminine(-) Positive (+) feminine(-) parainfluenza virus feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) Positive (+) human giant cell feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) Coxsackie feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) mumps feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) measles feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) Chlamydia pneumoniae feminine(-) feminine(-) feminine(-) feminine(-) feminine(-) feminine(-)

Each item has no cross-reaction with the above viruses.

Embodiment 6 Reagent detection application

591 samples were collected in the hospital, including 186 positive for influenza A, 69 positive for influenza B, 12 positive for adenovirus, 21 for syncytial virus, 10 for parainfluenza, 7 for mycoplasma pneumoniae, and 286 negative samples.

Sensitivity: 178 cases of influenza A were detected with a sensitivity of 95.70%; 66 cases of influenza B were detected with a sensitivity of 95.65%; 12 cases of adenovirus were detected with a sensitivity of 100%; 21 cases of syncytial virus were detected with a sensitivity of 100%; 10 copies were detected, with a sensitivity of 80%; 5 copies of Mycoplasma pneumoniae were detected, with a sensitivity of 71.43%.

Specificity: 280 out of 286 negative samples were detected, the specificity was 97.9%.

In summary, the present invention provides a convenient chromatographic reagent that can simultaneously detect six respiratory pathogens including influenza A, influenza B, adenovirus, syncytial virus, mycoplasma pneumoniae and parainfluenza virus, and only needs to collect nasal swabs and throat swabs It can be detected with just a few seeds, which is non-invasive and has high specificity. And it can meet the needs of grass-roots community hospitals and can be used for early diagnosis of diseases.

At the same time, the present invention uses the integrated pad + wet method mode, which can allow the marker and the target to fully react during the liquid phase reaction, so as to ensure that the sensitivity of the multi-joint inspection can be consistent with the single-test sample pad + binding pad mode; the integrated pad, All markers can reduce residues at different interfaces during the chromatography process, and more fluorescent particles will pass through the T line, and the reaction will be more thorough; the integrated pad + wet mode makes the membrane surface more uniform and clean.

Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we have described are only illustrative, rather than used to limit the scope of the present invention. Equivalent modifications and changes made by skilled personnel in accordance with the spirit of the present invention shall fall within the protection scope of the claims of the present invention.

Claims (6)

1. The utility model provides an immunity chromatography joint inspection kit of six respiratory disease pathogens of simultaneous detection, includes reagent card and fluorescent particle mixed liquid, its characterized in that:

the reagent card comprises a reagent strip, the reagent strip comprises a PVC bottom plate, an integrated pad, a nitrocellulose membrane and absorbent paper, and the integrated pad, the nitrocellulose membrane and the absorbent paper are adhered to the bottom plate in a lap joint mode;

the nitrocellulose membrane is sequentially provided with a quality control line, a detection line 1, a detection line 2, a detection line 3, a detection line 4, a detection line 5 and a detection line 6, the detection line and the quality control line are parallel to each other, and the spacing distance is 2.5cm +/-0.1 cm;

the quality control line is coated with DNP monoclonal antibodies, and the detection lines 1, 2, 3, 4, 5 and 6 are respectively coated with 1-2 mg/mL of influenza A monoclonal antibodies, influenza B monoclonal antibodies, adenovirus monoclonal antibodies, syncytial virus monoclonal antibodies, mycoplasma pneumoniae monoclonal antibodies and parainfluenza monoclonal antibodies;

the fluorescent particle mixed solution is prepared by diluting and mixing marked influenza A antibody-microsphere complex, influenza B antibody-microsphere complex, adenovirus antibody-microsphere complex, syncytial virus antibody-microsphere complex, mycoplasma pneumoniae antibody-microsphere complex, parainfluenza antibody-microsphere complex according to the proportion of 1-2.5% and DNP-BSA-microsphere complex according to the proportion of 0.02% by using a phosphate buffer solution containing 0.02mol/L and 0.5% of casein, wherein the microspheres are time-resolved fluorescent microspheres.

2. The kit of claim 1, wherein the test paper card is fixed on the plastic card shell by a test paper strip, the surface of the test paper strip is pressed by a card surface, and the card surface is provided with a sample hole and an observation window respectively at the parts corresponding to the integrated pad and the nitrocellulose membrane.

3. The kit of any one of claims 1 or 2, wherein the time-resolved fluorescent microspheres have a particle size of 300nm.

4. The kit of any one of claims 1 or 2, wherein the nitrocellulose membrane has a width of 2.5cm between the detection and quality control bands.

5. The kit according to any one of claims 1 or 2, wherein the integrated pad is prepared by freeze-drying a treatment solution comprising 0.02mol/L of phosphate buffer solution having PH = 7.2-7.6%, 5% sucrose and 0.8% triton-100 surfactant flat-laid on glass fibers of 21mm x 100 mm.

6. A method for preparing the kit of claim 1, comprising the steps of:

s1, time-resolved fluorescent microsphere labeling

1) Activation: adding a time-resolved fluorescent microsphere suspension with the solid content of 1% into an EP (EP) tube, adding 50mg/ml of N-hydroxysuccinimide solution with the volume of 100-1000 times of that of the suspension into the microsphere suspension for uniformly mixing, then adding 50mg/ml of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution with the volume equal to that of the N-hydroxysuccinimide solution into the microsphere suspension for uniformly mixing, and reacting for 0.5 hour at normal temperature;

2) Marking: centrifuging for 15min under the centrifugal condition of 14000r/min, removing supernatant, adding 0.02mol/L phosphate buffer solution with the pH = 5.0-9.0, washing and centrifuging, then dispersing uniformly by ultrasonic waves, and mixing the microspheres with an influenza A monoclonal antibody, an influenza B monoclonal antibody, an adenovirus monoclonal antibody, a syncytium virus monoclonal antibody, a mycoplasma pneumoniae monoclonal antibody, a parainfluenza monoclonal antibody and a DNP-BSA conjugate according to the mass ratio of 50:4, adding the marking raw materials in proportion, and reacting for 4 hours;

3) And (3) sealing: centrifuging for 15min under 14000r/min centrifugation condition. Removing supernatant, adding BSA to make the final concentration of the solution 0.50%, sealing for 1 hr, centrifuging the sealed microspheres at 14000r/min for 15min, removing supernatant, adding 250ul of preservation solution into the centrifuged microspheres, and ultrasonically dispersing the microspheres uniformly at 4 deg.C;

s2. Preparation of coated plate

Diluting another influenza A monoclonal antibody, another influenza B monoclonal antibody, another adenovirus monoclonal antibody, another syncytial virus monoclonal antibody, another mycoplasma pneumoniae monoclonal antibody, another parainfluenza monoclonal antibody and a DNP-BSA monoclonal antibody to 1mg/ml by using 0.01mmol/L PBS with the pH value of 7.4, and scribing on a nitrocellulose membrane by using a scribing instrument, wherein the DNP monoclonal antibody is used as a quality control line (line C); a test line 1 containing an influenza A monoclonal antibody; a detection line 2 containing an influenza B monoclonal antibody; using the adenovirus monoclonal antibody as a detection line 3; a detection line 4 containing a monoclonal antibody of the syncytial virus; a detection line 5 containing mycoplasma pneumoniae monoclonal antibodies; using the parainfluenza monoclonal antibody as a detection line 6, and then drying for 16h at 37 ℃ under the environment with the humidity of less than 20% to prepare a coated plate;

s3, preparation of fluorescent particle mixed solution

Diluting the labeled influenza A antibody-microsphere complex, influenza B antibody-microsphere complex, adenovirus antibody-microsphere complex, syncytial virus antibody-microsphere complex, mycoplasma pneumoniae antibody-microsphere complex, parainfluenza antibody-microsphere complex at a ratio of 1-2.5% and DNP-BSA-microsphere complex at a ratio of 0.02% with a phosphate buffer solution containing 0.02mol/L pH = 7.2-7.6 by 0.5% casein to mix into a desired fluorescent particle mixed solution;

s4. Preparation of integrated pad

Freeze-drying a glass fiber of 21mm × 100mm in a flat state with a treatment solution containing 0.02mol/L of phosphate buffer solution having pH =7.2 to 7.6, 5% of sucrose and 0.8% of Triton-100 surfactant;

s5. Preparation of reagent card

Attaching an integral blank pad to one end close to a quality control line, putting the integral blank pad on a nitrocellulose membrane for 1mm, attaching absorbent paper to one end close to a detection line 6, putting the absorbent paper on the nitrocellulose membrane for 1mm, cutting the absorbent paper into test strips with the thickness of 4mm +/-0.1 mm by a slitter, and putting the test strips into a plastic card shell for compaction.

Images