CN115453116A

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

Info

Publication number: CN115453116A
Application number: CN202211129489.3A
Authority: CN
Inventors: 林婷婷; 王薇; 阙素婷; 郭清顺; 曾文超; 郑丽娟
Original assignee: Xiamen Yingbomai Biotechnology Co ltd
Current assignee: Xiamen Yingbomai Biotechnology Co ltd
Priority date: 2022-09-16
Filing date: 2022-09-16
Publication date: 2022-12-09

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

Immunochromatography joint inspection kit for simultaneously detecting six respiratory pathogens and preparation method thereof

Technical Field

The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to an immunochromatography joint detection kit for simultaneously detecting six respiratory pathogens and a preparation method thereof

Background

Influenza virus (Influenza virus) is a single-stranded negative-strand RNA virus belonging to the orthomyxoviridae family. Influenza viruses are classified into 4 types, i.e., A, B, C, D, etc., according to the genetic characteristics and antigenicity of viral nucleoproteins and matrix proteins. Influenza a viruses are divided into subtypes based on differences in Hemagglutinin (HA) and Neuraminidase (NA) antigens on the viral envelope. The H5N1 and H7N9 avian influenza viruses infect human beings, so that the human beings have serious lesions and are called highly pathogenic avian influenza viruses. According to WHO statistics, the infection of influenza A virus and influenza B virus causes that the number of hospitalizations is up to 20 ten thousand every year, and 30000-50000 patients die, so that the infections become the main lethal cause after acquired immunodeficiency syndrome.

Parainfluenza virus (PIV) belongs to the family paramyxoviridae, the genus respiratory virus. The biological characteristics of the virus are similar to those of paramyxovirus, and the virus has the typical structural characteristics of paramyxovirus. There are 4 serotypes based on genetics and antigenicity, namely: PIV-1, PIV-2, PIV-3 and PIV-4, wherein PIV-1, PIV-2 and PIV-3 are the main types of human infection. HPIV-3 is often outbreak in spring and summer, often causing lower respiratory tract infections in infants, and is second only to RSV, the second largest pathogen causing bronchiolitis and pneumonia in infants up to 6 months. A survey from New Vaccine Surveillance Network (NVSN) over approximately 4 years showed that approximately 190 of all children ≦ 5 years old (approximately 2800) hospitalized for fever and acute respiratory illness were caused by HPIV infection, accounting for approximately 7% of all children infected with PIV-3, accounting for 50%.

Respiratory Syncytial Virus (RSV) is a non-segmented, single-stranded negative-strand RNA virus belonging to the family paramyxoviridae, the genus pneumovirus, one of the major pathogens responsible for acute Respiratory infections in infants and young children, with a latency period of typically 4-6d. Because the immune function development is not perfect, the infants of 2-6 months can cause severe bronchiolitis and pneumonia after being infected with RSV, and meanwhile, the RSV infection is also one of the risk factors of the bronchial asthma of children. Statistically, 24 out of every 1000 hospitalized infant patients worldwide are caused by direct infection with RSV, and 66000-199000 children die each year from RSV infection, with 99% of deaths occurring under the age of 5. Recent studies have shown that immunocompromised adults are also a susceptible population to RSV, and that 3% to 10% of adults are expected to become infected with RSV each year and develop severe lower respiratory tract infections. Therefore, the deep research on the early diagnosis technology of RSV has important and positive significance for the prevention and treatment thereof.

Adenovirus (AdV) is a double-stranded DNA virus without an envelope and can be transmitted by aerosol particle inhalation, fecal contamination or exposure to infected tissue or blood. After infection of the upper or lower respiratory tract by AdV, there is a high probability of developing pneumonia, acute Respiratory Distress Syndrome (ARDS) or disseminated infection. Patients with ARDS present with bilateral variable pulmonary opacity similar to pulmonary edema on chest X-ray or CT scans with mortality rates of 27% -45%. Early and timely determination of pathogens is of great help for treatment and improvement of prognosis.

Mycoplasma Pneumoniae (MP) belongs to the Mycoplasma family Mycoplasmatales, the order mollicutes, and is the smallest microorganism capable of self-replication and capable of surviving in vitro independently of living cells. The clinical manifestations of MP infection are diverse, from asymptomatic to nasopharyngitis, pharyngeal tonsillitis, tracheobronchitis, bronchiolitis and pneumonia, and severe pneumonia can occur in severe cases. MP pneumonia accounts for about 10% -40% of community-acquired pneumonia in children. Resulting in 10% of the children patients entering the ICU. Because the clinical symptoms of MP infection are not typical and mild, some patients show the first symptom outside the lung, the imaging examination lacks specificity, and MP infection is often misdiagnosed and missed.

Acute respiratory infection (ART) is one of the major causes of Acute and fatal diseases worldwide. The etiology can be diagnosed accurately in time at the early stage of virus infection, which is helpful for medical staff to master the illness state of the patient accurately. The current method for detecting respiratory pathogens mainly comprises 4 types of virus separation culture, antigen detection, serum immunological antibody detection and nucleic acid detection. Serotype detection cannot be used for early diagnosis of diseases, and nucleic acid detection reagents become important detection methods for respiratory virus infection at present, but nucleic acid detection generally requires at least about 2 hours. The requirement on the experimental environment is high, the operation is relatively complex, and an operator needs certain operation skills.

Respiratory tract infection is seasonal, infectious and regional, respiratory tract pathogens are various, most respiratory tract pathogen detection chromatographic reagents on the market can only detect a single pathogen, or a plurality of rapid detection test paper for detecting the single pathogen are spliced together, and no immunochromatography joint detection reagent for simultaneously screening six respiratory tract pathogens appears.

Disclosure of Invention

In view of the above, the present invention provides an immunochromatographic joint inspection kit for simultaneously detecting six respiratory pathogens and a preparation method thereof, which solve the problems in the background art.

In order to realize the purpose, the technical scheme of the invention is as follows:

in a first aspect, the invention provides an immunochromatographic joint inspection kit for simultaneously detecting six respiratory disease pathogens, which comprises a reagent card and a fluorescent particle mixed solution, wherein the reagent card comprises a reagent strip, the reagent strip comprises a PVC bottom plate, and an integrated pad, a nitrocellulose membrane and absorbent paper which are stuck on the bottom plate, and the integrated pad, the nitrocellulose membrane and the absorbent paper are sequentially lapped and stuck on the bottom plate;

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, wherein 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 a DNP monoclonal antibody, and the

detection lines

1, 2, 3, 4, 5 and 6 are respectively coated with 1-2 mg/mL of influenza A monoclonal antibody, influenza B monoclonal antibody, adenovirus monoclonal antibody, syncytial virus monoclonal antibody, mycoplasma pneumoniae monoclonal antibody and parainfluenza monoclonal antibody;

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.

Preferably, the time-resolved fluorescence test paper card is fixed on the plastic card shell by a detection test paper strip, the surface of the test paper is pressed by a card surface, and the card surface is respectively provided with a sample adding hole and an observation window at the part corresponding to the integrated pad and the nitrocellulose membrane.

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

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

Preferably, the integrated pad is prepared by spreading a treatment solution containing 0.02mol/L of phosphate buffer solution having pH = 7.2-7.6, 5% of sucrose and 0.8% of Triton-100 surfactant on a glass fiber of 21mm × 100mm and freeze-drying.

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

s1, time-resolved fluorescent microsphere marking

1. And (3) activation: taking a time-resolved fluorescent microsphere suspension with the solid content of 1%, adding the suspension into an EP (EP) tube, taking 50mg/ml N-hydroxysuccinimide solution with the volume of 100-1000 times of that of the suspension, adding the N-hydroxysuccinimide solution into the microsphere suspension, uniformly mixing, adding 50mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution with the volume same as that of the N-hydroxysuccinimide solution into the microsphere suspension, 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 PH = 5.0-9.0, washing and centrifuging, then dispersing uniformly by ultrasonic waves, and mixing the microspheres and the influenza A monoclonal antibody, the influenza B monoclonal antibody, the adenovirus monoclonal antibody, the syncytial virus monoclonal antibody, the mycoplasma pneumoniae monoclonal antibody, the parainfluenza monoclonal antibody and the 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 the centrifugation condition of 14000r/min, removing supernatant, adding BSA (bovine serum albumin) to enable the final concentration of the solution to be 0.50%, sealing for 1 hour, centrifuging the sealed microspheres for 15min under the centrifugation condition of 14000r/min, removing supernatant, adding 250ul of preservation solution into the centrifuged microspheres, and performing ultrasonic treatment to uniformly disperse the microspheres at 4 ℃ for later use;

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 performing membrane scribing on a nitrocellulose membrane by using a membrane scribing instrument to obtain a quality control line (line C) containing the DNP monoclonal antibody; the detection line 1 contains influenza A monoclonal antibodies; the detection line 2 contains influenza B monoclonal antibodies; 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

Spreading the mixture on a glass fiber of 21mm × 100mm in a treatment solution containing 0.02mol/L of phosphate buffer solution with pH = 7.2-7.6, 5% of sucrose and 0.8% of Triton-100 surfactant, and freeze-drying.

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 using a slitter, and putting the test strips into a plastic card shell for compaction.

The invention has the beneficial effects that: the current chromatographic reagent for detecting the upper respiratory tract pathogens more than the quadruple joint detection only comprises a nucleic acid detection reagent. The environment requirement of a nucleic acid detection laboratory is high, the time consumption is long, operators need certain operation skills, and the conditions for nucleic acid detection can be met only in Hospital, japan and Japan. Most respiratory tract pathogen detection chromatographic reagents on the market can only detect a single pathogen, or a plurality of rapid detection test papers for detecting a single pathogen are spliced together, and multiple times of sample loading are needed. Generally, the more items to be tested, the more problems such as the cross problem to be solved between items, the microsphere accumulation and the cleanness of the membrane surface. The invention overcomes the problems and technical obstacles in the prior art, provides a convenient chromatographic reagent which can simultaneously detect six respiratory pathogens of influenza A, influenza B, adenovirus, syncytial virus, mycoplasma pneumoniae and parainfluenza virus, can detect the pathogens by only collecting a nasal swab and a pharyngeal swab, and realizes non-invasiveness. And can meet the requirements of primary community hospitals and can be used for early diagnosis of diseases.

Meanwhile, the invention uses an integral pad + wet method mode, and can ensure that the marker and the target can fully react during liquid phase reaction so as to ensure that the sensitivity during multi-joint detection can be consistent with the single-detection sample pad + combined pad mode; by adopting the integrated pad, the residues at different interfaces can be reduced in the chromatographic process of all the markers, more fluorescent particles can pass through the T line, and the reaction is more thorough; the integrated pad and the wet method mode enable the film surface to be more uniform and clean.

Drawings

FIG. 1 is a schematic structural diagram of the joint test reagent test strip of the present invention.

Wherein: 1. a test strip; 11. a PVC base plate; 12. an integral pad; 13. a nitrocellulose membrane; 14. absorbent paper; 15. a quality control line (line C); 161. an influenza a detection line (T1); 162. an influenza B detection line (T2); 163. an adenovirus detection line (T3); 164. a syncytial virus detection line (T4); 165. a mycoplasma pneumoniae detection line (T5); 166. parainfluenza virus detection line (T6); 2. and (3) mixing the fluorescent particles.

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

Wherein: 3. a reagent card; 31. a plastic card shell; 32. a sample application hole; 33. observation window

Detailed Description

For better understanding of the present invention, the following embodiments and the accompanying drawings are used to describe the present invention in further detail, but those skilled in the art will appreciate that the following embodiments are not intended to limit the scope of the present invention, and any changes and modifications based on the present invention are within the scope of the present invention.

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

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 influenza A/influenza B/adenovirus/syncytial virus/parainfluenza/Mycoplasma pneumoniae antigen detection test paper kit

An embodiment of the immunochromatographic joint inspection kit for simultaneously detecting six respiratory disease pathogens comprises a test strip 1 and a fluorescent particle mixed solution, wherein the test strip 1 consists of a PVC (polyvinyl chloride) base plate 11, an integrated pad 12, a nitrocellulose membrane 13 and absorbent paper 14, which are stuck on the base plate, and the integrated pad 12, the nitrocellulose membrane 13 and the absorbent paper 14 are sequentially lapped and stuck on the base plate; the nitrocellulose membrane is sequentially provided with a quality control line (C line) 15, a detection line 1 (T1) 161, a detection line 2 (T2) 162, a detection line 3 (T3) 163, a detection line 4 (T4) 164, a detection line 5 (T5) 165 and a detection line 6 (T6) 166, wherein the detection lines and the quality control lines are parallel to each other, and the spacing distance is 2.5cm +/-0.1 cm; the quality control line is coated with a DNP monoclonal antibody, and the detection lines 1, 2, 3, 4, 5 and 6 are respectively coated with 1-2 mg/mL of influenza A monoclonal antibody, influenza B monoclonal antibody, adenovirus monoclonal antibody, syncytial virus monoclonal antibody, mycoplasma pneumoniae monoclonal antibody and parainfluenza monoclonal antibody;

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 and parainfluenza antibody-microsphere complex according to a proportion of 1-2.5% and DNP-BSA-microsphere complex according to a proportion of 0.02% by using 0.5% of phosphate buffer solution containing 0.02mol/L of PH = 7.2-7.6 and 0.5% of casein, wherein the microspheres are time-resolved fluorescent microspheres with the particle size of 300nm.

In order to shorten the chromatography time, a NC membrane with the width of 25cm is selected, 7 strips need to be coated on the NC membrane with the width of 25cm, and the distance between each detection strip and each quality control strip is 2.5cm +/-0.1 cm. The fluorescence detection instrument can well select the interval to ensure the position of the item strip.

As shown in fig. 2, the test paper card 3 is fixed on the plastic card shell 31 by the test paper strip 1, the surface of the test paper strip is pressed by the card surface, and the card surface is reserved with a sample hole 32 and an observation window 33 respectively at the part corresponding to the integrated pad 12 and the nitrocellulose membrane 13.

Example 2 preparation of influenza A/influenza B/adenovirus/syncytial virus/parainfluenza/Mycoplasma pneumoniae antigen detection kit

1. Time-resolved fluorescent microsphere labeling step

And (3) activation: and (3) carrying out ultrasonic dispersion on the fluorescent microspheres by using an ultrasonic cleaner, and uniformly mixing by using an oscillator. 50ul of the time-resolved fluorescent microsphere suspension with a solid content of 1% was diluted 20 times with ultra pure water, i.e., 1000ul, and added to the EP tube. Adding 1-10ul 50mg/ml N-hydroxysuccinimide (NHS) solution into the microsphere suspension, mixing uniformly, then adding 1-10ul 50mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution into the microsphere suspension, mixing uniformly, and reacting for 0.5 hour at normal temperature.

Marking: centrifuging for 15min under 14000 r/min. Discarding the supernatant, adding 0.02mol/L phosphate buffer solution with 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 syncytial virus monoclonal antibody, a mycoplasma pneumoniae monoclonal antibody, a parainfluenza monoclonal antibody and a DNP-BSA conjugate according to the mass ratio of 50: the labeled starting material was added at a ratio of 4 and reacted for 4 hours.

And (3) sealing: centrifuging for 15min under 14000 r/min. The supernatant was discarded, and BSA was added to make the solution a final concentration of 0.50% and blocking was performed for 1 hour. Centrifuging the sealed microspheres for 15min at 14000r/min, removing supernatant, adding 250ul of preservation solution into the centrifuged microspheres, and performing ultrasonic treatment to uniformly disperse the microspheres at 4 ℃ for later use.

In order to reduce the problems of non-specific cross and microsphere aggregation among projects, the reagent debugs the particle size of the microsphere marked by the fluorescent microsphere, the activation condition of the microsphere and the marking condition to reach the optimal state.

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

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

Preferably, the influenza a monoclonal antibody, the influenza B monoclonal antibody, the adenovirus monoclonal antibody, the syncytial virus monoclonal antibody, the mycoplasma pneumoniae monoclonal antibody and the DNP-BSA are labeled with a phosphate buffer at PH =7.0, and the parainfluenza monoclonal antibody is labeled with a phosphate buffer at PH = 9.0.

2. Preparation of coated plate

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 were each diluted to 1mg/ml with 0.01mmol/L PBS (PH = 7.4), and streaked on a nitrocellulose membrane with a streaking apparatus.

DNP monoclonal antibody-containing as a control line (line C);

as a detection line 1 (T1) containing an influenza a monoclonal antibody;

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

using the adenovirus monoclonal antibody as a detection line 3 (T3);

a detection line 4 (T4) containing a monoclonal antibody of the syncytial virus;

a detection line 5 (T5) containing a Mycoplasma pneumoniae monoclonal antibody;

detection line 6 (T6) containing parainfluenza monoclonal antibody was used.

Then dried for 16h at 37 ℃ and humidity <20% to prepare the coated plate.

3. Preparation of fluorescent particle mixture

Diluting the 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 and parainfluenza antibody-microsphere complex by 1-2.5% and diluting the DNP-BSA-microsphere complex by 0.02% to mix into the required fluorescent particle mixed solution by using 0.5% percent casein phosphate buffer solution containing 0.02mol/L of PH = 7.2-7.6.

4. Preparation of integral pad

Spreading on 21mm × 100mm glass fiber with a treatment solution containing 0.02mol/L phosphate buffer solution with pH = 7.2-7.6, 5% sucrose and 0.8% Triton-100 surfactant, and lyophilizing.

The reagent uses an integrated pad and wet method mode, and changes the conventional mode of a sample pad and a combined pad into the integrated pad and wet method mode. The integrated pad and wet method mode is a mode of liquid phase reaction and then solid phase reaction, and the marker and the target can fully react during the liquid phase reaction so as to ensure that the sensitivity during the multi-joint detection can be consistent with the single-detection sample pad and the combined pad mode; by adopting the integrated pad, the residues at different interfaces can be reduced in the chromatography process of all the markers, more fluorescent particles can pass through the T line, and the reaction is more thorough; the integrated pad and the wet method mode enable the film surface to be more uniform and clean.

5. Preparation of reagent cards

And sticking the integrated blank pad at one end close to the quality control line, and lapping the integrated blank pad on an NC film for 1mm. And (3) attaching the absorbent paper to one end close to the detection line 6 (T6) and lapping the absorbent paper on an NC film for 1mm. Then cutting into test strips with the thickness of 4mm +/-0.1 mm by using a slitter. The test paper is put into a polystyrene plastic shell to be compacted, then is put into a packaging bag to be added with a bag of drying agent, and is sealed. One aliquot of reagent contains one aliquot of reagent card plus one tube of fluorescent particle lyophilization reaction tube.

Example 3 detection

The detection card prepared in example 1 was used to detect a sample to be detected, and a nasal swab or a pharyngeal swab could be detected.

Collecting nasal swabs: the swab was inserted into the nasal cavity and gently rotated and pushed into the nasal cavity until the turbinate (2.0 cm-2.5cm from the nostril) was rotated 3 more times and the swab was removed.

Collecting throat swabs: the swab is inserted into the throat from the oral cavity, and the red part of the pharyngeal wall and the palatal tonsil is used as the center, and the pharyngeal tonsils and the pharyngeal wall on both sides are wiped with moderate force to avoid touching the tongue, and then the swab is taken out.

0.5ml of the sample extraction liquid was added to the plastic tube, and then a cotton swab of the collected nasal swab or pharyngeal swab was inserted into the sample extraction liquid, and the sample was spun and squeezed 5 times to dissolve the sample in the solution as much as possible. Finally, the swab head is squeezed to keep the liquid as much as possible in the tube, and the swab is removed and discarded.

And (3) dropwise adding 100ul of the dissolved sample into a fluorescent particle reaction tube, fully and uniformly mixing, dropwise adding 70ul of the dissolved sample into a sample adding port of the detection card, flatly placing for 15min, and inserting the detection card into a dry type fluorescence immunoassay analyzer to detect the result.

The sample extraction liquid has a phosphate buffer solution with 0.02mol/L PH =7.4, triton 100 with the volume fraction of 0.1% -1.0%, tween 20 with the volume fraction of 0.1% -1.0% and sodium chloride with the molar mass of 0.5-1.0 mol/L, wherein the sample extraction liquid has the function of cracking viruses to expose detection sites.

Example 4 integral pad + wet mode versus sample pad + conjugate pad mode

Using the reagents of example 1, and further using the sample pad + conjugate pad mode single detection reagent and the sample pad + conjugate pad mode joint detection reagent prepared from the same materials of example 1, the comparison results are shown in table 1 below:

table 1 table of results comparing the integrated pad + wet method mode with the sample pad + conjugate pad mode

From the results of the above-described measurements in table 1, the sensitivity of the one-pad + wet mode is consistent with the sample pad + conjugate pad (single assay) mode, and superior to the sample pad + conjugate pad (joint assay mode).

Example 5 evaluation of Properties

1. Minimum detection limit

(1) Lowest detection limit for influenza A

National reference products L1\ L2\ L5 of influenza A/B virus antigen detection reagents are prepared by extracting solutions of samples according to the following ratio of 1:80 and 1, 160, and the detection results are shown in the following table 2:

TABLE 2 Table of the results of the detection of influenza A in national reference samples of the detection reagent for detecting influenza A/B virus antigens by the detection card of the present invention

Numbering

T1：FluA

T2:FluB

T3:ADV

T4:RSV

T5：MP

T6：HPIV

L1(1:10)

Positive (+)

Negative (-)

L1(1:20)

Positive (+)

Negative (-)

L1(1:40)

Positive (+)

Negative (-)

L1(1:80)

Positive (+)

Negative (-)

L1(1:160)

Positive (+)

Negative (-)

L2(1:10)

Positive (+)

Negative (-)

L2(1:20)

Positive (+)

Negative (-)

L2(1:40)

Positive (+)

Negative (-)

L2(1:80)

Positive (+)

Negative (-)

L2(1:160)

Positive (+)

Negative (-)

L5(1:10)

Positive (+)

Negative (-)

L5(1:20)

Positive (+)

Negative (-)

L5(1:40)

Positive (+)

Negative (-)

L5(1:80)

Positive (+)

Negative (-)

L5(1:160)

Positive (+)

Negative (-)

Negative of(-)

Negative (-)

As can be seen from the test results in Table 2, the respiratory tract joint test card of the present invention can detect influenza A/B virus antigen test reagents with the lowest detection limit of national reference product influenza A, and influenza A has no cross with other items.

(2) Lowest detection limit for influenza B

National reference substance L3\ L4 used sample extraction solutions of the influenza A/B virus antigen detection reagent are respectively prepared according to the following steps of 1:80 and 1, 160 dilution, and the detection results are shown in the following table 3:

TABLE 3 Table of the results of the detection of influenza B virus antigen detection reagent of the present invention

Numbering

T1：FluA

T2:FluB

T3:ADV

T4:RSV

T5：MP

T6：HPIV

L3(1:10)

Negative (-)

Positive (+)

Negative (-)

L3(1:20)

Negative (-)

Positive (+)

Negative (-)

L3(1:40)

Negative (-)

Positive (+)

Negative (-)

L3(1:80)

Negative (-)

Positive (+)

Negative (-)

L3(1:160)

Negative (-)

Positive (+)

Negative (-)

L4(1:10)

Negative (-)

Positive (+)

Negative (-)

L4(1:20)

Negative (-)

Positive (+)

Negative (-)

L4(1:40)

Negative (-)

Positive (+)

Negative (-)

L4(1:80)

Negative (-)

Positive (+)

Negative (-)

L4(1:160)

Negative (-)

As can be seen from the test results in Table 3, the respiratory tract joint test card of the present invention can detect the lowest detection limit of national reference influenza B, which is an influenza A/B antigen test reagent, and has no cross with other items.

(3) Minimum detection limit of adenovirus

Adenovirus culture fluid is diluted to 2 x 10 by using a sample extract ^{^5} VP/ml(ADV1)、2×10 ^{^4} VP/ml(ADV2)、1×10 ^{^4} VP/ml (ADV 3), the results of the assay are shown in Table 4 below:

TABLE 4 test results of adenovirus test using the test card of the present invention

Numbering

T1：FluA

T2:FluB

T3:ADV

T4:RSV

T5：MP

T6：HPIV

ADV1

Negative (-)

Positive (+)

Negative (-)

ADV2

Negative (-)

Positive (+)

Negative (-)

ADV3

Negative (-)

Positive (+)

Negative (-)

As can be seen from the results shown in Table 4, the lower limit of detection of the adenovirus Carbamate for respiratory tract combination detection of the present invention is 1X 10 ^{^4} VP/ml, and adenovirus did not cross other items.

(4) Minimum detection limit of syncytial virus

Diluting the culture solution of the syncytial virus to 4.36X 10 by using a sample extract ^{^4} pfu/ml(RSV1)、8.72×10 ^{^3} pfu/ml(RSV2)、4.36×10 ^{^3} pfu/ml (RSV 3), the results of which are shown in Table 5 below:

TABLE 5 test result table for detecting syncytial virus by using the test card of the present invention

Numbering

T1：FluA

T2:FluB

T3:ADV

T4:RSV

T5：MP

T6：HPIV

RSV1

Negative (-)

Positive (+)

Negative (-)

RSV2

Negative (-)

Positive (+)

Negative (-)

RSV3

Negative (-)

Positive (+)

Negative (-)

As can be seen from the above results in Table 5, the lower limit of detection of the combined detection of the present invention for the carlsytial virus is 4.36X 10 ^{^3} pfu/ml, and syncytial virus did not cross other projects.

(5) Minimum detection limit of mycoplasma pneumoniae

The mycoplasma pneumoniae culture solution is diluted to 3.83 multiplied by 10 by using the sample extract ^{^4} pfu/ml(MP1)、1.532×10 ^{^} ⁴ pfu/ml(MP2)、7.66×10 ^{^3} pfu/ml (MP 3), the results of the assay are shown in Table 6 below:

TABLE 6 Table of the results of detection of Mycoplasma pneumoniae by the detection card of the present invention

Number of

T1：FluA

T2:FluB

T3:ADV

T4:RSV

T5：MP

T6：HPIV

MP1

Negative (-)

Positive (+)

Negative (-)

MP2

Negative (-)

Positive (+)

Negative (-)

MP3

Negative (-)

Positive (+)

Negative (-)

As can be seen from the results of the above Table 6, the lower limit of detection of the combined detection of Mycoplasma pneumoniae of the present invention is 7.66X 10 ^{^3} pfu/ml, and Mycoplasma pneumoniae did not cross other items.

(6) Minimum detection limit of parainfluenza virus

Parainfluenza virus culture broth diluted to 1.31X 10 with sample extract ^{^4} pfu/ml(HPIV1)、5.24×10 ^{^} ³ pfu/ml(HPIV)、2.62×10 ^{^3} pfu/ml (HPIV 3), the results of the measurements are shown in Table 7 below:

TABLE 7 detection results of parainfluenza virus by the detection card of the present invention

Numbering

T1：FluA

T2:FluB

T3:ADV

T4:RSV

T5：MP

T6：HPIV

HPIV1

Negative (-)

Positive (+)

HPIV2

Negative (-)

Positive (+)

HPIV3

Negative (-)

Positive (+)

As can be seen from the above results in Table 7, the lower limit of detection of the joint detection of the present invention for the parainfluenza virus of Cauloinfluenza virus is 2.62X 10 ^{^3} pfu/ml, and parainfluenza virus did not cross other items.

The final detection sensitivity results for the kits of the invention are shown in table 8:

TABLE 8 table of the test sensitivity results of the kit of the present invention

2. Cross reaction experiment

Cross article

T1：FluA

T2:FluB

T3:ADV

T4:RSV

T5：MP

T6：HPIV

H1N1

Positive (+)

Negative (-)

H3N2

Positive (+)

Negative (-)

H5N1

Positive (+)

Negative (-)

H7N9

Positive (+)

Negative (-)

Second stream V

Negative (-)

Positive (+)

Negative (-)

Stream B Y

Negative (-)

Positive (+)

Negative (-)

Adenovirus disease

Negative (-)

Positive (+)

Negative (-)

Syncytial virus

Negative (-)

Positive (+)

Negative (-)

Mycoplasma pneumoniae

Negative (-)

Positive (+)

Negative (-)

Parainfluenza virus

Negative (-)

Positive (+)

Human giant cell

Negative (-)

Coxsackie

Negative (-)

Parotitis

Negative (-)

Measles, measles and other diseases

Negative (-)

Chlamydia pneumoniae

Negative (-)

Each item did not cross-react with the above virus.

Example 6 reagent detection applications

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

Sensitivity: 178 parts of influenza A is detected, and the sensitivity is 95.70%; 66 parts of influenza B is detected, and the sensitivity is 95.65%; detecting 12 portions of adenovirus with sensitivity of 100%; 21 portions of syncytial virus are detected, and the sensitivity is 100%; detecting 10 parts of parainfluenza, and the sensitivity is 80%; 5 parts of mycoplasma pneumoniae is detected, and the sensitivity is 71.43%.

Specificity: 280 negatives were detected in 286 negative samples, with a specificity of 97.9%.

In conclusion, the invention provides the convenient chromatography reagent which 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 a nasal swab and a pharyngeal swab, and realizes non-wound and high specificity. And can meet the requirements of primary community hospitals and can be used for early diagnosis of diseases.

Meanwhile, the invention uses an integral pad + wet method mode, and the marker and the target can fully react during liquid phase reaction, so that the sensitivity during multi-joint detection can be ensured to be consistent with the single-detection sample pad + combined pad mode; by adopting the integrated pad, the residues at different interfaces can be reduced in the chromatography process of all the markers, more fluorescent particles can pass through the T line, and the reaction is more thorough; the integrated pad and wet method mode makes the film surface more uniform and clean.

Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.

Claims

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;

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

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.