Respiratory tract infectious pathogen detection kit based on fluorescence imaging as well as use method and application of respiratory tract infectious pathogen detection kit

Abstract

The invention discloses a respiratory tract infectious pathogen detection kit based on fluorescence imaging as well as a use method and application thereof, and belongs to the technical field of biological medicines. In order to solve the technical problems of long detection time, relatively low sensitivity and specificity and relatively high detection cost of a respiratory tract infectious pathogen detection method in the prior art, the invention provides a respiratory tract infectious pathogen detection kit based on fluorescence imaging. The detection kit comprises normal saline, a 1 * PBS buffer solution, an imaging buffer solution, lectin and a fluorescent probe labeled monoclonal antibody of respiratory tract infectious pathogens. The detection kit provided by the invention is relatively high in sensitivity and specificity, relatively high in imaging resolution, relatively short in detection time and relatively low in detection cost. The detection kit provided by the invention can be applied to fluorescence imaging detection of respiratory tract infectious pathogens.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a respiratory infectious pathogen detection kit and a use method and application thereof. Background Art

[0002] Respiratory infectious diseases are highly contagious and spread rapidly, with a significant impact on morbidity and mortality. The etiology of respiratory infections is complex, including bacteria, viruses, mycoplasma, chlamydia, fungi, etc.; viruses can be transmitted through droplets, mist droplets, or contaminated utensils, and can cause illness regardless of season and age. Its clinical manifestations are diverse, mainly symptoms such as rhinitis, pharyngitis, laryngitis, tonsillitis, etc., and the severity ranges from self-limiting upper respiratory tract infections to severe or even life-threatening lower respiratory tract infections, such as tracheitis, bronchitis and pneumonia. Common viruses that can cause respiratory infections in clinical practice include: influenza A virus, influenza B virus, new coronavirus, Haemophilus influenzae, universal human parainfluenza virus, respiratory syncytial virus, human metapneumovirus, enterovirus, Mycoplasma pneumoniae, group A streptococcus, Bordetella pertussis, common coronavirus, rhinovirus, bocavirus, adenovirus, Streptococcus pneumoniae, etc. Because the above viruses are highly contagious, spread quickly, have a short incubation period, and have similar clinical symptoms, clinicians are unable to accurately distinguish them, which makes clinical diagnosis and treatment extremely difficult. Therefore, it is extremely important to identify the type of infectious pathogen in order to control the transmission route, take preventive measures for the population, select targeted drugs for clinical use, and avoid the abuse of antibiotics.

[0003] At present, the common clinical respiratory pathogen detection methods have the following problems: (1) The virus isolation and culture method is cumbersome, the detection time is long, and the positive rate is low; (2) The sensitivity and specificity of methods such as direct specimen smear, Gram staining, and microscopy are low; (3) The pathogen culture method takes a long time to detect and is prone to false positives; (4) The serological identification method is prone to false positives and false negatives; (5) The polymerase chain reaction (PCR) detection used in molecular biology methods is expensive. Therefore, those skilled in the art are eager to develop a respiratory infectious pathogen detection method with short detection time, high sensitivity, high specificity, and low detection cost for clinical detection of respiratory infectious pathogens. Summary of the invention

[0004] The present invention aims to solve the technical problems in the prior art of respiratory infectious pathogen detection methods, such as long detection time, low sensitivity and specificity, and high detection cost, and provides a respiratory infectious pathogen detection kit based on fluorescence imaging, as well as a use method and application.

[0005] One of the purposes of the present invention is to provide a respiratory infectious pathogen detection kit based on fluorescence imaging, the detection kit comprising: physiological saline, 1×PBS buffer, imaging buffer, lectin and monoclonal antibodies of respiratory infectious pathogens labeled with fluorescent probes.

[0006] In a preferred embodiment of the present invention, the imaging buffer liquid is composed of: 5 mg / mL glucose oxidase, 40 μg / mL catalase and 140 mM β-mercaptoethanol.

[0007] In a preferred embodiment of the present invention, the lectin is Rhodamine-WGA.

[0008] In a preferred embodiment of the present invention, the fluorescent probe is an Alexa Fluor fluorescent probe, and the excitation wavelength of the Alexa Fluor fluorescent probe is one or more of Alexa Fluor 405, 488, 561 or 647.

[0009] In a preferred embodiment of the present invention, the monoclonal antibody against respiratory infectious pathogens is one or more of an anti-Streptococcus pneumoniae surface antigen antibody, an anti-Staphylococcus aureus antigen SpA antibody, an anti-Mycoplasma pneumoniae antigen P30 antibody, an adenovirus Adv antibody, or a respiratory syncytial virus RSV antibody.

[0010] The second object of the present invention is to provide a method for using the above-mentioned detection kit for non-diagnostic purposes, and the method for using comprises the following steps:

[0011] S1: Collect the respiratory tract sputum sample to be tested, wash the sputum sample with sterile saline, apply the washed yellow granules or opaque colored spots of sputum sample on a glass slide, and air-dry it under cold air from a hair dryer to obtain the original sputum smear;

[0012] S2: Add the monoclonal antibody of respiratory infectious pathogens labeled with lectin and fluorescent probe diluted with 1×PBS buffer in the detection system of the kit to the original sputum smear obtained in S1 for incubation, wash away the unbound antibody with 1×PBS buffer, then add the imaging buffer, cover with a coverslip, and seal with nail polish to obtain the sputum smear to be tested;

[0013] S3: The sputum smear obtained in S2 is placed under a fluorescence microscope for imaging observation.

[0014] In a preferred embodiment of the present invention, the sputum sample washing step described in S1 is: add 5 times the volume of sterile physiological saline to the sputum sample, shake it vigorously for 10 seconds, take out the purulent sputum precipitated at the bottom of the tube, put it into a new test tube, and wash it again, for a total of 3 times; the glass slide is pre-modified with a silanization reagent using an APTES reagent.

[0015] In a preferred embodiment of the present invention, the lectin in S2 is diluted with 1×PBS buffer at 1:500, and the monoclonal antibody against respiratory infectious pathogens is diluted with 1×PBS buffer at 1:100; the added amount of the imaging buffer liquid is 50 μl.

[0016] In a preferred embodiment of the present invention, the fluorescence microscope in S3 is one or more of a spinning disk confocal microscope, a high-resolution fluorescence microscope or a super-resolution fluorescence microscope.

[0017] The third object of the present invention is to provide the use of the above detection kit in the detection of respiratory infectious pathogens.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides a respiratory infectious pathogen detection kit based on fluorescence imaging, a method of use and an application, wherein the detection kit comprises: physiological saline, 1×PBS buffer, imaging buffer, lectin and monoclonal antibody of respiratory infectious pathogens labeled with fluorescent probe. The present invention uses rhodamine-WGA, a lectin labeled with rhodamine for identifying Gram-positive bacteria, and monoclonal antibody of respiratory infectious pathogens labeled with fluorescent probe to clarify the proportion of respiratory infectious pathogens to Gram-positive bacteria, thereby judging the infection degree of respiratory infectious pathogens.

[0020] The respiratory tract infectious pathogen detection method provided by the present invention has the following advantages compared with the prior art:

[0021] (1) Short detection time: The entire detection process can be completed within 30 minutes;

[0022] (2) High sensitivity and low cost: It can detect trace amounts of pathogens. By coupling monoclonal antibodies against different respiratory infectious pathogens with fluorescent dyes of different excitation wavelengths, multiple pathogens can be detected in a single sample at the same time, saving resources and time costs.

[0023] (3) High specificity: Specific antibodies can be used to specifically identify specific pathogens, and the type of pathogen can be accurately identified;

[0024] (4) High imaging resolution: The spinning disk confocal microscope used can resolve pathogens as small as 100 nm. Combined with a super-resolution / high-resolution microscope, it can resolve pathogens smaller than 100 nm, thereby maximizing the resolution of fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram of the imaging detection result of the spinning disk confocal microscope in Example 2;

[0026] Figure 2 This is a diagram of super-resolution microscope imaging detection results in Example 2;

[0027] Figure 3 This is a statistical result diagram of the size of Staphylococcus aureus under the spinning disk confocal microscope imaging in Example 2;

[0028] Figure 4 This is a diagram showing the size statistics of Staphylococcus aureus under super-resolution microscopy imaging in Example 2. DETAILED DESCRIPTION

[0029] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0030] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0031] Example 1: A respiratory infectious pathogen detection kit based on fluorescence imaging

[0032] The present embodiment provides a respiratory infectious pathogen detection kit based on fluorescence imaging, the detection kit comprising: physiological saline, 1×PBS buffer solution (pH=7.4), imaging buffer (5 mg / mL glucose oxidase, 40 μg / mL catalase and 140 mM β-mercaptoethanol), rhodamine-labeled lectin Rhodamine-WGA for identifying Gram-positive bacteria (purchased from Nanjing Wobo Biotechnology Co., Ltd.), and monoclonal antibody Anti-Protein A / AF647 for specific recognition of Staphylococcus aureus antigens (purchased from Bioss).

[0033] Example 2: Application of a respiratory infectious pathogen detection kit based on fluorescence imaging

[0034] S1: Sputum samples to be tested were collected from three patients suspected of being infected with respiratory pathogens. The collection steps were as follows: sputum samples were collected from patients after gargling with warm water several times in the morning. Patients without sputum could use 10% saline heated to 45°C for aerosol inhalation. After the sputum was coughed up, it was immediately placed in a clean and disinfected test tube, the lid was sealed, and it was immediately sent for inspection; the sputum samples were washed with sterile saline, and the sputum sample washing steps were as follows: 5 times the volume of sterile saline was added to the sputum sample, and after vigorous shaking for 10 seconds, the purulent sputum precipitated at the bottom of the tube was taken out, and the sputum was put into a new test tube and washed again, for a total of 3 times; the washed yellow granular or opaque colored spotted sputum sample was smeared on a glass slide (24×50mm, Fisherscientific), the glass slide needed to be pre-modified with a silanization reagent using an APTES reagent, and the area was marked with a marker pen, and it was blown with a hair dryer for 5 minutes until it was air-dried to obtain the original sputum smear;

[0035] S2: The lectin Rhodamine-WGA and monoclonal antibody Anti-Protein A / AF647 diluted with 1×PBS buffer in the detection system of the kit were added to the original sputum smear obtained in S1 for incubation, and incubated in a 37°C wet box for 15 minutes. The lectin was diluted with 1×PBS buffer at a ratio of 1:500, and the Anti-Protein A / AF647 was diluted with 1×PBS buffer at a ratio of 1:100; the unbound antibody was washed away with 1×PBS buffer for 3 times, and then an imaging buffer solution was added dropwise, and the amount of the imaging buffer solution added was 50 μl; a coverslip (22×22, Fisherscientific) was covered, and nail polish was used for sealing to obtain the sputum smear to be tested.

[0036] S3: The sputum smear to be tested obtained in S2 is placed under a spinning disk confocal microscope, and dual-channel imaging of the sputum smear to be tested is performed using 561 nm and 640 nm lasers at the same time.

[0037] The results are as follows Figure 1 As shown in Table 1, under a 100x microscope under 561nm excitation, a large number of green fluorescent spots were observed, representing the presence of Gram-positive bacteria, and the red fluorescent spots observed under 640nm excitation represented the presence of Staphylococcus aureus. The ratio of the number of Staphylococcus aureus to the number of Gram-positive bacteria represented the degree of infection with Staphylococcus aureus. It can be seen that in sample 1, the number of Staphylococcus aureus accounted for 91% of the number of Gram-positive bacteria, indicating that the degree of infection with Staphylococcus aureus was very serious; in sample 2, the number of Staphylococcus aureus accounted for 0.05% of the number of Gram-positive bacteria, indicating that the degree of infection with Staphylococcus aureus was relatively mild; in sample 3, the number of Staphylococcus aureus accounted for 29% of the number of Gram-positive bacteria, indicating that the degree of infection with Staphylococcus aureus was moderate to mild.

[0038] Table 1

[0039]

[0040] In order to obtain a more accurate size of Staphylococcus aureus, this embodiment further uses a 640nm laser to perform super-resolution imaging on the sputum smear (sample 1) obtained in S2 under a super-resolution microscope. The results are as follows: Figure 2 As shown; the size of Staphylococcus aureus observed by super-resolution imaging and confocal imaging was counted, and the results are as follows Figure 3-4 As shown, through comparative analysis, it was found that compared with confocal imaging, the size of Staphylococcus aureus observed using super-resolution imaging was more accurate, and the range was smaller than the size of Staphylococcus aureus observed by confocal imaging, indicating that super-resolution imaging can observe smaller single Staphylococcus aureus, and is more accurate in detecting whether Staphylococcus aureus exists in the sputum smear to be tested.

[0041] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A respiratory infectious pathogen detection kit based on fluorescence imaging, characterized in that: The detection kit comprises physiological saline, 1×PBS buffer, imaging buffer, lectin and monoclonal antibody of respiratory infectious pathogens marked by fluorescent probe.

2. The detection kit according to claim 1, characterized in that: The imaging buffer solution is composed of: 5 mg / mL glucose oxidase, 40 μg / mL catalase and 140 mM β-mercaptoethanol.

3. The detection kit according to claim 1, characterized in that: The lectin is Rhodamine-WGA.

4. The detection kit according to claim 1, characterized in that: The fluorescent probe is an Alexa Fluor fluorescent probe, and the excitation wavelength of the Alexa Fluor fluorescent probe is one or more of Alexa Fluor 405, 488, 561 or 647.

5. The detection kit according to claim 1, characterized in that: The monoclonal antibody against respiratory infectious pathogens is one or more of an antibody against the surface antigen of Streptococcus pneumoniae, an antibody against the antigen of Staphylococcus aureus SpA, an antibody against the antigen of Mycoplasma pneumoniae P30, an antibody against adenovirus Adv or an antibody against respiratory syncytial virus RSV.

6. A method for using the detection kit according to any one of claims 1 to 5 for non-diagnostic purposes, characterized in that: The method of use comprises the following steps: S1: Collect the respiratory tract sputum sample to be tested, wash the sputum sample with sterile saline, apply the washed yellow granules or opaque colored spots of sputum sample on a glass slide, and air-dry it under cold air from a hair dryer to obtain the original sputum smear; S2: Add the monoclonal antibody of respiratory infectious pathogens labeled with lectin and fluorescent probe diluted with PBS buffer in the detection system of the kit to the original sputum smear obtained in S1 for incubation, wash away the unbound antibody with 1×PBS buffer, then add the imaging buffer, cover with a coverslip, and seal with nail polish to obtain the sputum smear to be tested; S3: The sputum smear obtained in S2 is placed under a fluorescence microscope for imaging observation.

7. The method of use according to claim 6, characterized in that: The sputum sample washing steps described in S1 are: add 5 times the volume of sterile physiological saline to the sputum sample, shake vigorously for 10 seconds, take out the purulent sputum precipitated at the bottom of the tube, put it into a new test tube, and wash it again, for a total of 3 times; the glass slide is pre-modified with a silanization reagent using an APTES reagent.

8. The method of use according to claim 6, characterized in that: The lectin in S2 was diluted with 1×PBS buffer at 1:500, and the monoclonal antibody against respiratory infectious pathogens was diluted with 1×PBS buffer at 1:100; the added amount of the imaging buffer was 50 μl.

9. The method of use according to claim 6, characterized in that: The fluorescence microscope described in S3 is one or more of a spinning disk confocal microscope, a high-resolution fluorescence microscope or a super-resolution fluorescence microscope.

10. Use of the detection kit according to any one of claims 1 to 5 in the detection of respiratory infectious pathogens.

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