Kit and detection kit for detecting herpes simplex virus in tears

Abstract

The present invention discloses a kit and a detection kit for detecting herpes simplex virus in tears, the kit comprises: a housing, which comprises an internal cavity, and a sample application area and a detection window which are respectively communicated with the internal cavity; the immunochromatography test strip comprises a sample pad, a release pad and a nitrocellulose membrane which are connected in sequence, the immunochromatography test strip is arranged in the inner containing cavity, and part or all of the sample pad extends to the sample application area; the sampling assembly is in pivot connection with the shell and limits a first limit position, and when the sampling assembly is located at the first limit position, the sampling assembly is in surface contact with the sample pad; wherein herpes simplex virus type 1 G glycoprotein marked by fluorescence is embedded on the release pad; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with an anti-human IgA antibody, the quality control line is coated with a goat anti-rabbit IgG antibody, and the detection line and the quality control line are exposed to the detection window.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a kit and detection kit for detecting herpes simplex virus in tears. Background Technology

[0002] Herpes simplex keratitis (HSK) is one of the most serious infectious eye diseases in the world today, mainly caused by infection with herpes simplex virus (HSV) type 1. It accounts for 63.2% of all corneal blindness cases, and its incidence has been increasing year by year in recent years due to the widespread use of antibiotics, hormones, and other immunosuppressants. HSK is prone to recurrence, with a 2-year recurrence rate of 20%, a 5-year recurrence rate of 40%, and a 7-year recurrence rate as high as 67%. Each recurrence carries the risk of corneal scarring and even blindness, often requiring corneal transplantation. The risks of rejection, limitations, and high costs of surgery place a heavy burden on society. Early diagnosis of HSK using in vitro diagnostic techniques and identification of high-risk groups for recurrence are of great significance for guiding prophylactic medication and improving the overall prognosis of HSK.

[0003] Viral isolation and culture from corneal epithelial scrapings is the "gold standard" for detection. In corneal epithelial scrapings of herpes simplex keratitis (HSK), typical pathological features can be observed under a microscope, including multinucleated giant cells, intracellular eosinophilic inclusion bodies (Cowdry A bodies), and positive viral antigens. Combined with clinical manifestations, this can aid in diagnosis. However, cell culture is time-consuming and laborious, and corneal scrapings are difficult to obtain and risky, thus limiting its clinical application.

[0004] In conclusion, the lack of HSK testing tools has become a bottleneck restricting rapid clinical HSK screening. Summary of the Invention

[0005] In view of this, the present invention provides a rapid and efficient kit and test suite for detecting HSK, and the test sample is readily available tear fluid.

[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a kit for detecting herpes simplex virus in tears, the kit comprising: The housing includes an internal cavity, and a sampling area and a detection window respectively communicating with the internal cavity; An immunochromatographic test strip includes a sample pad, a release pad, and a nitrocellulose membrane connected in sequence. The immunochromatographic test strip is disposed in the internal cavity, and part or all of the sample pad extends into the spotting area. A sampling component, which is pivotally connected to the housing and defines a first extreme position, wherein the sampling component contacts the sample pad surface when it is in the first extreme position; in, The release pad is embedded with fluorescently labeled herpes simplex virus type 1 G glycoprotein; The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with anti-human IgA antibody, and the control line is coated with goat anti-rabbit IgG antibody. The detection line and the control line are exposed in the detection window.

[0007] Furthermore, the sampling assembly includes a rotating part and a sampling test strip connected to the rotating part. The rotating part is pivotally connected to the housing. When the sampling assembly is in the first extreme position, the sampling test strip is in contact with the sample pad surface.

[0008] Furthermore, the sampling test paper is absorbent filter paper.

[0009] Furthermore, the sampling test strip is equipped with indicator lines to help determine the minimum sampling amount required for the test.

[0010] Furthermore, the sampling area has a groove structure that conforms to the outer contour of the sampling component.

[0011] Furthermore, a positioning groove is provided on the housing on the other side relative to the sampling area. The positioning groove is adapted to the outer contour of the sampling component and defines the second extreme position of the sampling component.

[0012] Furthermore, the immunochromatographic test strip also includes an absorbent pad, which is attached to the nitrocellulose membrane at the other end opposite the release pad. Its main function is to absorb excess reagent and prevent backflow, ensuring the stability and accuracy of the detection process.

[0013] Furthermore, the herpes simplex virus type 1 G glycoprotein is labeled on europium fluorescent microspheres.

[0014] In a second aspect, the present invention provides an immunochromatographic assay kit for detecting herpes simplex virus in tears, comprising the above-described reagent kit and tear sample elution solution.

[0015] Furthermore, the tear sample elution solution is a PBS solution.

[0016] Compared with the prior art, the beneficial effects and significant progress of the present invention are as follows: The technical solution of this invention proposes for the first time that detecting herpes simplex virus-specific IgA antibodies in tears can provide early diagnosis of HSK and early warning of recurrence risk. Based on this, a kit suitable for tear collection and rapid detection of herpes simplex keratitis has also been designed.

[0017] The reagent kit and testing kit of the present invention have the advantages of safe and quantitative sampling, simple operation, rapid and accurate results, good patient compliance, and convenient outpatient follow-up. Attached Figure Description

[0018] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly introduced below.

[0019] Obviously, the accompanying drawings described below are only some of the drawings of the embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort, but these other drawings are also within the scope of the drawings required for the embodiments of the present invention.

[0020] Figure 1 This is a schematic diagram of the structure of a kit for detecting herpes simplex virus in tears according to an embodiment of the present invention, wherein the sampling component is in the second extreme position; Figure 2 yes Figure 1 A structural diagram of the reagent kit from another perspective, showing the back of the kit; Figure 3 yes Figure 1 A schematic diagram of the reagent kit in another state, wherein the sampling component is in the first extreme position; Figure 4 yes Figure 3 A structural diagram of the reagent kit from another perspective, showing the back of the kit; Figure 5 yes Figure 1 A structural breakdown diagram of the reagent kit; Figure 6 yes Figure 1 A schematic diagram of the reagent kit in another state, wherein the sampling component is close to the first limit position in the rotation direction; Figure 7 yes Figure 1 A schematic diagram of the reagent kit in another state, wherein the sampling component is close to the second limit position in the rotation direction; Figure 8 yes Figure 1 A schematic diagram of the structure of the immunochromatographic test strip in the kit; Figure 9The ROC curves are based on the tear detection scheme proposed in this invention, and are obtained from the detection results of tear samples from 105 outpatients diagnosed with HSK and 73 control group patients. Figure 10 Based on the tear detection scheme proposed in this invention, a bar chart was generated comparing the test results of tear samples from 105 outpatients diagnosed with HSK and 73 control group patients to compare the tear results of the affected eye and healthy eye of HSK patients with those of normal controls.

[0021] Explanation of reference numerals in the attached figures: 100—Shell 110—Sampling Area 120—Detection window, 130—Ball socket, 140—Positioning groove, 200—Immunochromatographic test strip, 210—Sample pad, 220—Release Pad 230—Nitrocellulose membrane, 231—Testing line, 232—Quality Control Line 240—Absorbent pad, 300—Sampling component, 310—Rotating part, 311—Ball head, 312—slot, 320—Sampling test strip, 321—Indicator line. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0023] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0024] It should be noted that the terms "first," "second," and "third" (if present), etc., in the specification, claims, and accompanying drawings of the embodiments of this invention, are only used to distinguish different objects and not to describe a specific order. Furthermore, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or devices.

[0025] The technical solution of the present invention will now be described in detail with reference to specific embodiments.

[0026] Figures 1 to 5 A schematic diagram of a kit for detecting herpes simplex virus in tears according to an embodiment of the present invention is shown, including a housing 100, an immunochromatographic test strip 200, and a sampling assembly 300.

[0027] refer to Figure 5 The housing 100 has an overall flat rectangular parallelepiped structure, including an internal cavity, a sample application area 110, and a detection window 120. The internal cavity is used to hold the immunochromatographic test strip 200; the sample application area 110 is configured as a recessed mechanism, with one side connected to the internal cavity, and one end of the immunochromatographic test strip 200 extends from the internal cavity to the sample application area 110; the detection window 120 connects the internal cavity to the outside, thereby exposing a corresponding portion of the immunochromatographic test strip 200 disposed in the internal cavity to the detection window 120.

[0028] like Figure 8 As shown, the immunochromatographic test strip 200 includes a sample pad 210, a release pad 220, a nitrocellulose membrane 230, and an adsorption pad 240 connected in sequence. The connections between adjacent pads partially overlap, allowing the sampling liquid loaded on the sample pad 210 to flow from the sample pad 210 to the adsorption pad 240 under chromatographic action and be absorbed by the adsorption pad 240. The release pad 220 is embedded with fluorescently labeled herpes simplex virus type 1 G glycoprotein; in this embodiment, the herpes simplex virus type 1 G glycoprotein is labeled with europium fluorescent microspheres. The nitrocellulose membrane 230 is provided with a detection line 231 and a control line 232. The detection line 231 is coated with anti-human IgA antibody, and the control line 232 is coated with goat anti-rabbit IgG antibody.

[0029] refer to Figure 5The sample pad 210 of the immunochromatographic test strip 200 extends partially or entirely into the sample application area 110, thereby allowing the sample liquid to be applied to the sample pad 210 from the sample application area 110. The nitrocellulose membrane 230 is positioned corresponding to the detection window 120, such that the nitrocellulose membrane 230 is partially exposed to the external environment through the detection window 120. In particular, the detection line 231 and control line 232 on the nitrocellulose membrane 230 are also exposed to the external environment through the detection window 120. The indicator letters "C" and "T" corresponding to the detection line 231 and control line 232 are respectively provided on the outer surface of the housing 100 near the detection window 120.

[0030] The sampling assembly 300 includes a rotating part 310 and a sampling test strip 320. The sampling test strip 320 is connected to the rotating part 310. In this embodiment, the rotating part 310 is provided with a slot 312, which is suitable for inserting the test strip 320 into the slot 312. In other embodiments, the sampling test strip 320 is bonded to the rotating part 310.

[0031] The sampling component 300 is pivotally connected to the housing 100. Specifically, the rotating part 310 is pivotally connected to one end of the housing 100 near the sampling area 110. In this embodiment, the rotating part 310 is provided with a ball head 311, and one end of the housing 100 is provided with a ball socket 130 that mates with the ball head 311, so that the two can rotate relative to each other about the axis defined by the ball head 311 and the ball socket 130. Alternatively, the ball socket can be provided in the rotating part 310, and the ball head 311 can be provided in one end of the housing 100. In practical applications, the pivotal connection between the sampling component 300 and the housing 100 can be achieved by various existing means, such as by pin connection, bolt connection, etc., and no limitation is made here.

[0032] Based on the rotation range of the sampling component 300 relative to the housing 100, a first limit position and a second limit position of the sampling component 300 are defined. When one end of the rotating part 310 of the sampling component 300, specifically the end connected to the sampling test strip 320, rotates into the sampling area 110 and abuts against the bottom of the sampling area 110 and can no longer rotate, the sampling component 300 reaches and is in the first limit position. Figure 3 and Figure 4 When the other end of the rotating part 310 rotates to the back of the housing 100 and abuts against the back of the housing 100 and can no longer rotate, that is, the sampling component 300 reaches and is in the second limit position, reference Figure 1 and Figure 2 For the process of the sampling component 300 rotating from the first limit position to the second limit position, please refer to the following steps: Figure 3 , Figure 6 , Figure 7 and Figure 2 ,vice versa.

[0033] In this embodiment, the outer contour of the sampling area 110 is adapted to one end of the sampling component 300, so that when the sampling component 300 is in the first extreme position, one end of the rotating part 310 can be engaged in the sampling area 110. Of course, the size of the engagement force can be limited according to the size matching of the two, so as to drive one end of the rotating part 310 into and out of the sampling area 110.

[0034] When the sampling component 300 is in the first extreme position, the sampling test strip 320 is also positioned in the sampling area 110 along with one end of the rotating part 310. It is positioned above the sample pad 210 in the sampling area 110. Preferably, the sampling test strip 320 can be in surface contact with the sample pad 210 at the same time. It is also feasible to set them to line contact or to have a small gap.

[0035] In this embodiment, the back of the housing 100 is provided with a positioning groove 140 that is adapted to the outer contour of the other end of the sampling component 300 (i.e., the other end of the rotating part 310). When the sampling component 300 is in the second extreme position, the other end of the rotating part 310 can be engaged in the positioning groove 140. Of course, the size of the engagement force can be limited according to the size matching of the two, so as to drive the other end of the rotating part 310 into and out of the positioning groove 140.

[0036] The sampling test strip 320 uses absorbent filter paper, such as existing ophthalmic test strips. One end of the sampling test strip 320 is inserted into the slot 312 of the rotating part 310, and the other end is a free end exposed to collect the patient's tears. The corners of the free end are rounded to prevent unnecessary scratches during use. In order to ensure that the amount of tears collected is sufficient for testing, the sampling test strip 320 is provided with an indicator line 321 to help determine the minimum sampling amount required for testing. Sampling can be stopped when the tears are immersed in the sampling test strip 320 and exceed the indicator line 321.

[0037] The kit for detecting herpes simplex virus in tears in this embodiment, together with the tear sample elution solution, can form a detection kit for detecting herpes simplex virus in tears. In this embodiment, the tear sample elution solution is PBS solution.

[0038] The detection of herpes simplex virus in tears using the above-mentioned detection kit includes the following steps: Remove the reagent kit of this embodiment and press the sampling component 300 to rotate it to the second limit position; Place the free end of the sampling test strip 320 at the tear outflow point of the patient's eye (such as the corner of the eye) to collect tears until the sampling test strip absorbs a sufficient amount of tears (i.e., the collected tears exceed the indicator line 321 on the sampling test strip 320). Press the sampling component 300 to rotate it to the first limit position; PBS buffer is added to the sampling test strip 320 in the sampling area 110, so that the tear fluid collected by the sampling test strip 320 is washed away and immersed in the sample pad 210 below. Under capillary action, the tear fluid sample continues to diffuse laterally along the length of the immunochromatographic test strip 200 and reaches the adsorption pad 240. The reagent kit was used to excite fluorescence display of the detection line 231 and control line 232 exposed to the detection window 120 by irradiating with ultraviolet light, and the fluorescence intensity of the detection line 231 and control line 232 was read using a fluorescence counter.

[0039] The principles and result interpretation of the above tests: If the tear sample contains the HSV IgA to be tested, the europium-labeled herpes simplex virus type 1 G glycoprotein embedded in the release pad 220 will recognize and bind to the IgA antibody, forming an antigen-antibody complex. Under chromatography, the tear sample continues to move forward. When it reaches the test line 231, where there is anti-human IgA monomer, a "europium-labeled HSV1 type G glycoprotein-HSV1 IgA-anti-human IgA antibody" complex will be formed. Therefore, a large amount of europium-labeled HSV1 type G glycoprotein will accumulate at the test line 231. Excess tear sample will continue to flow from the test line 231 to the control line 232, where there is goat anti-rabbit IgG antibody. Here, a "goat anti-rabbit IgG antibody-europium-labeled rabbit IgG" complex will be formed and accumulate in large quantities. Ultimately, a large amount of europium-labeled material will accumulate at both the test line 231 and the control line 232. Fluorescence is excited by ultraviolet light, and fluorescent lines appear at both the test line 231 and the control line 232. The fluorescence intensity at the test line 231 and the control line 232 is read using a fluorescence counter. If no fluorescent line appears at the control line 232, the test is invalid.

[0040] For cases where the detection is valid, the fluorescence intensity values ​​at the detection line 231 and the control line 232 read by the fluorescence counter are recorded as T and C, respectively. The ratio of T / C is used as the detection result, and an appropriate threshold is set to determine whether HSV-IgA is present in the tear sample.

[0041] In an experiment based on the detection kit and method of this embodiment, tear samples from 105 outpatients diagnosed with HSK and 73 control group patients were tested. These included tear samples from the affected eyes of HSK patients, tear samples from the healthy eyes of HSK patients, and normal tear samples from the control group. The fluorescence intensity values ​​at the detection line and control line were recorded as T and C, respectively. The T / C ratio was used as the detection result, and ROC curve analysis was performed. Figure 9 As shown, based on the trend of the ROC curve, T / C=0.4 was selected as the optimal cutoff point, where the sensitivity was 72.38% and the specificity was 84.93%, indicating that the method has good diagnostic performance.

[0042] Figure 10 The results of tear film testing in the affected eye, healthy eye, and normal control group of HSK patients are shown. The range and median of the test results are shown. It can be seen from the figure that the HSV-IgA level in the affected eye is significantly higher than that in the unaffected eye and the control group.

[0043] In the description process of the above instruction manual: The terms "this embodiment," "an embodiment of the present invention," "as shown," "further," and "further improved technical solutions," etc., indicate that the specific features, structures, materials, or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example, and the specific features, structures, materials, or characteristics described can be combined or combined in any suitable manner in one or more embodiments or examples. Furthermore, without causing contradiction, those skilled in the art can combine or combine the different embodiments or examples described in this specification and the features of the different embodiments or examples.

[0044] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. A kit for detecting herpes simplex virus in tears, characterized in that, include: The housing includes an internal cavity, and a sampling area and a detection window respectively communicating with the internal cavity; An immunochromatographic test strip includes a sample pad, a release pad, and a nitrocellulose membrane connected in sequence. The immunochromatographic test strip is disposed in the internal cavity, and part or all of the sample pad extends into the sample spotting area. A sampling component, which is pivotally connected to the housing and defines a first extreme position, wherein the sampling component contacts the sample pad surface when it is in the first extreme position; in, The release pad is embedded with fluorescently labeled herpes simplex virus type 1 G glycoprotein; The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with anti-human IgA antibody, and the control line is coated with goat anti-rabbit IgG antibody. The detection line and the control line are exposed in the detection window.

2. The kit according to claim 1, characterized in that, The sampling assembly includes a rotating part and a sampling test strip connected to the rotating part. The rotating part is pivotally connected to the housing. When the sampling assembly is in the first extreme position, the sampling test strip is in contact with the sample pad surface.

3. The kit according to claim 2, characterized in that, The sampling test paper is absorbent filter paper.

4. The kit according to claim 2, characterized in that, The sampling test strip is equipped with indicator lines to help determine the minimum sampling amount required for the test.

5. The kit according to claim 1, characterized in that, The sampling area has a groove structure that conforms to the outer contour of the sampling component.

6. The kit according to claim 1, characterized in that, A positioning groove is provided on the housing on the other side opposite to the sampling area. The positioning groove is adapted to the outer contour of the sampling component and defines the second extreme position of the sampling component.

7. The kit according to claim 1, characterized in that, The immunochromatographic test strip also includes an adsorption pad, which is connected to the other end of the nitrocellulose membrane opposite to the release pad.

8. The kit according to claim 1, characterized in that, The herpes simplex virus type 1 G glycoprotein is labeled on europium fluorescent microspheres.

9. A detection kit for detecting herpes simplex virus in tears, characterized in that, Includes the kit and tear sample elution solution as described in any one of claims 1-8.

10. The detection kit as claimed in claim 9, characterized in that, The tear sample elution solution was PBS solution.

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