Kit for detecting group B streptococcus

Abstract

The invention relates to a kit for group B streptococcus detection, and relates to the technical field of biomedical detection, the kit comprises a reagent strip, the reagent strip is provided with a binding antibody and a capture antibody, and the binding antibody and the capture antibody can specifically recognize and bind group B streptococcus; the binding antibody is a nano antibody and comprises a CDR sequence as shown in SEQ ID NO: 2-4; the capture antibody is a nano antibody and comprises a CDR sequence as shown in SEQ ID NO: 6-8. The method has the effects of high detection sensitivity, good specificity and short detection time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical detection, and particularly relates to a colloidal gold test strip for detecting group B streptococcus. BACKGROUND

[0002] Group B Streptococcus (GBS) is a kind of facultative anaerobic gram-positive coccus, which can be intermittently, once or continuously colonized in the respiratory tract, digestive tract and reproductive tract of human body. It is a kind of conditional pathogenic bacteria, which can be converted from the colonization state to the pathogenic bacteria under certain conditions, causing maternal and neonatal infections. The bacterial carriage rate of the population is about 30%, and the carriage rate of pregnant women is 11.3%.

[0003] According to the difference of bacterial membrane polysaccharide, 10 serotypes have been identified and classified: Ia, Ib, II, III, IV, V, VI, VII, VIII and IX. The six serotypes of Ia-V are more common, which can cause maternal puerperal infection, neonatal meningitis and neonatal pneumonia and other serious diseases. Since the bacteria can cause premature rupture of membranes and premature birth, the vertical transmission rate can reach 50%, therefore, most hospitals at home and abroad have included the screening of the bacteria into the routine prenatal examination, and the screening object in China is the pregnant women at 35-37 weeks. For pregnant women with bacteria, if there is no infection sign, only the bacteria colonization, generally only the prophylactic temporary antibacterial before delivery is needed, so as to reduce the risk of neonatal infection of the bacteria.

[0004] At present, the detection methods of group B streptococcus mainly include: 1. bacterial culture method, through collecting vaginal and rectal swabs of pregnant women in culture medium for separation and identification, the method is the gold standard and can further conduct drug sensitivity test, but the time consumption is long (3-5 days), the detection rate is low, and the detection is easily disturbed by external conditions; 2. fluorescent quantitative PCR method: the target gene of bacteria is detected by expansion, which is accurate, rapid and has higher detection rate than the culture method, but needs to send the detection reagent and instrument, and the cost is slightly high; 3. immunological method: the bacterial antigen is detected by using latex agglutination and immunochromatography technology, which has the characteristics of rapidness and simplicity. However, the existing immunological detection methods still have the shortcomings of low detection sensitivity, poor specificity, unstable detection results and slightly long detection time.

[0005] Therefore, there is an urgent need in the field to develop a new kit for detecting group B streptococcus. SUMMARY

[0006] In order to overcome the problems existing in the prior art, the purpose of the present application is to provide a kit for detecting group B streptococcus.

[0007] In a first aspect, the application provides a kit for detecting group B streptococcus, comprising a reagent strip, wherein the reagent strip is provided with a binding antibody and a capture antibody, and the binding antibody and the capture antibody can specifically recognize and bind to group B streptococcus. The binding antibody is a nanobody, and comprises CDR sequences as shown in SEQ ID NOs: 2-4. The capture antibody is a nanobody, and comprises CDR sequences as shown in SEQ ID NOs: 6-8.

[0008] Optionally, the binding antibody comprises a variable region sequence as shown in SEQ ID NO: 1.

[0009] Optionally, the capture antibody comprises a variable region sequence as shown in SEQ ID NO: 5.

[0010] Optionally, the reagent strip comprises, in sequence, a sample pad, a colloidal gold binding pad, and a nitrocellulose membrane, wherein the nitrocellulose membrane is provided with a detection line, and the detection line comprises the capture antibody.

[0011] Optionally, the colloidal gold binding pad contains colloidal gold particles labeled with the binding antibody.

[0012] Optionally, the nitrocellulose membrane is further provided with a quality control line, and the quality control line is farther away from the colloidal gold binding pad than the detection line.

[0013] Optionally, the quality control line comprises at least one of a mouse anti-sheep antibody, an avidin, and a biotin-BSA.

[0014] Optionally, the kit further comprises a sample processing solution, and the sample processing solution is divided into A liquid and B liquid, the A liquid is sodium nitrite and phenol red, and the B liquid is acetic acid and Tween-20.

[0015] In a second aspect, the application provides a method for detecting group B streptococcus, comprising: detecting a sample to be tested by using the kit of the first aspect; optionally, the sample to be tested is selected from a vagina.

[0016] In summary, the application has at least one of the following beneficial technical effects: (1) suitable for direct detection of a vaginal swab, without sample pretreatment, simple operation, result interpretation within 5 minutes, suitable for rapid detection of a large number of samples; (2) significantly improved sensitivity, with a minimum detection limit of 1.73 x 10 4 CFU / mL; (3) strong specificity, no cross-reaction with common vaginal bacteria, etc. (4) The kit has high stability, high application value and good commercial prospect, and provides technical support for rapid detection of group B streptococcus. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The figure is a structural schematic diagram of the reagent strip of the present application. DETAILED DESCRIPTION

[0018] The term "antibody" is used herein in the broadest sense to refer to a protein comprising an antigen binding site, and encompasses both natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, single-chain antibodies, intact antibodies and antibody fragments. Preferably, the antibody of the present application is a nanobody.

[0019] The term "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). The CDRs are primarily responsible for binding an antigenic epitope, and include, in order from N-terminus, CDR1, CDR2, and CDR3. The precise amino acid sequence boundaries of each CDR in a given variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment systems, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering with a large number of crystal structures. Unless otherwise specified, in the present application, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the manners described above. CDRs can also be determined based on having the same AbM numbered positions as a reference CDR sequence, such as any of the CDRs exemplified in the present application. In one embodiment, the CDRs of the single domain antibodies of the present application are determined positions according to the AbM numbering scheme. Unless otherwise specified, in the present application, when referring to residue positions in antibody variable regions and CDRs, including heavy chain variable region residues, the numbered positions are according to the AbM numbering system.

[0020] The following examples are intended to be illustrative only and should not be viewed as limiting the application in any way.

[0021] Example 1: Preparation of raw materials A solution: a solution containing 10-20% sodium nitrite and a small amount of phenol red; B solution: a solution containing 1-4% acetic acid and 1-2% Tween-20; Polyester film: a solution containing 0.5-1.0% Tris, 0.1-0.5% casein, 0.5-1% PVP10, and 0.05-0.1% Tween-20; after soaking the polyester film, dry the treatment solution: a solution containing 0.1-1% Tris, 0.1-0.5% casein, 0.1-1% PVP-10, 0.1-0.2% sodium carbonate, and 1-2% Tritox X-405; Colloidal gold: boil a solution containing 0.02% gold chloride, add sodium citrate solution, continue boiling for 10 min after the solution turns wine red, and cool at room temperature.

[0022] Example 2: Screening of anti-group B streptococcus antibodies Sheep were immunized with the capsular polysaccharide of group B streptococcus as an antigen to obtain high-titer antisera. After the end of animal immunization, 50 mL of fresh blood was taken from the sheep, peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Paque density gradient separation solution, RNA was extracted, and after reverse transcription, universal primers were used for amplification and cloned into phagemids, transformed into TG1 strain, and a phage library was established for screening of monoclonal antibodies. Five monoclonal antibodies Nb1-Nb5 with high specificity and affinity were obtained by screening. Further study of antibody combinations found that using Nb1 as the binding antibody and Nb2 as the capture antibody had the highest binding value, indicating that the two antibodies bind to different antigen sites and can be used for double-antibody sandwich immunoassay.

[0023] Analysis of the sequences of Nb1 and Nb2 showed that the amino acid sequence of VHH of antibody Nb1 is shown in SEQ ID NO: 1, the amino acid sequences of CDR1, CDR2, and CDR3 defined by AbM are shown in SEQ ID NOs: 2-4, respectively; the amino acid sequence of VHH of antibody Nb2 is shown in SEQ ID NO: 5, and the amino acid sequences of CDR1, CDR2, and CDR3 defined by AbM are shown in SEQ ID NOs: 6-8, respectively.

[0024] Example 3: Preparation of detection kit The detection kit is composed of the following components: a detection reagent strip sealed in an aluminum foil bag, an A solution bottle, a B solution bottle, a sampling cotton swab, an extraction tube, an operation table, and an instruction manual.

[0025] 3.1 Reagent strip a) Production of nitrocellulose membrane: mouse anti-goat antibody and group B streptococcus antibody Nb2 were diluted to 0.5-1.0 mg / mL and 0.5-1.5 mg / mL respectively with PBS solution with sugar, and the solutions were uniformly sprayed on the NC membrane with a dot membrane instrument, and dried in an oven at 37°C x 12 h. The nitrocellulose membrane was provided with a detection line (T line), and the detection line was anchored with a coated capture antibody Nb2. The nitrocellulose membrane was also provided with a quality control line (C line), and the quality control line was anchored with a mouse anti-goat antibody, and the quality control line was farther away from the colloidal gold binding pad than the detection line.

[0026] b) Production of colloidal gold binding pad: Antibody coupling colloidal gold: colloidal gold was taken, and the pH was adjusted to 7.8-8.2 with a potassium carbonate solution, and group B streptococcus antibody Nb1 was added at a dosage of 5-10 μg / mL, and after stirring for 30 min, 10% BSA solution was added for blocking, and after 30 min, the solution was centrifuged, and the precipitate was resuspended with a solution containing 0.1-0.5% Tris, 1-2% BSA (pH 8.0), and the concentration of the resuspension was measured with a spectrophotometer.

[0027] Gold spraying: the colloidal gold resuspension was diluted to an OD20-40 concentration with a solution containing 0.1-0.5% Tris, 1-2% BSA, 10-20% sucrose, and 1-5% trehalose, and was uniformly sprayed on the polyester film with a gold spraying machine at 1.0-2.0 μL / cm, and was dried in an oven at 37°C x 12 h.

[0028] c) Production of sample pad: the treatment liquid was uniformly sprayed on 8964 glass fiber at a ratio of 50 g / piece, and was dried in an oven at 37°C x 12 h.

[0029] 3.2 Assembly and cutting of reagent strip First, the filter paper was cut to 17*301 mm, the colloidal gold binding pad was cut to 10*301 mm, and the sample pad was cut to 17*301 mm, and the sample pad, colloidal gold binding pad, and nitrocellulose membrane were sequentially pasted, and then a PVC bottom sheet and the filter paper were assembled into a large card as shown. Figure 1 The assembled large card was cut into 3-4 mm reagent strips, which were then loaded into a card board, and together with a desiccant were placed in an aluminum foil bag, and heat-sealed.

[0030] 3.3 Production of A liquid bottle and B liquid bottle A liquid and B liquid were respectively filled into bottles, and the bottle caps were covered, and labels were attached. A liquid was red, and its bottle cap was red; B liquid was colorless, and its bottle cap was white.

[0031] 3.4 Instructions for use of kit In use, open the aluminum foil bag, place the reagent strip on the operating table, add 4 drops of A liquid and 4 drops of B liquid in the extraction tube, and mix gently; use a cotton swab to take samples by rotating in the patient's vagina several times, then put the cotton swab into the extraction tube, lyse for 1 min, then rotate the cotton swab 5-10 times, and then take out the cotton swab while pressing the wall of the tube as much as possible to make the liquid in the cotton swab remain in the extraction tube as much as possible; invert the extraction tube and add 3 drops of liquid to the test reagent sample hole, start timing, and if the C line and the T line develop color after 5 min, it is positive regardless of the color depth; if the C line develops color and the T line does not develop color, it is negative; if the C line does not develop color, it is an invalid result regardless of whether the T line develops color, indicating that the reagent is invalid or there is an error in the operation process.

[0032] Example 4: Performance evaluation of the test kit 4.1 Detection limit experiment, dilute the group B streptococcus culture to different concentrations, and the lowest concentration that can obtain a positive result is the minimum detection limit. The results are shown in Table 1, and the minimum detection limit is 1.73 x 10 4 CFU / mL. In the following table, "+" indicates that the antibody specifically reacts with group B streptococcus, and "-" indicates that the antibody does not react with group B streptococcus.

[0033] Table 1 4.2 Specificity experiment To test the specificity of the test kit, we selected common vaginal colonizing bacteria, and the results are shown in Table 2, which shows that the kit for detecting group B streptococcus of the present application has no cross interference with common vaginal colonizing bacteria and has good specificity.

[0034] Table 2 4.3 Clinical sample experiment The test kit for detecting group B streptococcus of the present application was used to test clinical vaginal and rectal swabs collected from hospitals, and these samples have been confirmed to be negative (Nos. N1-N20) or positive (Nos. P1-P10). The test results are shown in Table 3, the positive coincidence rate is 10 / 10, the negative coincidence rate is 20 / 20, and the kit of the present application has excellent clinical performance.

[0035] Table 3 Number Results Number Results Number Results Number Results N1 - N11 - P1 + N2 - N12 - P2 + N3 - N13 - P3 + N4 - N14 - P4 + N5 - N15 - P5 + N6 - N16 - P6 + N7 - N17 - P7 + N8 - N18 - P8 + N9 - N19 - P9 + N10 - N20 - P10 + 4.4 Stability experiment The test kit for detecting group B streptococcus of the present application was accelerated at 55°C to simulate the stability at room temperature for 2 years. The results are shown in Table 4, which can be inferred that the kit has good stability within a 2-year use cycle.

[0036] Table 4 While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alterations to the details can be made within the scope of the teachings disclosed herein and that the scope of the application is not limited to the specific embodiments described herein. The scope of the protection is given by the appended claims and any equivalents thereof.

Claims

1. A kit for group B streptococcus detection comprising a reagent strip, characterized in that, The reagent strip is provided with a binding antibody and a capture antibody, both of which can specifically recognize and bind to group B streptococcus; The binding antibody is a nanobody, comprising CDR sequences as shown in SEQ ID NO: 2-4; The capture antibody is a nanobody, comprising CDR sequences as shown in SEQ ID NO: 6-8.

2. The kit of claim 1, wherein The binding antibody comprises a variable region sequence as shown in SEQ ID NO:

1.

3. The kit of claim 1, wherein The capture antibody comprises a variable region sequence as shown in SEQ ID NO:

5.

4. The kit of claim 1, wherein The reagent strip comprises a sample pad, a colloidal gold binding pad and a nitrocellulose membrane connected in sequence. The nitrocellulose membrane is provided with a detection line comprising a capture antibody.

5. The kit of claim 4, wherein The colloidal gold binding pad contains colloidal gold particles labeled with a binding antibody.

6. The kit of claim 4, wherein The nitrocellulose membrane is further provided with a quality control line, which is farther away from the colloidal gold binding pad than the detection line.

7. The kit of claim 6, wherein The quality control line comprises at least one of mouse anti-sheep antibody, avidin, and biotin-BSA.

8. The kit of claim 1, wherein The kit further comprises a sample processing solution, which is divided into A and B liquids, the A liquid is sodium nitrite and phenol red; the B liquid is acetic acid and Tween-20.

9. A method for detecting group B streptococci, characterized by, Comprise: The kit of any one of claims 1-8 is used for detecting a sample to be tested; optionally, the sample to be tested is selected from a vagina.

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