A group b streptococcal antigen detection kit

Abstract

The application provides a group B streptococcus antigen detection kit, and belongs to the technical field of antigen detection, and comprises the following components: a reagent card, an ID card, an extraction solution and an extraction tube; the reagent card comprises a reagent strip, a light transmission film and a card shell; the reagent strip comprises a sample pad, a binding pad, a nitrocellulose film, an absorption pad and a PVC bottom plate; the binding pad contains latex microsphere-labeled group B streptococcus antibody a and fluorescent microsphere-labeled chicken IgY; and the nitrocellulose film contains group B streptococcus antibody b and goat anti-chicken IgY polyclonal antibody. The technical scheme provided by the application realizes preparation of an antibody with high sensitivity and strong specificity while reducing false positive judgment.

Description

Technical Field

[0001] This invention relates to the field of antigen detection technology, specifically to a group B streptococcal antigen detection kit. Background Technology

[0002] Group B Streptococcus (GBS) is a Gram-positive bacterium named for its chain-like arrangement under microscopic observation. It is a normal parasite in the human body, typically residing in the gastrointestinal tract, perineum, and vagina. Its pathogenicity is related to its surface structure, and it is the leading cause of neonatal sepsis. Approximately one-quarter of healthy adult women carry Group B Streptococcus in their reproductive tract. Carriers are mostly asymptomatic. Adult infection mainly occurs in pregnant women or those with weakened immune systems, manifesting as sepsis, endocarditis, meningitis, cellulitis, lung infections, or urinary tract infections. Group B Streptococcus primarily infects newborns through vertical transmission during birth or horizontal transmission after birth, manifesting as sepsis without a focal infection site, pneumonia, and meningitis. It can also cause otitis media and empyema. GBS neonatal sepsis can sometimes progress rapidly, catching patients off guard. Prevention is worse than cure; prenatal screening is more effective than postnatal diagnosis and treatment.

[0003] Chinese patent application CN105063173A, entitled "A Method and Kit for Detecting Group B Streptococcus Infection," discloses a detection reagent prepared using hippuric acid or a hippurate analog as a chromogenic substrate and buffer solution. Group B Streptococcus utilizes hippuric acid enzymes to specifically recognize the binding of glycine and benzoic acid in the chromogenic substrate and hydrolyze the amide bond linking the two, causing a color change in the chromogenic substrate. This color change is used to determine whether the sample is infected with Group B Streptococcus. The application also discloses a kit for detecting Group B Streptococcus infection. The above technical solution is simple to implement, requires no specialized training in bacterial streak plating techniques, and has a short detection time.

[0004] Chinese Patent No. CN104328212B, entitled "Primer Set and Kit for Detection of Group B Streptococcus by Loop-Mediated Isothermal Amplification," discloses a primer kit kit. The primer set includes a pair of specific inner primers and a pair of specific outer primers. The kit includes several reaction tubes containing 18 μL of reaction solution, one positive control tube containing 10 μL of GBS DNA, and one negative control tube containing 100 μL of sterile ultrapure water. The kit uses primers designed for the highly conserved and specific cAMP gene in the GBS gene, employing loop-mediated isothermal amplification (LAMP) technology for amplification and detection. The DNA extraction process is simple, requiring no chemical reagents; only a 10-minute boiling water bath followed by centrifugation is needed. This avoids the influence of chemical reagents on subsequent nucleic acid amplification. Furthermore, it has low requirements for instruments and equipment, simplifying the operation and reducing detection costs.

[0005] However, the antibodies screened by the above two technical solutions and existing technologies such as WB and enzyme-linked immunosorbent assay (ELISA) are mostly immunized against a single antigen or immunized by inactivated pathogenic microbial cells. The antibodies prepared from existing recombinant proteins are only simple combinations of multiple sequences. Therefore, antibodies against this bacterium still have the characteristics of poor specificity and low sensitivity. At the same time, the stability of existing group B streptococcal antibodies between different batches is still defective, and weak false positives are easy to occur. Summary of the Invention

[0006] In view of this, the present invention provides a group B streptococcal antigen detection kit, which enables the preparation of highly sensitive and specific antibodies while reducing false positives.

[0007] To achieve the above objectives, the present invention provides a group B streptococcal antigen detection kit, comprising the following components: a reagent card, an ID card, an extraction solution, and an extraction tube; the reagent card includes a reagent strip, a transparent membrane, and a card shell; the reagent strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate; the conjugate pad contains group B streptococcal antibody a labeled with latex microspheres and chicken IgY labeled with fluorescent microspheres; the nitrocellulose membrane contains group B streptococcal antibody b and goat anti-chicken IgY polyclonal antibody.

[0008] Optionally, the group B streptococcal antibody a is derived from rabbits and has a concentration of 0.22~0.27 mg / mL; the chicken IgY is derived from chickens and has a concentration of 0.9~1.1 mg / mL; the group B streptococcal antibody b is derived from rabbits and has a concentration of 1.8~2.2 mg / mL; and the goat anti-chicken IgY polyclonal antibody is derived from goats and has a concentration of 0.38~0.42 mg / mL.

[0009] Optionally, the cartridge includes an upper cartridge and a lower cartridge, the reagent strip is located between the lower cartridge and the light-transmitting film, and the light-transmitting film is embedded between the reagent strip and the upper cartridge.

[0010] Optionally, the upper casing includes a sample loading window and a display window. The sample loading window overlaps with the sample pad. The extract contacts the sample pad through the drop window. The display window overlaps with the light-transmitting membrane. The light-transmitting membrane overlaps with the nitrocellulose membrane. The nitrocellulose membrane includes a T-line and a C-line. The display window shows the detection results through the light-transmitting membrane.

[0011] The light-transmitting film can be one of the following: 100% light transmittance, 90% light transmittance, 80% light transmittance, 70% light transmittance, 60% light transmittance, and 50% light transmittance.

[0012] Optionally, the group B streptococcal antibody a is an antibody prepared by immunization of the fusion protein and screened by immunomagnetic beads combined with real-time quantitative qPCR.

[0013] Optionally, the preparation of the fusion protein includes the following steps:

[0014] S1. Selection of Group B Streptococcus immunogen: The coding region of Group B Streptococcus capsular polysaccharide biosynthetic protein Cps4B was truncated using the equal division method, and polypeptides were synthesized from the truncated coding regions. The polypeptides were then combined with capsular polysaccharide biosynthetic protein Cps4B, surface immunogenic protein, and cAMP factor in different arrangements. The Cps4B gene sequence is shown in SEQ ID NO.1, the amino acid sequence of Cps4B is shown in SEQ ID NO.2, the amino acid sequence of surface immunogenic protein is shown in SEQ ID NO.3, and the amino acid sequence of cAMP factor is shown in SEQ ID NO.4.

[0015] S2. Construction of fusion protein expression vector: The fusion protein expression vector was constructed using the seamless cloning method to obtain the recombinant plasmid;

[0016] S3. Expression and purification of fusion protein: The recombinant plasmid was transformed into E. coli BL21-DE3, and the protein expression was induced by IPTG. The fusion protein was obtained by affinity purification using a Ni column.

[0017] Optionally, after the S1 equipartition method truncates the coding region of the Group B Streptococcus capsular polysaccharide biosynthetic protein Cps4B, each resulting coding region contains a 183bp sequence.

[0018] Optionally, the fusion protein is used as an antigen to prepare antibodies through immunization. The obtained antibodies are then biotin-labeled, and the labeled antibodies are used to prepare immunomagnetic beads. The immunomagnetic beads are uniformly mixed with group B streptococcal bacterial suspension and then magnetically separated. The immunomagnetic beads are washed with washing solution, and then the immunomagnetic beads are suspended in washing solution. The supernatant is collected, stained with PMAxx, and DNA is extracted. The DNA is then amplified and quantified by qPCR, and the number of colonies captured by the immunomagnetic beads is calculated using a standard curve.

[0019] Optionally, the biotin labeling method involves diluting the antibody, mixing it with biotin, reacting it on a shaker, and then adding it to a labeling chromatography column for column chromatography.

[0020] Optionally, the standard curve is determined by serially diluting the logarithmic-phase Group B Streptococcus bacterial suspension with sterile physiological saline and counting the bacteria using the plate method, thereby determining the standard curve between the Ct value and Log CFU / mL.

[0021] The above-described technical solution of the present invention has at least the following beneficial effects:

[0022] 1. The technical solution of this invention uses methods such as truncation analysis and fusion expression to prepare antibodies through immunization. Subsequently, immunomagnetic beads combined with quantitative real-time PCR are used to screen group B streptococcal antibodies. The prepared antibodies and the screened antibodies are bound to each other. The steps are simple and the cost is low. It can intuitively reflect the capture efficiency of the antibodies and obtain antibodies with high sensitivity and strong specificity.

[0023] 2. In the technical solution of this invention, an adjustable transparent membrane is embedded in the test kit to optimize the color development and observation of the chromatography reagent card. By uniformly dispersing the light, the test line signal is made clearer, avoiding false positive judgments caused by local light reflection. Attached Figure Description

[0024] Figure 1 This is the standard curve diagram in Embodiment 2 of the present invention;

[0025] Figure 2 This is a graph showing the capture efficiency of different antibodies prepared in Example 2 against Group B Streptococcus using the immunomagnetic bead-qPCR method of the present invention.

[0026] Figure 3 This is a schematic diagram of the reagent kit structure in Example 3 of the present invention;

[0027] Figure 4 This is a picture of the reagent kit used in Example 3.

[0028] Figure 5 This is a picture of the actual reagent kit used in Comparative Example 2. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.

[0030] Example 1

[0031] Group B streptococcal immunogen selection: The Cps4B coding region was truncated using an aliquot method, with each segment containing a 183bp sequence. Further, peptides were chemically synthesized from each segment using conventional liquid-phase synthesis. Commercialization services for peptide synthesis were provided by Genscript Biotech Inc. and Beijing Bio-Sens Biotechnology Co., Ltd. The Cps4B gene sequence is shown in SEQ ID NO.1, the amino acid sequence of Cps4B is shown in SEQ ID NO.2, the amino acid sequence of the surface immunogenic protein is shown in SEQ ID NO.3, and the amino acid sequence of the cAMP factor is shown in SEQ ID NO.4. The peptides were mixed with capsular polysaccharide biosynthetic protein Cps4B, surface immunogenic protein, and cAMP factor in different arrangements. The synthesized immunogenic proteins were diluted to a working concentration of 10 μg / mL with PBS buffer (purchased from Sangon Biotech (Shanghai) Co., Ltd.) and stored at -20°C for later use.

[0032] Configure the following combinations:

[0033] GBS-Ag1: Capsular polysaccharide biosynthetic protein Cps4B.

[0034] GBS-Ag2: A fusion protein of the N-terminal region of capsular polysaccharide biosynthetic protein Cps4B, surface immunogenic protein, and cAMP factor.

[0035] GBS-Ag3 is a fusion protein of the middle region of capsular polysaccharide biosynthetic protein Cps4B, surface immunogenic protein, and cAMP factor.

[0036] GBS-Ag4 is a fusion protein of the middle region of capsular polysaccharide biosynthetic protein Cps4B, surface immunogenic protein, and cAMP factor.

[0037] GBS-Ag5: Surface immunogenic protein.

[0038] GBS-Ag6: cAMP factor.

[0039] The method for constructing fusion protein expression vectors is as follows:

[0040] (1) Primer design: The fusion protein expression vector was constructed using the seamless cloning method. NcoI and XhoI were selected as primers for amplifying the fusion protein gene.

[0041] (2) Obtaining the insert fragment: Using genomic DNA as a template, the gene fragment was amplified by F and R. The correctness of the gene fragment band was verified by agarose gel electrophoresis. The gene fragment was then recovered by the Novizan gel recovery kit to obtain the purified insert fragment.

[0042] (3) Obtaining the linearized vector: Following the instructions of the Novizumab plasmid extraction kit, the pET-28a plasmid was extracted and then digested with NcoI and XhoI. The 50 μL digestion system consisted of: 5 μL buffer, 5 μL plasmid, 1 μL each of NcoI and XhoI, and 36 μL ultrapure water. The reaction time was 30 minutes. The plasmid bands were verified to be correct using agarose gel electrophoresis, and the gene fragments were recovered using the Novizumab gel recovery kit to obtain the purified linearized vector.

[0043] (4) Recombination reaction: The purified linearized vector and the insert fragment are mixed in a molar ratio of 1:2. 5 μL of 2 x ClonExpress Mix (containing recombinase) is added, and ultrapure water is added to make the reaction system 10 μL. The reaction is carried out at 50℃ for 15 min to complete the recombination reaction and obtain the recombinant product.

[0044] (5) Transformation of DH5-α competent cells with recombinant product: Thaw competent cells on ice, add 2 μL of purified recombinant product to 100 μL of competent cells, gently tap the tube wall to mix (do not shake), incubate on ice for 30 minutes, heat shock in a 42°C water bath for 90 seconds, and quickly remove and place on ice for 2-5 minutes. Add 1 mL of LB liquid medium, incubate at 37°C for 15 minutes, and then shake on a 37°C shaker for 45 minutes. After centrifugation at 8000 rpm for 2 minutes, discard 900 μL of the supernatant, resuspend the bacterial pellet in the remaining medium, and spread it on LB solid medium containing 34 μg / μL kanamycin. Incubate upside down at 37°C for 12-16 h to obtain positive clones on transformation plates. Extract the recombinant plasmid using a plasmid extraction kit.

[0045] The fusion protein expression and purification methods are as follows:

[0046] (1) Transformation of Escherichia coli BL21-DE3 with recombinant plasmid: Thaw BL21-DE3 competent cells on ice, add 2 μL of purified recombinant product to 100 μL of competent cells, gently tap the tube wall to mix (do not shake), incubate on ice for 30 minutes, heat shock in a 42℃ water bath for 90 seconds, and quickly remove and place on ice for 2-5 minutes. Add 1 mL of LB liquid medium, incubate at 37℃ for 15 minutes, and then shake on a 37℃ shaker for 45 minutes. After centrifugation at 8000 rpm for 2 minutes, discard 900 μL of the supernatant, resuspend the bacterial pellet in the remaining medium, and spread it on LB solid medium containing 34 μg / mL kanamycin. Incubate upside down at 37℃ for 12-16 h.

[0047] (2) IPTG-induced protein expression and identification: Single clones from the transformation plate were picked and inoculated into test tubes containing LB medium with 34 μg / mL kanamycin, and cultured overnight at 37℃ and 220 r / min. The next day, they were inoculated at a 1:100 ratio into 100 mL of LB medium with 34 μg / mL kanamycin, and cultured at 37℃ and 220 r / min until the bacterial OD600 reached 0.5-0.8. IPTG was added to a final concentration of 0.2 mM, and the cells were cultured at 30℃ and 16℃ for 10 hours and 18 hours, respectively, to induce fusion protein expression. The bacterial pellet was resuspended in PBS, and PMSF with a final concentration of 1 mM was added to the resuspended solution for ultrasonic disruption. The supernatant and pellet were resuspended in PBS. 12% SDS-PAGE analysis was performed, and Coomassie brilliant blue staining was used for banding.

[0048] (3) Ni column affinity purification: Load the supernatant onto the column, fill the Ni-NTA affinity chromatography column pre-equilibrated with Ni-NTA Binding-Buffer (20 mM PBS, pH 7.4), (2) wash with Binding-Buffer at a flow rate of 1 mL / min until the OD280 value of the eluent reaches the baseline. Prepare Ni-NTA Elution-Buffer containing 25 mM, elute the target protein at a flow rate of 1 mL / min, collect the eluent and dialyze overnight with PBS to obtain the fusion protein, and store it in a -80℃ freezer. Subsequent antibody immunoassay services were provided by Genscript Biotech Co., Ltd.

[0049] The Cps4B gene sequence (SEQ ID NO.1) is as follows:

[0050] ATGATTGATATTCATTCTCATATCGTTTTTGATGTAGATGATGGGCCCAAAACGCTTGAAGAGAGTTTATCTTTAATAGAAGAAAGTTATCGACAAGGTGTTAGAATAATTGTTTCGACATCACATAGAAGAAAAGGTATGTTTGAAACACCTGAAGATATTATCTTCAAAAACTTTTCTATAGTTAAACACGAAGCGGAAAAAAGATTTGAACATCTTCAAATCTTATATGGTGGAGAATTATACTATACAAGTGATATGTTAGAAAAACTGAAGTTAAAGCAAATTCCAACTTTAAACAATACGAAATTTGCTTTAATTGAATTTTCTATGCAAACTTCTTGGAAAGATATTCATACAGCTTTGTCAAATGTTTTAATGCTTGGTATTACACCAGTCGTTGCGCATATAGAGAGGTATAACGCTTTAGAGAATCAAAAAGAACGGGTGAAGGAAATTATTAATATGGGGTGTTACACACAAATAAATAGTTTCCATATTTTGAAACAAAAACTTTTTAATGATAAGCATAAACGCTTTAAGAAAAGAGCCCGTTATTTTTTAGAGGAAAATTTAGTGCATTTTGTAGCGAGTGATATGCATAACCTTGATGTTAGACCGCCATTTTTAGCAGAAGCTTATAAGATTATCTGTAGAGATTTCGGTAAAGAACGTGCTAACCAACTTTTTATTGAAAATGCTCAATCTATATTAAAAAACCATTACATTTAG

[0051] The amino acid sequence of Cps4B (SEQ ID NO.2) is:

[0052] MIDIHSHIVFDVDDGPKTLEESLSLIEESYRQGVRIIVSTSHRRKGMFETPEDIIFKNFSIVKHEAEKRFEHLQILYGGELYYTSDMLEKLKLKQIPTLNNTKFALIEFSMQTSWKDIHTA LSNVLMLGITPVVAHIERYNALENQKERVKEIINMGCYTQINSSHILKQKLFNDKHKRFKKRARYFLEENLVHFVASDMHNLDVRPPFLAEAYKIICRDFGKERANQLFIENAQSILKNHYI

[0053] The amino acid sequence of the linker peptide is: GGGGSGGGSGGSGS

[0054] The surface immunogenic protein sequence (SEQ ID NO.3) is as follows:

[0055] LLTSTMAASLLSVASVQAQETDTTWTARTVSEVKADLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLT

[0056] The cAMP factor sequence is (SEQ ID NO.4):

[0057] MNVKHMMYLSGTLVAGALLFSPAVLEVHADQVTTPQVVNHVNSNNQAQQMAQKLDQDSIQLRNIKDNVQGTDYEKPVNEAITSVEKLKTSLRANPETVYDLNSIGSRVEALTDVIEAITFSTQHLAN KVSQANIDMGFGITKLVIRILDPFASVDSIKAQVNDVKALEQKVLTYPDLKPTDRATIYTKSKLDKEIWNTRFTRGKKVLNVKEFKVYNTLNKAITHAVGVQLNPNVTVQQVDQEIVTLQAALQTALK

[0058] Example 2

[0059] The synthesized fusion protein was used as an antigen, and antibodies were prepared through immunization. The antibody preparation service was outsourced to Wuhan GenScript Biotech Co., Ltd.

[0060] The prepared antibodies (GBS-Ab1, GBS-Ab2, GBS-Ab3, GBS-Ab4, GBS-Ab5, GBS-Ab6) were labeled with biotin as follows: The antibodies to be labeled were diluted to 1 mg / mL with PBS buffer, and 200 μg was placed in a 1.5 mL EP tube for later use. Biotin was added to the antibody tube at a ratio of antibody mass to biotin mass of 10:1, and the mixture was reacted at 37 °C with shaking for 2 h. PBS was added to the antibody system after the reaction with biotin at 37 °C to a total volume of 1 mL, and the mixture was then fed into a labeling chromatography column. The flow of labeled material continued until approximately 1 mL of antibody was collected. After antibody collection, the column was washed with PBS, keeping the column moist, for 1 h. After washing, the column was sealed with 20% ethanol and stored at 4 °C. A certain volume of glycerol was added to achieve a final glycerol concentration of 30%, and the column was stored at -20 °C.

[0061] The labeled antibody was used to prepare immunomagnetic beads as follows: The magnetic beads were mixed by inverting, and 25 µL (20 mg / mL) of streptavidin magnetic beads were transferred to a sterile 1.5 mL centrifuge tube. Before conjugation, the streptavidin magnetic beads were washed 2-3 times with sterile PBST solution (100 mL 0.01 M PBS, 50 µL Tween 20, pH 7.4), followed by magnetic adsorption. The supernatant was discarded, and 400 µL of biotinylated Vibrio parahaemolyticus polyclonal antibody (0.15 mg / mL) was added. The mixture was gently vortexed and incubated at room temperature for 45 min on a vertical mixer. Then, magnetic separation was performed for 5 min, and the supernatant was discarded. The magnetic beads were washed 2-3 times with 1 mL of PBST solution for 5 min each time, followed by magnetic adsorption. The supernatant was discarded, and the prepared magnetic beads were resuspended in PBST solution containing 0.1% BSA and NaN3 and stored at 4 °C for later use.

[0062] Antibody screening using immunomagnetic beads: The concentration of Group B Streptococcus bacterial suspension was adjusted to 10⁵ CFU / mL. 150 μL of immunomagnetic beads were transferred to a new 1.5 mL sterile centrifuge tube and washed 2-3 times with PBST solution. Then, 0.5 mL of bacterial suspension was taken and incubated at room temperature for 30 min to ensure homogeneity of the immunomagnetic beads and bacterial suspension. Magnetic separation was then performed for 5 min. After discarding the supernatant, the tube was washed 3 times with PBST, resuspended in 0.5 mL of PBST, and the supernatant was stained with PMAxx. DNA was extracted according to the bacterial genomic DNA kit instructions and finally quantified by qPCR amplification. The number of colonies captured by the immunomagnetic beads was calculated using a standard curve.

[0063] The system and procedure for qPCR detection are as follows: qPCR analysis was performed using a Bio-Rad CFX96Touch real-time quantitative PCR instrument. The total volume was 25 μL, containing 12.5 μL of AceQ® qPCR premix, 1 μL each of 10 μM forward and reverse primers, 0.5 μL of 10 μM probe, 0.5 μL of DNA template, and 5 μL of ultrapure water. The amplification program first performed pre-denaturation at 95℃ for 10 min, followed by 45 cycles of 95℃ for 30 s and 58℃ for 1 min.

[0064] The standard curve was determined as follows: Group B Streptococcus bacteria in the logarithmic growth phase were serially diluted 10-fold with sterile physiological saline, and counted using the plate method to determine the standard curve between the Ct value and Log CFU / mL. The standard curve was Y = -3.16X + 40.50, with a fitting coefficient R0. 2 =0.9968, see Figure 1 .

[0065] The capture efficiency is calculated as follows: Capture rate (%) = (1-B / A) * 100%, where A is the total number of bacteria in the sample (CFU / mL), and B is the number of bacteria linked to the immunomagnetic beads, present in the supernatant and washing solution, and detected by plate counting method.

[0066] The capture efficiency of different antibodies prepared in Example 2 against Group B Streptococcus by immunomagnetic bead-qPCR is shown in the figure. Figure 2 .Depend on Figure 2 It can be seen that the antibody screening method provided by the present invention has a high antibody capture effect.

[0067] Example 3

[0068] A group B streptococcal antigen detection kit includes the following components: a reagent card, an ID card, an extraction solution, and an extraction tube; the ID card is mainly used to record and identify the test results, the extraction solution is the sample, and the extraction tube is used for sampling.

[0069] The reagent card includes a reagent strip, a transparent film, and a card case; the card case includes an upper card case and a lower card case, and the reagent strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate.

[0070] The reagent strip is positioned between the lower housing and the transparent membrane, which is embedded between the reagent strip and the upper housing. The upper housing includes a sample application window and a display window. The sample application window overlaps with the sample pad, allowing the extract to contact the sample pad through the application window. The display window overlaps with the transparent membrane, which in turn overlaps with a nitrocellulose membrane. The nitrocellulose membrane includes T-lines and C-lines. The display window shows the test results through the transparent membrane. The transparent membrane has 80% light transmittance.

[0071] The conjugate pad contained latex microsphere-labeled group B streptococcal antibody a and fluorescent microsphere-labeled chicken IgY; the nitrocellulose membrane contained group B streptococcal antibody b and goat anti-chicken IgY polyclonal antibody. Group B streptococcal antibody a was prepared by immunoassay of a fusion protein, obtained after screening using immunomagnetic beads combined with quantitative real-time qPCR, derived from rabbits, with a concentration of 0.22–0.27 mg / mL; the chicken IgY was derived from chickens, with a concentration of 0.9–1.1 mg / mL; group B streptococcal antibody b was derived from rabbits, with a concentration of 1.8–2.2 mg / mL; the goat anti-chicken IgY polyclonal antibody was derived from goats, with a concentration of 0.38–0.42 mg / mL. Group B streptococcal antibody b was commercially available, purchased from Beijing Huaxinhang Biotechnology Co., Ltd., product number M-StrepB-01.

[0072] Comparative Example 1

[0073] Commercially available Group B Streptococcus antigen detection kit.

[0074] Comparative Example 2

[0075] Compared to Example 3, the only difference is that no light-transmitting film is added.

[0076] The detection limit (LOD) of the Group B Streptococcus antigen detection kit in Example 3 was determined. The method involved diluting five different Group B Streptococcus strains in 0.01M PBS to determine the LOD. Each concentration gradient was tested at least three times. The lowest concentration level with 100% detectability was used as the estimated LOD. Several samples with gradient concentrations were prepared near this concentration, and each concentration was tested at least 20 times. The lowest concentration level with a 95% positive detection rate was calculated through direct detection and determined as the confirmed LOD. The results are shown in Table 1. The LOD of Comparative Example 1, i.e., the commercially available kit, was confirmed, and the results are shown in Table 1.

[0077] Table 1. Detection limit results of the kits in Example 3 and Comparative Example 1.

[0078]

[0079] As shown in Table 1, the Group B Streptococcus antigen detection kit prepared in Example 3 of this invention has a positive detection rate of no less than 95% and a limit of detection (L) of 50,000 CFU / mL when the bacterial concentration is 50,000 CFU / mL. Comparative Example 1, i.e., the commercially available Group B Streptococcus antigen detection kit, has a positive detection rate of no less than 95% and a limit of detection (L) of 100,000 CFU / mL when the bacterial concentration is 100,000 CFU / mL. Therefore, the detection kit prepared in this invention has higher sensitivity than commercially available detection kits.

[0080] Example 3 and Comparative Example 2 were used to simulate false positives:

[0081] False positive and positive samples were tested using the kits from Example 3 and Comparative Example 2, respectively. The results are shown in Table 2. A photograph of the kit from Example 3 is shown below. Figure 4 The actual image of the reagent kit for Comparative Example 2 can be found in the image below. Figure 5 , Figure 4 and Figure 5 In the middle, samples added on the left are false positives, while samples added on the right are true positives.

[0082] Table 2. False positive verification results of the kits in Example 3 and Comparative Example 2

[0083]

[0084] It is evident that the kit provided by this invention can avoid false positives.

[0085] The above are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A group B streptococcal antigen detection kit, characterized in that, It includes the following components: The reagent card includes a reagent card, an ID card, an extraction solution, and an extraction tube. The reagent card includes a reagent strip, a transparent membrane, and a card case. The reagent strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and a PVC base plate. The conjugate pad contains latex microspheres labeled with group B streptococcal antibody a and fluorescent microspheres labeled with chicken IgY. The nitrocellulose membrane contains group B streptococcal antibody b and goat anti-chicken IgY polyclonal antibody. The group B streptococcal antibody a is an antibody prepared by immunization of fusion protein and screened by immunomagnetic beads combined with real-time quantitative qPCR. The preparation of the fusion protein includes the following steps: S1. Selection of Group B Streptococcus immunogen: The coding region of the Group B Streptococcus capsular polysaccharide biosynthetic protein Cps4B was truncated using the equal division method, and polypeptides were synthesized from the truncated coding regions. The polypeptides were then combined with surface immunogenic protein and cAMP factor in different arrangements. The Cps4B gene sequence is shown in SEQ ID NO.1, the amino acid sequence of Cps4B is shown in SEQ ID NO.2, the amino acid sequence of surface immunogenic protein is shown in SEQ ID NO.3, and the amino acid sequence of cAMP factor is shown in SEQ ID NO.

4. S2. Construction of fusion protein expression vector: The fusion protein expression vector was constructed using the seamless cloning method to obtain the recombinant plasmid; S3. Expression and purification of fusion protein: The recombinant plasmid was transformed into E. coli BL21-DE3, and the protein expression was induced by IPTG. The fusion protein was obtained by affinity purification using a Ni column. The S1 equipartition method truncates the coding region of Group B Streptococcus capsular polysaccharide biosynthetic protein Cps4B, resulting in each coding region containing a 183bp sequence.

2. The group B streptococcal antigen detection kit according to claim 1, characterized in that, The group B streptococcal antibody a was derived from rabbits and had a concentration of 0.22–0.27 mg / mL; the chicken IgY was derived from chickens and had a concentration of 0.9–1.1 mg / mL; the group B streptococcal antibody b was derived from rabbits and had a concentration of 1.8–2.2 mg / mL; and the goat anti-chicken IgY polyclonal antibody was derived from goats and had a concentration of 0.38–0.42 mg / mL.

3. The group B streptococcal antigen detection kit according to claim 1, characterized in that, The cartridge includes an upper cartridge and a lower cartridge, and the reagent strip is located between the lower cartridge and the light-transmitting film. The light-transmitting film is embedded between the reagent strip and the upper cartridge.

4. The group B streptococcal antigen detection kit according to claim 3, characterized in that, The upper casing includes a sample loading window and a display window. The sample loading window overlaps with the sample pad. The extract contacts the sample pad through the drop window. The display window overlaps with the light-transmitting membrane, which overlaps with the nitrocellulose membrane. The nitrocellulose membrane includes a T-line and a C-line. The display window shows the detection results through the light-transmitting membrane.

5. The group B streptococcal antigen detection kit according to claim 1, characterized in that, The fusion protein was used as an antigen, and antibodies were prepared by immunization. The obtained antibodies were then biotin-labeled, and the labeled antibodies were used to prepare immunomagnetic beads. The immunomagnetic beads were uniformly mixed with group B streptococcal bacterial suspension and then magnetically separated. The immunomagnetic beads were washed with washing solution, and then the immunomagnetic beads were suspended in washing solution. The supernatant was collected, stained with PMAxx, and DNA was extracted. The DNA was amplified and quantified by qPCR, and the number of colonies captured by the immunomagnetic beads was calculated by converting the standard curve.

6. The group B streptococcal antigen detection kit according to claim 5, characterized in that, The biotin labeling method involves diluting the antibody, mixing it with biotin, reacting it on a shaker, and then adding it to a labeling chromatography column for column chromatography.

7. The group B streptococcal antigen detection kit according to claim 5, characterized in that, The standard curve was determined by serially diluting the logarithmic-phase Group B Streptococcus bacterial suspension with sterile physiological saline and counting the bacteria using the plate method, thereby determining the standard curve between the Ct value and Log CFU / mL.

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