Kits and detection methods for detecting Siglec fusion mutant genes and GBS pathogenic genes

Abstract

This invention provides a kit and detection method for detecting the Siglec fusion mutation gene and the GBS pathogenic gene. It is used for rapid combined detection of the Siglec5 / 14 fusion mutation gene, a preterm birth susceptibility gene, and the GBS pathogenic gene. It can effectively assess the risk of gynecological reproductive tract infections, preterm birth in pregnant women, and infection in newborns. It solves the problem that existing kits only detect the conditionally pathogenic bacteria and have poor specificity for disease association. The detection using this kit is faster and more sensitive. The detection process is closed-loop, avoiding the additional operation of electrophoresis differentiation. It is low-cost and easy to promote.

Description

Technical Field

[0001] This invention relates to the field of chemical biology, specifically to a kit and detection method for detecting the Siglec fusion mutant gene and the GBS pathogenic gene. Background Technology

[0002] When the activating receptor Siglec-14, which activates the immune response on the surface of neutrophils in a woman's innate immune system, undergoes a fusion mutation with the similar inhibitory receptor Siglec-5 during homologous recombination, resulting in neutrophils containing only the Siglec-5 gene, such as... Figure 1 As shown, the Siglec5 / 14 receptor is a key receptor in the immune response to Group B Streptococcus (GBS), a critical pathogen in preterm birth, fetal infection, and fetal injury. Traditionally, risk has been assessed solely by detecting the presence of GBS, neglecting the role of the immune system in the immune response to the pathogen.

[0003] Preterm birth is defined as delivery between 28 and 37 weeks of gestation. Complications of preterm infants include various physical problems such as visual or hearing impairment, chronic lung disease, cardiovascular disease, neurodevelopmental delay, and behavioral defects. In my country, the incidence of preterm birth is approximately 10%, and the perinatal mortality rate among preterm infants is 4-6 times higher than that of full-term infants. Approximately 40% of preterm births are due to vaginal microbial imbalance or pathogenic bacteria, such as Gardnerella vaginalis, Atorva vaginalis, Prevotella vaginalis, and GBS, which are opportunistic pathogens closely associated with preterm birth risk. GBS, in particular, has a carrier rate of approximately 35% in non-pregnant women and approximately 20-26% in pregnant women. The 2021 "Expert Consensus on the Prevention of Perinatal Group B Streptococcal Infection (China)" explicitly recommended GBS screening for all pregnant women between 35 and 37 weeks of gestation for the first time. However, the GBS carrier rate is much higher than the incidence rate. Assessing risk solely by detecting the presence of GBS ignores the role of the immune system in the immune response to pathogens and cannot effectively predict the occurrence of preterm birth. High detection rates can lead to indiscriminate medication use, exacerbating the problem of antibiotic resistance and significantly restricting medication use during pregnancy, limiting it to only a few antibiotics such as erythromycin and clindamycin. Currently, GBS resistance to these antibiotics is around 50%. Clinically, there is an urgent need to select appropriate treatment plans through accurate diagnosis, which can effectively reduce antibiotic abuse and alleviate the problem of antibiotic resistance.

[0004] GBS virulence factors play important roles in pathogenicity. For example, the composition of genes related to CPS capsule formation, the CBA gene associated with immune escape, and the LMB gene, a laminin-binding protein on the basal cell surface, exhibits significant polymorphism. Furthermore, GBS pathogenicity is closely related to the host's innate immune system. For instance, it can activate or suppress the host's immune response by interacting with a pair of sialic acid adhesion protein receptors (Siglec-5 and Siglec-14) on the neutrophil surface through the interaction of the surface protein βProtein encoded by the CBA gene with these receptors.

[0005] Siglec-5 and Siglec-14 are located on the same chromosome, with the two genes arranged sequentially. Their 5' ends, encoding the extracellular sequence, are identical, while their 3' ends, encoding the intracellular tail sequence, differ significantly. Therefore, their functions are quite different. Siglec-5 primarily functions as an immunosuppressant, while Siglec-14 primarily functions as an immune activator. Their promoter sequences also differ considerably. During gene replication, due to the long homologous sequences between Siglec-5 and Siglec-14, homologous recombination easily occurs. This leads to Siglec-5's tail recombination into their identical sequence, causing a deletion mutation in Siglec-14. Siglec-5 then utilizes the promoter of Siglec-14 for expression, producing only the Siglec-5 phenotype. Figure 1 ).

[0006] Differences in host genes and vaginal flora contribute to the risk of preterm birth. Among GBS-positive pregnant women, the preterm birth rate is significantly higher in those with the Siglec-14 deletion mutation than in those with the Siglec-14 wild-type. In preterm infants, GBS colonization is statistically significantly correlated with the Siglec-14 deletion mutation allele compared to the Siglec-14 wild-type allele.

[0007] Because Siglec-14 and Siglec-5 wild-types share a large number of identical sequences with the Siglec-14 deletion mutant, with fusion regions exceeding 1299 bp and a similarity greater than 99%, their detection faces significant challenges. Conventional qPCR methods cannot amplify them effectively. Current detection methods mostly rely on conventional PCR amplification, designing primers specifically for the differences in promoter and tail sequences, and then performing electrophoresis to distinguish wild-type, mutant, and heterozygous genes based on sequence length and band count. However, PCR experiments are complex, requiring electrophoresis and visual band analysis, hindering large-scale deployment; therefore, no such in vitro diagnostic test has yet been developed. While Siglec-5 and Siglec-14 exhibit some differences in their extracellular receptors, allowing for identification using specific antibodies, antibody incubation is lengthy, prone to interference and non-specific binding, and requires intact cell isolation, making the process difficult and unsuitable for medical diagnosis and large-scale deployment.

[0008] Currently, most tests for premature birth rely on symptoms or metabolic markers for diagnosis, such as arachidonic acid ethanolamide (200580014584.7) and IL-1β (201610330318.5). However, these can only be detected after symptoms appear, failing to provide early diagnosis and intervention. Other methods use susceptibility cfRNA markers like CLCN3 and DAPP1 for auxiliary diagnosis (201910890694.), but cfRNA levels are low, requiring invasive testing involving isolation from blood, which is complex and has low sensitivity. Another example is detecting the RANTES gene polymorphism site IN1.1T / C (201510050767.X). These methods ignore the pathogen-host correlation, only detecting gene polymorphism changes, easily leading to missed or false positives. Furthermore, these methods require sequencing to identify related site changes, making the process complex and time-consuming. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a kit and detection method for detecting Siglec fusion mutant genes and GBS pathogenic genes, which can effectively assess the risk of gynecological reproductive tract infections, premature birth in pregnant women and infection in newborns, and solve the problem that existing kits only detect conditionally pathogenic bacteria and have poor specificity for disease association, thus effectively reducing the risk of antibiotic abuse.

[0010] To address the aforementioned problems, the present invention proposes a kit for detecting Siglec fusion mutation genes and GBS pathogenic genes, used for the detection of fusion mutations in preterm birth susceptibility genes and their rapid combined detection with GBS pathogenic genes. This kit contains:

[0011] The primer-probe mixture for detecting the I-Siglec5 / 14 mutation includes: Siglec-GAP-F, Siglec-GAP-R, Siglec-GAP-P, Siglec-LF, Siglec-LR, Siglec-SF, Siglec-SR, and Siglec-SP.

[0012] Reaction solution for II-GBS detection primers, probes and internal control mixture: GBS-F, GBS-R, GBS-P, RNP-F, RNP-R, RNP-P;

[0013] Reaction Solution III - Primer and probe mixture for quantitative PCR detection of GBS: CBA-F, CBA-R, LMB-F, LMB-R, CBA-P, LMB-P;

[0014] The primer and probe mixture for IV-qPCR detection of GBS virulence gene is as follows: CPS1-F, CPS1-R, CPS2-F, CPS2-R, CPS3-F, CPS3-R, CPS4-F, CPS4-R, CPS1-P, CPS2-P, CPS3-P, CPS4-P;

[0015] Enzyme mixture: II Probe qPCR SuperMix;

[0016] Negative control: Ultrapure water;

[0017] Positive control: Artificially synthesized plasmids containing fragments of the target gene being amplified.

[0018] Specifically, the sequences of the primers and probes used to detect the Siglec fusion mutant gene are shown in Table 1:

[0019] Table 1 Primers and probes used for Siglec fusion gene detection

[0020]

[0021]

[0022] Furthermore, in each reaction solution within the kit, the molar ratio of primer to probe is 1:1 to 4:1, preferably 1:1.

[0023] Specifically, the primer and probe sequences used for the detection of GBS virulence genes (cba, lmb, cps1-4) are shown in Table 2:

[0024] Table 2 Primers and probes for GBS and its related pathogenic genes

[0025]

[0026]

[0027] The method for detecting the Siglec fusion mutant gene using a kit is as follows:

[0028] First, the different reaction solutions of the kit were placed in the corresponding templates to prepare the system. The volume ratio of each component in the resulting system was as follows:

[0029] Reaction solution I: Primer / Probe: Enzyme mixture: Template addition: Ultrapure water ratio is 0.32:10:2:7.68;

[0030] Reaction solution II: Primer / Probe: Enzyme mixture: Template addition ratio: Ultrapure water = 0.25:10:2:7.75;

[0031] Reaction solution III: Primer / Probe: Enzyme mixture: Template addition: Ultrapure water ratio is 0.25:10:2:7.75;

[0032] The reaction solution IV primer probe: enzyme mixture: template addition amount: ultrapure water is 0.5:10:2:7.75.

[0033] In this process, reaction solution I was subjected to two rounds of PCR and qPCR reactions. The Tm value of the primers in the first round of PCR was higher than that in the second round of qPCR. Both rounds of reactions involved denaturation, annealing, and extension processes, with the annealing temperature of the first round of PCR being higher than that of the second round of qPCR. In the second round of qPCR, the different fluorescence signals were used to effectively distinguish the wild-type, mutant, and heterozygous types of the Siglec gene.

[0034] Specifically, reaction solution I underwent two rounds of PCR and qPCR reactions. The reaction steps were as follows: 1) Pre-denaturation at 94℃ for 2 min; 2) Initiation of the first round of PCR amplification reaction, denaturation at 94℃ for 5 s; 3) Annealing and extension at 72℃ for 120 s, with a total of 30 cycles for the first round of PCR reaction; 4) Initiation of the second round of qPCR detection reaction, denaturation at 94℃ for 5 s; 5) Annealing and extension at 60℃ for 30 s, with a total of 45 cycles for the second round of qPCR reaction, during which signals were collected.

[0035] For reaction solution I, by adjusting the reaction temperature, two rounds of PCR and qPCR were performed consecutively. The different types of the Siglec gene were distinguished by the different fluorescence signals. Figures 2-5As shown, the upstream primer in the first round of PCR amplification targets the promoter region of the Siglec14 gene, and the downstream primer targets the downstream coding region of the Siglec5 gene. Only genes that have undergone fusion can be amplified normally, with an amplification length of 2672 bp. Genes that have not undergone fusion have a length of 16933 bp between the two primers, which cannot be amplified normally within the set extension time. The downstream PCR primer consists of three parts: the R part is a Siglec-5 gene-specific primer; the A part is the probe targeting region for the second round of qPCR, which is an artificially added DNA sequence that is not homologous to Siglec; and the O part is the complementary sequence of the downstream primer for the second round of qPCR detection, also an artificially added non-Siglec homologous DNA sequence. The primer R part binding sequence is present in both wild-type and Siglec-14 deletion mutants. However, because the length of the non-fused gene is much greater than the length of the fused gene, under optimized annealing and extension conditions, only the Siglec-14 deletion mutant fragment, with a length of 2672 bp, can be amplified. The newly generated PCR product incorporates artificially added sequences into the downstream primers. Because the Tm values ​​of the primers designed for the first round of PCR are much higher than those for the second round, it ensures that the primers and probes used for the second-round qPCR detection cannot amplify during the first round of amplification. The downstream primers in the second round of PCR are added simultaneously with the initial system preparation, but they are only detected by the second-round qPCR after the annealing temperature has decreased following the actual first round of PCR. During the second round of qPCR, the genotype of Siglec in the sample is determined based on the collected fluorescence signal.

[0036] The second round of qPCR amplification can amplify the sequence between wild-type Siglec-14 and Siglec-5. If a normal amplification fluorescence signal is present, it indicates the presence of the wild-type gene. If the Siglec14 deletion mutant fusion gene is amplified normally, it is used as a template. The upstream primer recognizes the 3' end sequence of the Siglec14 / 5 mutant gene, the downstream primer recognizes the artificially added sequence O region, and the probe recognizes the artificially added sequence A region for amplification. If a normal amplification fluorescence signal is present, it indicates the presence of the mutant gene. If both wild-type and mutant gene signals are present, it indicates a heterozygous genotype.

[0037] Specifically, in the second round of qPCR testing, the FAM fluorescently labeled probe Siglec-GAP-P targets the non-coding region between the wild-type and normal Siglec14 and Siglec5 genes to detect the wild-type gene, while the VIC fluorescently labeled probe Siglec-SP targets the O region of the fusion mutant gene to detect the mutant gene. A negative control using H2O as a template is used for quality control throughout the testing process. Results are considered acceptable if the following conditions are met:

[0038] (1) The negative control (using H2O as the template) had no amplification curve.

[0039] (2) Samples with a typical S-shaped amplification curve only in the FAM channel were wild-type, those with a typical S-shaped amplification curve only in the VIC channel were mutant, and those with typical S-shaped amplification curves in both the FAM and VIC channels were heterozygous, as Figure 6 shown.

[0040] The method for detecting the GBS pathogenic gene using the kit was to continuously perform qPCR reactions on the reaction solutions II, III, and IV systems by the fluorescence probe method to detect the GBS virulence gene.

[0041] The qPCR reaction steps for the reaction solutions II, III, and IV systems were as follows: 1) Pre-denaturation at 94°C for 2 min; 2) Initiate the qPCR detection reaction, denaturation at 94°C for 5 s; 3) Annealing and extension at 60°C for 30 s. The qPCR reaction was cycled 45 times, and signals were collected during the annealing and extension process.

[0042] Among them, for the GBS detection using the probe-based fluorescence quantitative PCR method, the GBS probe was fluorescently labeled with FAM, RNase P was used as an internal reference, the Rnase P probe was fluorescently labeled with VIC, and the negative control using H2O as the template was used to quality control the entire detection process. The result interpretation was considered qualified when the following conditions were met:

[0043] (1) There was a typical S-shaped amplification curve in the VIC channel, and its Ct amplification curve;

[0044] (2) The negative control (using H2O as the template) had no amplification curve.

[0045] (3) For the FAM channel for GBS-specific gene detection: If there was a typical S-shaped curve in the FAM channel, and Ct curve, and, then the GBS in the sample was positive; If there was a typical S-shaped curve in the FAM channel, and Ct > 40, re-perform qPCR detection on the sample. If the results were the same, it was determined as GBS positive, otherwise it was determined as GBS negative, as Figure 7 shown.

[0046] For the GBS virulence gene detection using the fluorescence probe method qPCR detection, the negative control using H2O as the template was used to quality control the entire detection process. The result interpretation was considered qualified when the following conditions were met:

[0047] (1) The negative control (using H2O as the template) had no amplification curve.

[0048] (2) Samples with a typical S-shaped amplification curve were positive for the relevant待测 gene.

[0049] The test samples used to rapidly assess the risk of preterm birth with the kit can be vaginal secretions, blood, amniotic fluid, urine, or feces.

[0050] Advantages and beneficial effects of the present invention:

[0051] The kit described in this application can be used for the combined detection of Siglec5 / 14 receptor fusion mutations and GBS pathogenic genes. It involves adding PCR amplification primers for the first round of Siglec14 / 5 fusion mutation gene, qPCR detection primers and probes targeting the fusion mutation gene for the second round, and qPCR detection primers and probes targeting the non-coding region between wild-type normal Siglec14 and Siglec5 genes to the same tube. By adjusting the temperature changes in the denaturation, annealing, and extension steps, two rounds of PCR and qPCR reactions are performed continuously without interference. The different fluorescence signals are utilized for a single detection. This invention effectively distinguishes between wild-type, mutant, and heterozygous Siglec14 / 5 genes. The entire detection process is conducted in a closed tube, avoiding the additional operation of electrophoresis differentiation. Simultaneously, it uses fluorescent probe qPCR to detect GBS pathogens and their pathogenicity-related genes, enabling rapid and effective assessment of gynecological reproductive tract infections, premature birth in pregnant women, and neonatal infection risks. This addresses the problem of existing kits that only detect conditionally pathogenic bacteria and have poor specificity for disease association, effectively reducing the risk of antibiotic overuse. Furthermore, this invention performs host and pathogenicity detection simultaneously through a single vaginal and anal swab sampling, resulting in low cost and ease of promotion. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the wild-type and mutant gene sequences of Siglec-5 and Siglec-14.

[0053] Figure 2 This is a schematic diagram illustrating the principle of the method for detecting the Siglec14 / 5 genotype.

[0054] Figure 3 This is a schematic diagram showing the location of the Siglec14 / 5 fusion mutant gene-specific primers (for the first round of PCR amplification).

[0055] Figure 4 This is a schematic diagram showing the location of the Siglec fusion gene mutation-specific primers and probes (for the second round of qPCR detection).

[0056] Figure 5 This is a schematic diagram showing the positions of the primers and probes for detecting the Siglec14 and Siglec-5 wild-type genes (second round of qPCR detection).

[0057] Figure 6 This is a schematic diagram of qPCR amplification curves for different Siglec genotypes.

[0058] Figure 7 This is a schematic diagram of the qPCR amplification curves for GBS positive and negative results.

[0059] Figure 8 This is the result of a routine PCR test for the Siglec genotype in a vaginal swab sample from a patient suspected of having GBS infection.

[0060] Figure 9 The amplification curves for Siglec genotype detection and GBS pathogenic gene detection of sample 1 in Example 1 are shown.

[0061] Figure 10 The amplification curves for Siglec genotype detection and GBS pathogenic gene detection of sample 3 in Example 1 are shown.

[0062] Figure 11 The amplification curves for Siglec genotype detection and GBS pathogenic gene detection of sample 9 in Example 1 are shown.

[0063] Figure 12 This is the result of routine PCR detection of the Siglec genotype in the amniotic fluid sample of Example 2.

[0064] Figure 13 These are the Siglec genotype detection and GBS gene detection amplification curves of sample Y1 in Example 2.

[0065] Figure 14 These are the Siglec genotype detection and GBS gene detection amplification curves of sample Y2 in Example 2.

[0066] Figure 15 These are the Siglec genotype detection and GBS gene detection amplification curves of sample Y6 in Example 2. Detailed Implementation

[0067] The specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0068] Methods for detecting the Siglec14 / 5 genotype (conventional PCR + qPCR):

[0069] The enzyme used is II. Probe qPCR SuperMix (Transgen, AQ711-01) is used, but not limited to this enzyme. The optimal reaction system is selected based on the enzyme, as shown in Table 3.

[0070] Table 3. System of a single qPCR reaction (20 μl reaction volume)

[0071]

[0072] Set the qPCR reaction conditions according to Table 4.

[0073] Table 4 qPCR reaction conditions

[0074]

[0075] Simultaneously, conventional PCR was designed to detect the Siglec gene type. The primer sequences are shown in Table 5, and the selected enzyme is... PCR SuperMix (-dye) (Transgen, AS111-02) is used, but not limited to this enzyme. The optimal reaction system is selected based on the enzyme as shown in Table 6. The reaction is carried out according to the conditions in Table 7, and the results are compared with those of qPCR.

[0076] Table 5 Primer sequences for conventional PCR

[0077]

[0078] Table 6. Standard PCR reaction system (20 μl system)

[0079]

[0080] Table 7 Reaction conditions for conventional PCR

[0081]

[0082] Methods for detecting GBS and its virulence factors (qPCR detection using fluorescent probes)

[0083] GBS detection was performed using a probe-based quantitative real-time PCR reaction. The reaction system was prepared according to Table 8. The GBS probe was labeled with FAM fluorescence, RNase P was used as an internal control, RNase P probe was labeled with VIC fluorescence, and H2O was used as a negative control for quality control throughout the detection process. The reaction conditions are shown in Table 9. Table 8: Reaction system for GBS detection using probe-based quantitative real-time PCR.

[0084]

[0085]

[0086] Table 9. Reaction conditions for probe-based quantitative PCR detection of GBS

[0087]

[0088] The reaction system for GBS virulence gene detection (fluorescent probe qPCR detection) was prepared according to Tables 10-1 and 10-2. A negative control using H2O as a template was used for quality control throughout the detection process. Reaction conditions are shown in Tables 11-1 and 11-2.

[0089] Table 10-1 Detection reaction system for CBA and LMB

[0090]

[0091] Table 10-2 Detection reaction systems for cps1 to cps4

[0092]

[0093]

[0094] Table 11-1 Reaction conditions for qPCR detection of CBA and LMB

[0095]

[0096] Table 11-2 Reaction conditions for qPCR detection of cps1–cps4

[0097]

[0098] Example 1: Siglec genotyping and GBS pathogenic gene detection from vaginal swabs

[0099] To investigate the correlation between the Siglec14 / 5 fusion mutation and GBS infection, as well as the severity of infection, we collected vaginal swab samples from 35 suspected GBS-positive patients. Following the detection method of this invention, the patients underwent Siglec14 / 5 genotyping. Then, the GBS infection status of the samples was detected using fluorescent probe PCR. GBS-positive samples were further subjected to qPCR detection of virulence genes (cba, lmb, and cps1-4). Furthermore, to further verify the accuracy of this invention, the detection of Siglec14 / 5 was compared with the conventional PCR-electrophoresis method. The methods and steps for detecting the Siglec14 / 5 gene type (conventional PCR + qPCR) are the same as those described in the above detection procedure section.

[0100] In conventional PCR testing, the wild-type gene is characterized by a single band of 1622 bp, meaning that both Siglec14 and Siglec5 have normal double copies, denoted as Wt / Wt. The mutant gene is characterized by a single band of 1790 bp, meaning that both copies of Siglec14 undergo homologous recombination with Siglec5, forming a Siglec14 / 5 fusion mutant gene, denoted as Mt / Mt. If both 1622 bp and 1790 bp double bands are present, the gene is heterozygous, denoted as Wt / Mt.

[0101] The FAM fluorescently labeled probe Siglec-GAP-P targets the non-coding region between the wild-type normal Siglec14 and Siglec5 genes to detect the wild-type gene. The VIC fluorescently labeled probe Siglec-SP targets the O region of the fusion mutant gene to detect the mutant gene. Wild-type genes exhibit a typical S-shaped amplification curve only in the FAM channel (denoted as Wt / Wt); mutant genes exhibit a typical S-shaped amplification curve only in the VIC channel (denoted as Mt / Mt); and heterozygous genes exhibit typical S-shaped amplification curves in both the FAM and VIC channels (denoted as Wt / Mt).

[0102] Thirty-five vaginal swab samples suspected of being GBS-positive were subjected to qPCR and conventional PCR testing for the Siglec14 / 5 genotype. The results were statistically analyzed and compared. The qPCR test results are shown in Table 12. Figures 9-11 As shown, the results of the routine PCR test are detailed in Table 12 and... Figure 8 The results of the two methods are 100% consistent, but the PCR method is faster and easier to operate than the traditional PCR electrophoresis method. The entire detection process is closed-tube, which makes it less likely to generate aerosol pollution and is very easy to promote on a large scale.

[0103] Table 12 Summary of the results of qPCR and conventional PCR detection of the Siglec fusion gene

[0104]

[0105]

[0106] In addition, as shown in Table 13, we have statistically analyzed the fusion distribution of the Siglec gene. Homozygous mutations account for 22.86% of the total, and together with heterozygous genotypes, they account for 77.14% of the total.

[0107] Table 13 Statistical table of Siglec fusion gene detection results

[0108] genotype Number of examples percentage Wt / Wt 8 22.86％ Wt / Mt 19 54.28％ Mt / Mt 8 22.86％

[0109] Further investigation was conducted on the GBS pathogen and its pathogenic genes in these clinical samples exhibiting significant inflammatory responses to clarify the association between host gene mutations and pathogenic genes. The methods and procedures for detecting GBS virulence factors (qPCR detection using fluorescent probes) are the same as those described in the detection procedure section above.

[0110] Table 14 qPCR detection results of GBS

[0111]

[0112]

[0113] Table 15 Results of GBS Virulence Gene Detection

[0114]

[0115]

[0116] Table 16 Summary of final test results for samples

[0117]

[0118] GBS virulence factors play an important role in pathogenesis, but not all GBS carry the relevant virulence genes. To further investigate the relationship between the severity of GBS infection and the Siglec14 / 5 genotype, we performed qPCR detection of several important virulence genes (cba, lmb, and cps-related genes) on GBS-positive samples. The results are shown in Tables 14-15. Figures 9-11 As shown, the cba and lmb genes are almost always present in GBS-positive patients. Unlike previous analyses of all GBS genomes in NCBI, which showed genotypic polymorphisms in cba and lmb, the paired occurrence of cba and lmb is significantly associated with vaginal infection. Joint detection of these two genes can help predict the occurrence of infection and adverse outcomes such as premature birth.

[0119] Based on a comprehensive analysis of the above test results, as shown in Table 16, further analysis leads to the following conclusions:

[0120] Among GBS-positive samples, the Siglec14 / 5 fusion mutant genotype was present in a high proportion; 18 out of 23 positive samples carried the Siglec14 / 5 fusion gene, accounting for 83%. This indicates that individuals carrying the Siglec14 / 5 fusion gene are susceptible to GBS.

[0121] In patients infected with GBS, both the cba and lmb genes are positive, indicating that double-positive GBS has a higher probability of causing genital tract infections.

[0122] The three Siglec wild-type patients were positive for all six pathogenic genes of GBS, which may indicate that cps1 and cps4 are more pathogenic; it may also indicate that the "threshold" for Siglec wild-type individuals to be infected with GBS is higher, requiring GBS to be highly pathogenic in order to infect this group.

[0123] In both homozygous and heterozygous GBS-infected individuals with Siglec14 / 5, most lacked cps1 or cps4, but all had cps2, indicating that cps2 may be a constitutively expressed gene and does not exhibit genotypic differences.

[0124] By differentiating the Siglec14 / 5 genotype differences among patients and the composition of GBS pathogenic genes, it is possible to effectively determine the patient's susceptibility to the pathogen and the severity of the disease.

[0125] Example 2: Siglec genotyping and GBS pathogenic gene detection in amniotic fluid samples

[0126] The method and steps for detecting the Siglec14 / 5 gene type (using conventional PCR and qPCR) are the same as those described in the detection procedure section above.

[0127] The method and steps for detecting GBS virulence factors (qPCR detection using fluorescent probes) are the same as those described in the detection procedure section above.

[0128] Table 17. Siglec typing results of the sample using the method of this invention.

[0129]

[0130] The results of GBS virulence factor detection (qPCR using fluorescent probe method) are shown in Table 18. Six out of seven amniotic fluid samples showed the Siglec mutation genotype. Among the GBS virulence genes, both cba and lmb were positive, indicating their close association with infection and pathogenicity. In the only preterm patient with the Siglec wild-type virulence, all six GBS virulence genes were positive.

[0131] Table 18 Summary of pathogenic gene and Siglec fusion gene typing in amniotic fluid samples

[0132]

[0133]

[0134] In conjunction with Table 18 and Figure 13 It is known that GBS with all virulence genes positive can significantly increase the infection rate in Siglec wild-type patients. For women with Siglec14 / 5 deletion mutations, GBS is more likely to cause adverse consequences such as premature birth during pregnancy. Even if the Siglec14 / 5 genotype is wild-type, further analysis of the GBS pathogenic gene type is needed. If all virulence genes are positive, it is necessary to pay more attention and intervene and treat the disease.

[0135] The foregoing has described several embodiments of the invention in detail, but these are merely preferred embodiments and should not be construed as limiting the scope of the invention. All equivalent variations and improvements made within the scope of the claims of this invention should still fall within the patent coverage of this invention.

Claims

1. A kit for detecting the Siglec fusion mutant gene and the GBS pathogenic gene, characterized in that, This kit is used for the detection of the Siglec5 / 14 fusion mutation, a susceptibility gene for preterm birth, and for rapid combined detection with the GBS pathogenic gene. It contains: The primer-probe mixture for detecting the I-Siglec5 / 14 mutation includes: Siglec-GAP-F, Siglec-GAP-R, Siglec-GAP-P, Siglec-LF, Siglec-LR, Siglec-SF, Siglec-SR, and Siglec-SP. The specific sequence is as follows: Siglec-GAP-F: 5'-ACTTGATCTGTGGCCTGTATACAAT-3'; Siglec-GAP-R: 5'-CAGGTATGAGCCACCGCACCC-3'; Siglec-GAP-P: 5'-Fluorescent reporter group-CACCCCTCACTCCCACTG-MGB-3'; Siglec-LF: 5'-CAGCCCAGCTCTGTGGTTCTTCTCTCACCCTT-3'; Siglec-LR sequences are formed by sequentially connecting the O region, A region to R region, from the 5' end to the 3' end: R region: 5'-AGGTCAGGAGTTCAAGATCAGCTTGGCCGACAT-3'; O region: 5'-GAGTCTAGAATCGATCAGTAACTCAGCT-3'; A region: 5'-CTCCACCACTCTACTCATGCCACCCCTACC-3'; Siglec-SF: 5'-GGATTACAGGTGCACATCATCACGCCTGGCTA-3'; Siglec-SR: 5'-GAGTCTAGAATCGATCAGTAACTCAGC-3'; Siglec-SP: 5'-Fluorescent reporter group -CCACCACTCTACTCATGCCACCCCT AC-BHQ1-3'; The fluorescent reporter groups in the Siglec-GAP-P and Siglec-SP probes can be any two different labels from FAM, VIC, CY5, and ROX; Reaction solution for II-GBS detection primers, probes and internal control mixture: GBS-F, GBS-R, GBS-P, RNP-F, RNP-R, RNP-P; The specific sequence is as follows: GBS-F: 5'-CGCAAGTGTTCAAGCACAAGA-3'; GBS-R: 5'-GTCTTGCTTTACCAAATCAGCCT-3'; GBS-P: 5'-Fluorescent reporter group-ACCTCTGAA ACAGTACGTGC-MGB -3'; RNP-F: 5'-GCAGACATTCTCATATTTTGCTCGT-3'; RNP-R: 5'-GAATAGTTGTTGCCAAATTACCCAC-3'; RNP-P: 5'-Fluorescent reporter group-ACTGTGTAAATGAGAACAGTCCC-MGB -3’； The fluorescent reporter groups in GBS-P and RNP-P probes can be any two different labels from FAM, VIC, CY5, and ROX; Reaction Solution III - Primer and probe mixture for quantitative PCR detection of GBS: CBA-F, CBA-R, CBA-P, LMB-F, LMB-R, LMB-P; The specific sequence is as follows: CBA-F: 5'-AACTGTTCGTCTCGCACTTGGTC-3'; CBA-R: 5'-TGTCTTAATCGCAAACAAACTGAAGTGGT-3'; CBA-P: 5'-Fluorescent reporter group-ACTGATTCGGATGTTCAAGTC-MGB-3'; LMB-F: 5'-ATTCTTTTTGAACCATCAGCCAACGA-3'; LMB-R: 5'-GCAACATCTTCTAATCCCGGCACAC -3'; LMB-P: 5'-Fluorescent reporter group -TAGCAGCTATCTATGATGCAGAT-MGB -3’； The fluorescent reporter groups in the CBA-P and LMB-P probes can be any two different labels from FAM, VIC, CY5, and ROX; The primer and probe mixture for IV-qPCR detection of GBS virulence gene is as follows: CPS1-F, CPS1-R, CPS1-P, CPS2-F, CPS2-R, CPS2-P, CPS3-F, CPS3-R, CPS3-P, CPS4-F, CPS4-R, CPS4-P; The specific sequence is as follows: CPS1-F: 5'-CAACGACTTCTACAAATCTAGCCAT-3'; CPS1-R: 5'-GTCGCACCAGCTAAATAATCTGTCA-3'; CPS1-P: 5'-Fluorescent reporter group -CTTTTGCACGCTCAGGATATAATACTTT AC-MGB-3'; CPS2-F: 5'-AATTTCTCATCGTTGTGACCGCTT-3'; CPS2-R: 5'-AAGTATGTCCCAGCTTGAAGGTCT-3'; CPS2-P: 5'-Fluorescent reporter group -CATTGGCTATTGTTGCTTTAGGCTA- MGB-3'; CPS3-F: 5'-TGTGCTCCGTTTAGGAATTACACC-3'; CPS3-R: 5'-TTCACCAAATAACTTCGGTTTGAGG-3'; CPS3-P: 5'-Fluorescent reporter group -TTGTAGCTCATATTGAGCGTTATCATT- MGB-3'; CPS4-F: 5'-TATGGTCCCGTTTCATCTGTATCTCG-3'; CPS4-R: 5'-CGCTTCCACGCCATAAATACCAG-3'; CPS4-P: 5'-Fluorescent reporter group-CGGATGTGAATATCATCATGAC-MGB-3'; The fluorescent reporter groups in the CPS1-P, CPS2-P, CPS3-P, and CPS4-P probes can be any four different labels from FAM, VIC, CY5, and ROX; Enzyme mixture: PerfectStart ® II Probe qPCR SuperMix; Negative control: Ultrapure water; Positive control: Artificially synthesized plasmids containing fragments of the target gene being amplified.

2. The kit for detecting the Siglec fusion mutant gene and the GBS pathogenic gene according to claim 1, characterized in that, Siglec-GAP-F and Siglec-GAP-R are primers for detecting non-coding regions between normal Siglec genes; Siglec-GAP-P is a probe for detecting non-coding regions between normal Siglec genes, and is only used in the second round of qPCR. Siglec-LF targets the promoter region of the Siglec14 gene and is the upstream amplification primer for the first round of PCR. Siglec-LR targets the downstream coding region of the Siglec5 gene and is the downstream amplification primer for the first round of PCR. Siglec-SF is the upstream detection primer for the second round of qPCR targeting fusion mutant genes; Siglec-SR is the downstream detection primer for the second round of qPCR targeting fusion mutant genes; and Siglec-SP is the detection probe for the second round of qPCR targeting fusion mutant genes.

3. The kit for detecting the Siglec fusion mutant gene and the GBS pathogenic gene according to claim 1, characterized in that, The primers and probes used for detecting GBS were GBS-F, GBS-R, and GBS-P; the primers and probes used for detecting the internal reference gene RNaseP were RNP-F, RNP-R, and RNP-P; the primers and probes used for detecting the GBS virulence gene cba were CBA-F, CBA-R, and CBA-P; the primers and probes used for detecting the GBS virulence gene lmb were LMB-F, LMB-R, and LMB-P; and the primers and probes used for detecting the GBS virulence genes cps1–4 were CPS1-F, CPS1-R, CPS1-P, CPS2-F, CPS2-R, CPS2-P, CPS3-F, CPS3-R, CPS3-P, CPS4-F, CPS4-R, and CPS4-P.

4. The kit for detecting the Siglec fusion mutant gene and the GBS pathogenic gene according to any one of claims 1 to 3, characterized in that, In each reaction solution within the kit, the molar ratio of primer to probe is 1:1 to 4:

1.

5. The kit for detecting the Siglec fusion mutant gene and the GBS pathogenic gene according to claim 4, characterized in that, The different reaction solutions in the kit are placed in the corresponding templates, and the volume ratio of each component in the resulting system is: Reaction solution I: Primer / Probe: Enzyme mixture: Template addition: Ultrapure water ratio is 0.32:10:2:7.68; Reaction solution II: Primer / Probe: Enzyme mixture: Template addition ratio: Ultrapure water = 0.25:10:2:7.75; Reaction solution III: Primer / Probe: Enzyme mixture: Template addition ratio: Ultrapure water = 0.25:10:2:7.75; The reaction solution IV primer probe: enzyme mixture: template addition amount: ultrapure water is 0.5:10:2:7.

75.

6. A method for detecting the Siglec fusion mutant gene and the GBS pathogenic gene using the kit described in claim 1 for non-disease diagnosis and treatment purposes, characterized in that, The reaction solution I was subjected to two rounds of PCR and qPCR reactions consecutively. The Tm value of the primers in the first round of PCR was higher than that in the second round of qPCR. Both rounds of reactions underwent denaturation, annealing, and extension processes, and the annealing temperature of the first round of PCR was higher than that of the second round of qPCR. In the second round of qPCR, the difference in fluorescence signal was used to effectively distinguish the wild-type, mutant, and heterozygous types of the Siglec gene in a single reaction. Reaction solutions II, III, and IV were used to perform qPCR reactions using fluorescent probes to detect GBS and its virulence genes. The mutant type is characterized by a typical S-shaped amplification curve in the channel of the fluorescent reporter group carried by the probe targeting the fusion mutant gene in the second round. The wild type is characterized by a typical S-shaped amplification curve in the channel of the fluorescent reporter group carried by the probe targeting the wild-type normal Siglec14 and Siglec5 genes in the second round. The heterozygous type is characterized by an S-shaped amplification curve in both channels.

7. The method for detecting the Siglec fusion mutant gene and the GBS pathogenic gene using a kit according to claim 6, characterized in that, Reaction solution I underwent two rounds of PCR and qPCR reactions. The reaction steps were as follows: 1) Pre-denaturation at 94℃ for 2 minutes; 2) Start the first round of PCR amplification reaction, denature at 94℃ for 5 seconds; 3) Anneal at 72℃ and extend for 120s, repeat the first round of PCR reaction 30 times; 4) Start the second round of qPCR detection reaction, denature at 94℃ for 5 seconds; 5) Anneal at 60℃ and extend for 30s. The second round of qPCR reaction was repeated for a total of 45 cycles. Signals were collected during the annealing and extension process.

8. The method for detecting the Siglec fusion mutant gene and the GBS pathogenic gene using a kit according to claim 6, characterized in that, The qPCR reaction steps for reaction solution II, reaction solution III, and reaction solution IV are as follows: 1) Pre-denaturation at 94℃ for 2 minutes; 2) Start the qPCR detection reaction and denature at 94℃ for 5 seconds; 3) Anneal and extend at 60℃ for 30s, and repeat the qPCR reaction for a total of 45 cycles. Collect signals during the annealing and extension process.

9. The method for detecting the Siglec fusion mutant gene and the GBS pathogenic gene using a kit according to claim 6, characterized in that, The reaction conditions for qPCR detection of GBS and GBS-related genes cba, lmb, and cps are as follows: 1) Pre-denaturation at 94℃ for 30 seconds; 2) Start the qPCR detection reaction and denature at 94℃ for 5 seconds; 3) Anneal and extend at 60℃ for 30s, and repeat the qPCR reaction for a total of 45 cycles. Collect signals during the annealing and extension process.

10. The method for detecting the Siglec fusion mutant gene and the GBS pathogenic gene using a kit according to claim 6, characterized in that, Test samples can be any of the following: vaginal secretions, blood, amniotic fluid, urine, or feces.

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