A method for rapid detection of GBS-related susceptibility genes Siglec14 / 5 and their fusion mutations

Abstract

The present invention provides a method for rapidly detecting the susceptible gene Siglec14 / 5 and its fusion mutation associated with GBS, namely, adding a first-round PCR amplification primer for the Siglec14 / 5 fusion mutation gene, a second-round qPCR detection primer and probe targeting the fusion mutation gene, and a qPCR detection primer and probe targeting the non-coding region sequence between the wild-type normal Siglec14 and Siglec5 genes to the same tube, and by adjusting the temperature changes of each reaction step of denaturation-annealing-extension, continuously performing two rounds of PCR and qPCR reactions without interfering with each other, and utilizing the difference in fluorescent signals to effectively distinguish the wild type, deletion mutation type, and heterozygous type of the Siglec14 / 5 gene at one time. This application is faster and more convenient to operate than the traditional PCR plus electrophoresis method. The detection process is closed throughout the tube, and aerosol contamination is not easily generated, making it easy to promote on a large scale.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, and in particular to a method for rapidly detecting GBS-related susceptibility genes Siglec14 / 5 and fusion mutations thereof. Background Art

[0002] Group B Streptococcus (GBS) is a common opportunistic pathogen in women of childbearing age, with a carriage rate of approximately 35% in non-pregnant women and 20-26% in pregnant women. GBS infection in pregnant women can lead to serious consequences such as miscarriage, premature birth, fetal infection, and fetal injury. The pathogenicity of GBS is closely linked to the host's innate immune system. GBS can activate or inhibit the host immune response through interaction between its surface protein (the surface protein β protein encoded by the cba gene) and a pair of sialoadhesion protein receptors (Siglec-5 and Siglec-14) with opposing functions on the surface of neutrophils.

[0003] Siglec-5 and Siglec-14 are located on the same chromosome, with the two genes arranged in tandem. The genes encoding the extracellular sequences at their 5' ends are completely identical, while the sequences encoding the intracellular tails at their 3' ends are quite different. Therefore, the functions they perform are very different. Siglec-5 mainly plays an immunosuppressive role, while Siglec-14 mainly plays an immune-activating role. There are also significant differences in their promoter sequences. During gene replication, due to the long homologous sequences between Siglec-5 and Siglec-14, homologous recombination is prone to occur, resulting in the recombination of the tail of Siglec-5 to their consensus sequence, causing a deletion mutation in Siglec-14. Siglec-5 is expressed using the Siglec-14 promoter, producing only the Siglec-5 phenotype (Figure 1).

[0004] When the activating receptor Siglec-14 that exercises immune response on the surface of neutrophils in a woman's innate immune system fuses and mutates with the similar inhibitory receptor Siglec-5 during homologous recombination, only the Siglec-5 gene exists in the neutrophils. GBS combines with Siglec-5, inhibiting the phagocytic activity of neutrophils against GBS, causing GBS to escape the immune system, causing it to cause serious invasive infection in the host or be transmitted to newborns, resulting in more serious consequences.

[0005] Wild-type Siglec-5 and Siglec-14 are located on the same chromosome, with the two genes arranged in tandem. The non-coding region between them is greater than 15,000 bp long. This fragment is deleted after the fusion of Siglec5 and Siglec14, forming the Siglec-14 deletion mutant. Due to the large number of identical sequences between the wild-type Siglec-14 and Siglec-5 and the Siglec-14 deletion mutant, the fusion region is greater than 1299 bp long and has an identity greater than 99%, which poses a significant obstacle to their detection. Conventional qPCR methods cannot be used for normal detection. Most current detection methods use conventional PCR amplification, with primers designed for amplification based on differences in promoter and tail sequences. The PCR products are then subjected to electrophoresis detection, and wild-type, mutant, and heterozygous genes are distinguished by sequence length and number of bands. However, PCR experiments are complex and require electrophoresis detection and visual identification of bands. This is not only time-consuming and labor-intensive, but also prone to contamination, making it very unfavorable for large-scale promotion. Currently, such in vitro diagnostic tests have not yet been developed. Since there are some differences in the extracellular receptors of Siglec-5 and Siglec-14, specific antibodies can be used for identification. However, the antibody incubation time is long, the interference is large, and it is easy to cause non-specific binding. In addition, intact cells need to be separated for detection, which is difficult to operate and is not conducive to medical diagnosis and large-scale promotion. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for rapid detection of GBS-related susceptibility genes Siglec14 / 5 and their fusion mutations, which is rapid, sensitive, low-cost and easy to promote.

[0007] Based on the above problems, the technical solution proposed in the present invention is to provide a method for rapidly detecting GBS-related susceptibility genes Siglec14 / 5 and their fusion mutations.

[0008] Three sets of primers and probes were added to the same tube, and two rounds of PCR and qPCR reactions were performed consecutively. The primers included the first-round Siglec14 / 5 fusion mutation gene PCR amplification primers, the second-round qPCR detection primers and probes targeting the fusion mutation gene, and the qPCR detection primers and probes targeting the non-coding region sequence between the wild-type normal Siglec14 and Siglec5 genes.

[0009] Among them, the Tm value of the first-round PCT primer is higher than the Tm value of the second-round qPCR primer. Both rounds of reactions undergo high-temperature denaturation and annealing extension processes, and the annealing temperature of the first-round PCR is higher than the annealing temperature of the second round.

[0010] In the second round of qPCR, the wild type, deletion mutant type, and heterozygous type of the Siglec gene were effectively distinguished at one time using the difference in fluorescence signals.

[0011] Among them, the primers and probes used for Siglec fusion gene detection can be selected from the primers and probes shown in Table 1 or Table 2. The specific sequences of each primer and probe are shown in Table 1 or Table 2:

[0012] Table 1 Primers and probes used for Siglec fusion gene detection

[0013]

[0014]

[0015] Table 2 Another set of primers and probes used for Siglec fusion gene detection

[0016]

[0017] The probe can be a common Taqman probe or an MGB probe with the same sequence, and the probe can be any two different labels among FAM, VIC, CY5, ROX, and SYTO-9 channels.

[0018] The specific reaction process is as follows Figure 2As shown, the upstream primer in the first round of PCR amplification targets the Siglec14 gene promoter region, and the downstream primer targets the downstream coding region of the Siglec5 gene. Only the fused gene was amplified, with an amplified length of 2672 bp. The unfused gene, however, had a length of 16933 bp between the two primers, which prevented normal amplification within the specified extension time. The downstream PCR primer consists of three parts: the R portion is a specific primer for the Siglec-5 gene; the A portion is the probe targeting region for the second round of qPCR, which is an artificially added DNA sequence with no Siglec homology; and the O portion is the complementary sequence of the downstream primer used in the second round of qPCR detection, also an artificially added DNA sequence with no Siglec homology. The R portion of the primer binds to both the wild-type and Siglec-14 deletion mutant. However, because the unfused gene is much longer than the fused gene, only the 2672 bp fragment of the Siglec-14 deletion mutant can be amplified under the optimized annealing and extension conditions. The newly generated PCR products incorporate the artificially added sequences in 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 of qPCR, the primer probes used in the second round of qPCR cannot amplify during the first round of amplification. The downstream primers in the second round of qPCR are added during the initial system preparation, but they are only detected in the second round of qPCR after the first round of PCR has actually been performed and the annealing temperature has been lowered. During the second round of qPCR, the fluorescence signal collected is used to determine the Siglec genotype in the sample being tested.

[0019] The second round of qPCR amplification can amplify the sequence between wild-type Siglec-14 and Siglec-5. If there is a normal amplified fluorescent signal, it is judged that the wild-type gene exists; if the Siglec14 deletion mutation fusion gene is amplified normally, it is used as a template, the upstream primer recognizes the 3' end sequence of the Siglec14 / 5 deletion fusion gene, the downstream primer recognizes the artificially added sequence O region, and the probe recognizes the artificially added sequence A region for amplification. If there is a normal amplified fluorescent signal, it is judged that the mutant gene exists; if both wild-type and mutant gene signals exist, it is judged to be a heterozygous genotype.

[0020] The steps of the two-round PCR and qPCR reactions required for detecting the susceptibility gene Siglec14 / 5 are shown in Table 3-1:

[0021] Table 3-1 Reaction conditions

[0022]

[0023] The preferred steps for the two-round PCR and qPCR reactions required for detecting the susceptibility gene Siglec14 / 5 are shown in Table 3-2:

[0024] Table 3-2 Reaction conditions

[0025]

[0026] Another preferred procedure for the two-round PCR and qPCR reactions for detecting the susceptibility gene Siglec14 / 5 is shown in Table 3-3:

[0027] Table 3-3 qPCR reaction conditions

[0028]

[0029] In the second round of qPCR detection, the FAM fluorescently labeled probe Siglec-GAP-P targets the non-coding region sequence between the wild-type normal Siglec14 and Siglec5 genes to detect the wild-type gene, and the VIC fluorescently labeled probe Siglec-SP targets the O region of the fusion mutant gene to detect the mutant gene. The negative control with H2O as the template is used to control the entire detection process. The cells with a typical S-shaped amplification curve only in the FAM channel are wild-type, the cells with a typical S-shaped amplification curve only in the VIC channel are mutant, and the cells with a typical S-shaped amplification curve in both the FAM and VIC channels are heterozygous, such as Figure 3 shown.

[0030] The test samples relied upon by the above methods may be vaginal secretions, blood, amniotic fluid, urine, and feces.

[0031] Advantages and beneficial effects of the present invention:

[0032] The present invention provides a method for rapidly detecting the susceptible gene Siglec14 / 5 and its fusion mutation associated with GBS, namely, adding the first round of PCR amplification primers of the Siglec14 / 5 fusion mutation gene, the second round of qPCR detection primers and probes targeting the fusion mutation gene, and the qPCR detection primers and probes targeting the non-coding region sequence between the wild-type normal Siglec14 and Siglec5 genes to the same tube, and by adjusting the temperature changes of each reaction step of denaturation-annealing-extension, the two rounds of PCR and qPCR reactions are performed continuously without interfering with each other, and the wild type, deletion mutation type, and heterozygous type of the Siglec14 / 5 gene are effectively distinguished at one time by using the difference in fluorescence signals. Compared with the traditional PCR plus electrophoresis method, this application is faster and more convenient to operate. The detection process is closed throughout the tube, and it is not easy to generate aerosol pollution, which is easy to promote on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the wild-type and mutant gene sequences of Siglec-5 and Siglec-14.

[0034] Figure 2 This is a schematic diagram of the principle of the method for detecting Siglec fusion mutant genes.

[0035] Figure 3 Schematic diagram of the qPCR amplification curves for different Siglec genotypes.

[0036] Figure 4 This is the result of detecting Siglec genotype by conventional PCR in Example 1.

[0037] Figure 5 This is the amplification result of Siglec genotype detected by qPCR of some samples in Example 1.

[0038] Figure 6 This is the result of detecting Siglec genotype by conventional PCR in Example 2.

[0039] Figure 7 This is the amplification result of qPCR detection of Siglec genotype of some samples in Example 2. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are described in detail below in conjunction with specific embodiments.

[0041] Example 1 Siglec genotype detection of vaginal swab

[0042] Take 10 vaginal swab samples, number them from 1, 2, 3 to 10, and extract the sample genome. In the qPCR laboratory, prepare the qPCR reaction system according to Table 4 and set the qPCR reaction conditions according to Table 5. The enzyme used in this example is II Probe qPCR SuperMix (Transgen, AQ711-01) but is not limited to this enzyme. The optimal reaction system is selected according to the enzyme as shown in Table 4.

[0043] Table 4 Single qPCR reaction system (20 μl reaction system)

[0044]

[0045] Table 5 qPCR reaction conditions

[0046]

[0047] The FAM-labeled probe Siglec-GAP-P targets the noncoding region between the wild-type Siglec14 and Siglec5 genes and is used to detect the wild-type gene. The VIC-labeled probe Siglec-SP targets the O region of the fusion mutant gene and is used to detect the mutant gene. A negative control using H2O as a template serves as a quality control for the entire assay process. A typical S-shaped amplification curve only in the FAM channel indicates the wild-type, designated Wt / Wt; a typical S-shaped amplification curve only in the VIC channel indicates the mutant, designated Mt / Mt; and a typical S-shaped amplification curve in both the FAM and VIC channels indicates the heterozygous genotype, designated Wt / Mt.

[0048] At the same time, a conventional PCR was designed to detect the type of Siglec14 / 5 gene, and the enzyme selected was PCRSuperMix(-dye) (Transgen, AS111-02) is not limited to this enzyme. The optimal reaction system is selected according to the enzyme as shown in Table 7, the primer sequences are shown in Table 6, and the reaction conditions are shown in Table 8. The reaction is performed and compared with the results of qPCR.

[0049] Table 6 Primer sequences for conventional PCR

[0050]

[0051] Table 7 Common PCR reaction system (20 system)

[0052]

[0053] Table 8 Reaction conditions of common PCR

[0054]

[0055] In the ordinary PCR reaction, only a single band of 1622bp in length is amplified, which is the wild type, that is, Siglec14 and Siglec5 each have normal double copies, recorded as Wt / Wt; and a single band of 1790bp is amplified, which is the mutant type, that is, both copies of Siglec14 undergo homologous recombination with Siglec5 to form a Siglec14 / 5 fusion mutant gene, recorded as Mt / Mt; if double bands of 1622bp and 1790bp appear at the same time, it is a heterozygous type, recorded as Wt / Mt.

[0056] Table 9 Siglec typing results of samples by the method of the present invention

[0057]

[0058] Table 10 Siglec typing results of samples by conventional PCR-electrophoresis

[0059]

[0060] The results of conventional PCR tests are shown in Table 10 and Figure 4 As shown in Table 9, the PCR+qPCR test results of this application are shown in Table 9 and Figure 5 As shown in Figure 2, the results of the two tests in this application are 100% consistent. qPCR is faster and more convenient to operate than traditional PCR electrophoresis, and the entire detection process is closed-tube, which is not prone to aerosol contamination and is very easy to promote on a large scale.

[0061] Example 2 Siglec genotype detection in EDTA anticoagulated whole blood samples

[0062] Five EDTA-anticoagulated whole blood samples were collected and numbered sequentially from X1, X2, X3 to X6, and the sample genomes were extracted.

[0063] The enzyme used in this example is Platinum II Hot-Start PCR Master Mix (2X) (Thermo, 14000012), but is not limited to this enzyme. The optimal reaction system is selected according to the enzyme as shown in Table 11, and the qPCR reaction conditions are set according to Table 12.

[0064] Table 11 Single qPCR reaction system (20 volume reaction system)

[0065] name Sample volume (μl) Siglec-GAP-F-2 0.8 Siglec-GAP-R-2 0.8 Siglec-LF-2 0.8 Siglec-LR-2 0.8 Siglec-SF-2 0.8 Siglec-SR-2 0.8 Siglec-SP-2 0.2 Siglec-GAP-P-2 0.2 Platinum II Hot-Start PCR Master Mix(2X) 10 Sample genome template 2 <![CDATA[H2O]]> 2.8

[0066] Table 12 qPCR reaction conditions

[0067]

[0068] At the same time, a conventional PCR was designed to detect the type of Siglec14 / 5 gene, and the enzyme selected was PCRSuperMix(-dye) (Transgen, AS111-02) is not limited to this enzyme. The optimal reaction system is selected based on the enzyme as shown in Table 14, the primer sequences are shown in Table 13, and the reaction conditions are shown in Table 15. The reaction results are compared with the qPCR results. If the amplified fluorescent signal is only in the FAM channel, it is wild type, recorded as Wt / Wt. If the amplified fluorescent signal is only in the VIC channel, it is mutant type, recorded as Mt / Mt. If both signals are present, it is heterozygous, recorded as Wt / Mt.

[0069] Table 13 Primer sequences for conventional PCR

[0070]

[0071] Table 14 Common PCR reaction system (20 μl system)

[0072]

[0073] Table 15 Reaction conditions of common PCR

[0074]

[0075] The results of conventional PCR tests are shown in Table 16 and Figure 6 As shown in Table 16 and Table 17, the PCR+qPCR test results of this application are shown in Table 16 and Table 17. Figure 7 As shown, the consistency of the results of the two tests in this application is 100%.

[0076] Table 16 Comparison of Siglec typing results of samples by conventional PCR and qPCR methods

[0077]

[0078] The above describes several embodiments of the invention in detail. However, the contents described are only preferred embodiments of the invention and should not be considered to limit the scope of the invention. All equivalent changes and improvements made within the scope of the invention should still fall within the scope of the patent.

Claims

1. A method for rapid detection of GBS-associated susceptibility genes Siglec14 / 5 and their fusion mutations for non-disease diagnosis and treatment purposes, characterized by: Three sets of primers and probes were added to the same tube, and two rounds of PCR and qPCR reactions were performed consecutively, wherein the primers included PCR amplification primers for the Siglec14 / 5 fusion mutation gene in the first round, qPCR detection primers and probes targeting the fusion mutation gene in the second round, and qPCR detection primers and probes targeting the non-coding region sequence between the wild-type normal Siglec14 and Siglec5 genes; The specific sequences of the primers for PCR amplification of the Siglec14 / 5 fusion mutant gene in the first round are as follows: Siglec-LF-1: 5'-CAGCCCAGCTCTGTGGTTCTTCTCACCCTT-3'; The Siglec-LR-1 sequence is composed of the O region, A region, and R region in series from the 5' end to the 3' end. The specific sequence is as follows: R region: 5'-AGGTCAGGAGTTCAAGATCAGCTTGGCCGACAT-3'; O region: 5′-GAGTCTAGAATCGATCAGTAACTCAGCT-3′; A region: 5'-CTCCACCACTCTACTCATGCCACCCCTACC-3'; The specific sequences of the second round of qPCR detection primers and probes targeting the fusion mutation gene are as follows: Siglec-SF-1: 5'-GGATTACAGGTGCACATCATCACGCCTGGCTA-3'; Siglec-SR-1: 5'-GAGTCTAGAATCGATCAGTAACTCAGC-3'; Siglec-SP-1: 5'-fluorescent reporter-CCACCACTCTACTCATGCCACCCCTAC -BHQ1-3'; The specific sequences of the second round of qPCR detection primers and probes targeting the non-coding region between the wild-type normal Siglec14 and Siglec5 genes are as follows: Siglec-GAP-F-1: 5'-ACTTGATCTGTGGCCTGTATACAAT-3'; Siglec-GAP-R-1: 5'-CAGGTATGAGCCACCGCACCC-3'; Siglec-GAP-P-1: 5'-fluorescent reporter gene - CACCCCTCACTCCCACTG- MGB-3'; or, The specific sequences of the primers for PCR amplification of the Siglec14 / 5 fusion mutant gene in the first round are as follows: Siglec-LF-2: 5'-CTAAGGACCACTCCATGCCCCTCTCATCTCAGTC A-3’； The Siglec-LR-2 sequence is composed of the O region, A region, and R region in series from the 5' end to the 3' end. The specific sequence is as follows: R region: 5′-AGAGAGGCCAAGAGATATAAAAGCGCACCAA-3′; O region: 5'-CTAGCTTAGGCCATAGCAGAATCACGTGCA-3'; Region A: 5′-ATCGCTGCAGTCGATTGGACCATGCAA-3′; The specific sequences of the second round of qPCR detection primers and probes targeting the fusion mutation gene are as follows: Siglec-SF-2: 5'-GTATGAGCAACATAGCAAGACTCTCGCC-3'; Siglec-SR-2: 5'-GCACGTGATTCTGCTATGGCCTAAG-3'; Siglec-SP-2: 5'-fluorescent reporter gene-TCGCTGCAGTCGATTGGACCATGCA -BHQ1-3'; The specific sequences of the second round of qPCR detection primers and probes targeting the non-coding region between the wild-type normal Siglec14 and Siglec5 genes are as follows: Siglec-GAP-F-2: 5'-CTTCTTGGCTATGATGCATAACACT-3'; Siglec-GAP-R-2: 5'-ACTGAATTTCCATCATATAGAACCC-3'; Siglec-GAP-P-2: 5'-fluorescent reporter-CTATGACCATTCCTGCCATGGTTTT GT-MGB-3'; The probe can be a common Taqman probe or an MGB probe with the same sequence; the fluorescent reporter group in the probe can be any two different labels from FAM, VIC, CY5, ROX, and SYTO-9 channels; Among them, the Tm value of the first-round PCR primer is higher than the Tm value of the second-round qPCR primer. The two-round PCR reactions are subjected to high-temperature denaturation and annealing extension processes respectively, and the annealing temperature of the first-round PCR is higher than the annealing temperature of the second-round qPCR. In the second round of qPCR, the wild type, mutant type, and heterozygous type of the Siglec gene were effectively distinguished at one time using the difference in fluorescence signals; When there is a typical S-shaped amplification curve in the channel of the fluorescent reporter group carried by the second-round targeted fusion mutant gene probe, it is a mutant type; when there is a typical S-shaped amplification curve in the channel of the fluorescent reporter group carried by the second-round targeted wild-type normal Siglec14 and Siglec5 gene probe, it is a wild type; when there are S-shaped amplification curves in both channels, it is a heterozygous type.

2. The method for rapid detection of GBS-related susceptibility genes Siglec14 / 5 and their fusion mutations according to claim 1, characterized in that: The steps for the two rounds of PCR and qPCR reactions are as follows: 1) Pre-denaturation at 90-98°C for 2-10 min; 2) Start the first round of PCR amplification reaction and denature at 90-98°C for 5-30 seconds; 3) Anneal and extend at 72-65°C for 60-200 seconds. The first round of PCR reaction is cycled 30-45 times. 4) Start the second round of qPCR detection reaction at 90-98°C for 5-30 seconds; 5) Anneal and extend at 55-60°C for 30-60 seconds. The second round of qPCR reaction is cycled 30-45 times, and the signals are collected during the annealing and extension process.

3. The method for rapid detection of GBS-related susceptibility genes Siglec14 / 5 and their fusion mutations according to claim 2, characterized in that: The steps for the two rounds of PCR and qPCR reactions are as follows: 1) Pre-denaturation at 94°C for 2 min; 2) Start the first round of PCR amplification reaction and denature at 94°C for 5 seconds; 3) Annealing and extension at 72°C for 120 seconds, and the first round of PCR reaction was cycled 30 times; 4) Start the second round of qPCR detection reaction and denature at 94°C for 5 seconds; 5) Anneal and extend at 60°C for 30 seconds. The second round of qPCR reaction was cycled 45 times, and the signals were collected during the annealing and extension process.

4. The method for rapid detection of GBS-associated susceptibility genes Siglec14 / 5 and their fusion mutations according to claim 2, characterized in that: The steps for the two rounds of PCR and qPCR reactions are as follows: 1) Pre-denaturation at 98°C for 2 min; 2) Start the first round of PCR amplification reaction and denature at 98°C for 15 seconds; 3) Annealing and extension at 68°C for 60 seconds, and the first round of PCR reaction was cycled 30 times; 4) Start the second round of qPCR detection reaction and denature at 98°C for 5 seconds; 5) Anneal and extend at 60°C for 30 seconds. The second round of qPCR reaction was cycled 45 times, and the signals were collected during the annealing and extension process.

5. The method for rapid detection of GBS-related susceptibility genes Siglec14 / 5 and fusion mutations thereof according to claim 1, characterized in that: The test samples can be vaginal secretions, blood, amniotic fluid, urine, and feces.

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