Primer probe composition and kit for group A streptococcus species identification and M1UK and M1global subtype typing and application of primer probe composition and kit

Abstract

The invention discloses a primer probe composition and a kit for group A streptococcus species identification and M1UK and M1global subtype typing and application of the primer probe composition and the kit. According to the primer probe composition, quadruple target amplification primers and probes are designed, so that specific amplification can be achieved in a single tube, and cross interference is avoided; the primer probe group forms a species-type-double SNP subtype three-stage detection system and is used for preparing detection kits for GAS species identification, em1 type confirmation, M1UK / M1global subtype typing and the like, interference of other em1 derived sublines is eliminated according to the double SNP co-occurrence requirement, and the M1UK typing accuracy is remarkably improved. The kit has the advantages of high specificity, high throughput, simplicity and convenience in operation and low cost, and is suitable for clinical diagnosis, high-virulence clone screening and public health emergency monitoring.

Description

Technical Field

[0001] This invention relates to the field of detection kit technology, specifically to a kit for identifying Group A Streptococcus species and M1. UK With M1 global Primer-probe compositions, kits, and applications for subtype typing. Background Technology

[0002] Group A Streptococcus ( Group A Streptococcus GAS), also known as Streptococcus pyogenes (GAS). Streptococcus pyogenes Streptococcal toxic shock syndrome (STSS) is an important Gram-positive human pathogen that can cause a wide range of diseases, from pharyngitis and skin infections to necrotizing fasciitis and streptococcal toxic shock syndrome (STSS). It can also induce acute rheumatic fever and rheumatic heart disease, constituting a significant global public health burden. In recent years, invasive GAS (iGAS) infections have rebounded significantly in many countries worldwide, including highly virulent ones. emm Type 1 lineage is dominant.

[0003] exist emm Inside the Type 1, with M1 UK (A new type of highly toxic) emm 1. Derivative lineage, also referred to in some studies as emm Emerging subtypes, represented by M1 (1.76), have attracted widespread attention due to their significantly enhanced toxicity phenotype. UK Carrying a set of characteristic single nucleotide polymorphisms (SNPs) can lead to upregulated expression of the toxin SpeA, resulting in faster clinical progression and higher mortality. However, M1 UK It does not exist in isolation, but is related to M1 13SNPs M1 19SNPs M1 23SNPs Multiple derivative subtypes together form a complex genetic network. These subtypes exhibit partially shared mutations at key regulatory sites: for example, those associated with SpeA overexpression. ssrA SNPs in the 5′ untranslated region of the gene are also present in M1. 23SNPs ;and pstB The missense mutation H123N in the gene is mainly limited to M1 in the currently available global GAS genome database. UK The pedigree (as of 2024) is well-documented, but its association with the core virulence phenotype is weak. This pattern of genetic overlap dictates that no single SNP can serve as a representative of M1. UK Its exclusive logo, relying solely on pstB or ssrA Typing at any single site can easily lead to the identification of other sites. emm 1. Derivative subtypes or non- emm Type 1 strain misidentified as M1 global .

[0004] Current molecular typing methods struggle to meet the combined demands of accuracy, speed, accessibility, and low cost in clinical and public health settings. While whole-genome sequencing (WGS) is comprehensive, its high cost and long turnaround time make it unsuitable for routine screening. Sanger sequencing or multiplex PCR are cumbersome, have low throughput, and require open-tube electrophoresis, increasing the risk of contamination. Although real-time quantitative PCR (qPCR) offers advantages such as speed, closed operation, and automation, making it the mainstream platform for pathogen detection, existing qPCR protocols still suffer from the following shortcomings: (1) Lack of species and phylogenetic verification: Most methods directly detect SNP sites in unknown samples without simultaneously confirming the species and phylogenetic of GAS. emm Type 1 background leads to non- emm Type 1 GAS (such as emm 12. emm (89, etc.) was misclassified as M1 because it naturally exhibits a wild-type appearance. global ; (2) Determination based on a single site: If limited to emm 1. Background, detection only pstB or ssrA No single SNP can rule out other shared partial mutations. emm 1. Derived subtypes lead to unreliable typing results; (3) Cannot achieve three-level discrimination with a single tube: Existing products usually require step-by-step detection (first species, then type, and finally subtype), which is lengthy and difficult to be embedded in high-throughput clinical testing processes.

[0005] In summary, existing technologies either rely on expensive and time-consuming sequencing methods or employ single-target qPCR methods lacking species and type validation, failing to achieve high specificity while simultaneously enabling M1 sequencing. UK With M1 global Precise differentiation is crucial. Crucially, no known molecular diagnostic product can simultaneously identify Group A streptococci in a single-tube closed reaction. emm1 Type confirmation and SNP based on dual features ( ssrA and pstB M1 of co-occurrence logic UK Subtype discrimination. Therefore, developing an integrated, highly specific, easy-to-operate, and cost-effective multiplex qPCR detection system has become crucial for the rapid clinical identification of highly virulent M1. UK There is an urgent need for cloning and guidance for precise prevention and control. Summary of the Invention

[0006] This invention provides a primer-probe composition based on quadruple target synergistic interpretation to achieve simultaneous identification of GAS species in a single-tube closed reaction. emm1 Type confirmation and M1 UK / M1global Precise subtyping of subtypes is used to meet clinical testing needs.

[0007] In view of this, the solution of the present invention is as follows: A first aspect of the present invention is to provide a primer-probe composition comprising a primer for detecting... speB The first primer and probe set for the gene is used for detection. emm Primer-probe set two with type 1 specific sequence, used for detection pstB and ssrA Primer and probe set three for gene SNP sites; The primer-probe set includes primers with sequences as shown in SEQ ID NO. 1-2, and probes as shown in SEQ ID NO. 3; The primer-probe set two includes primers with sequences as shown in SEQ ID NO.4-5, and probes as shown in SEQ ID NO.6; The primer-probe set three is selected from any one of the following: a) Primers with nucleotide sequences as shown in SEQ ID NO.7-8, 10-11, and probes as shown in SEQ ID NO.9, 12; b) Primers with nucleotide sequences as shown in SEQ ID NO. 13-14, 16-17, and probes as shown in SEQ ID NO. 15, 18, wherein the probes are modified with LNA; c) Primers with nucleotide sequences as shown in SEQ ID NO.19-20, 22-23, and probes as shown in SEQ ID NO.21, 24, wherein the probes are modified with MGB.

[0008] Furthermore, each probe in the primer-probe composition is labeled with a fluorescent group, and different probes are labeled with different fluorescent groups.

[0009] Preferably, each probe is labeled with a different fluorescent reporter group (selected from FAM, VIC, ROX, Cy5) at its 5′ end and a quencher group (selected from BHQ1, BHQ2 or MGB-NFQ) at its 3′ end to achieve four-channel signal separation.

[0010] Furthermore, the LNA modification method of the probe shown in SEQ ID NO.15 is: modification of the 8th, 10th, 12th and 14th bases starting from the 5' end; and / or, the LNA modification method of the probe shown in SEQ ID NO.18 is: modification of the 8th, 11th and 15th bases starting from the 5' end; and / or, the 3' end of the probes shown in SEQ ID NO.21 and 24 is modified with NFQ-MGB.

[0011] A second aspect of the invention is to provide an application of the primer-probe composition described in the first aspect, said application being the preparation of a detection product for at least one of the following uses: 1) Used for identification of Group A Streptococcus species; 2) Used for emm 1. Type identification; 3) Used for M1 UK Subtype identification; 4) Used for M1 global Subtype identification.

[0012] Furthermore, the detection product is a reagent kit, chip, system, or device.

[0013] A third aspect of the present invention is to provide a detection kit comprising the primer-probe composition described in the first aspect.

[0014] Furthermore, the detection kit also includes Taq DNA polymerase, dNTPs, and Mg. At least one of UNG enzyme and PCR buffer.

[0015] Furthermore, the test kit also includes nucleic acid extraction and / or purification reagents.

[0016] A fourth aspect of this invention is to provide a method for identifying Group A Streptococcus species and M1. UK With M1 global Subtype typing methods, for non-diagnostic purposes, include: 1) Nucleic acid extraction is performed on the sample; 2) The extracted nucleic acid was amplified using the detection kit described in the third aspect, and the amplification signal was collected; 3) Based on speB Gene, emm Under the premise of a positive type 1 specific sequence, according to ssrA and pstB The subtype is determined by the amplification signal of the gene SNP site.

[0017] Furthermore, in the above detection method, the sample is selected from pharyngeal swabs, blood, cerebrospinal fluid, pus, or culture.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a primer-probe set that, through the design of quadruple-target amplification primers and probes, enables specific amplification within a single tube without cross-interference. This primer-probe set forms a three-level detection system of "species-type-double SNP subtype," used for the preparation of GAS species identification, emm1 Type confirmation and M1 UK / M1 globalSubtype typing and other detection kits improve specificity, sensitivity and accuracy.

[0019] The detection method described in this invention is based on a primer-probe set or kit for quadruple target amplification, logically blocking non-target amplification. emm1 GAS was misclassified as M1 global The risk is mitigated by excluding other SNPs through the co-occurrence requirement of two SNPs. emm1 Derivative subsystem interference significantly improves M1 UK It offers high typing accuracy and combines the advantages of high specificity, high throughput, ease of operation, and low cost, making it suitable for clinical diagnosis, screening of highly virulent clones, and public health emergency surveillance. Attached Figure Description

[0020] Figure 1 The present invention is used for the identification of Group A Streptococcus species and M1. UK / M1 global Flowchart of three-level logical interpretation for subtype classification.

[0021] Figure 2 This is the amplification curve of the ARMS method quadruple real-time PCR in Example 1.

[0022] Figure 3 This is the amplification curve of the LNA probe method in Example 1, using quadruple real-time PCR.

[0023] Figure 4 This is the amplification curve of the quadruple real-time PCR using the MGB probe method in Example 1.

[0024] Figure 5 The amplification curve (a) and linear fitting graph (b) are the amplification efficiency verification experiments of the kit (ARMS method) described in Example 2.

[0025] Figure 6 This is a quality control amplification curve of the kit (ARMS method) described in Example 2.

[0026] Figure 7 The results are for sensitivity determination of the kit (ARMS method) described in Example 3.

[0027] Figure 8 The results are from the accelerated stability test of the kit (ARMS method) described in Example 4. Detailed Implementation

[0028] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] In the first embodiment of the present invention, a method for identifying Group A Streptococcus species and M1 is provided. UK With M1 global A combination of molecular marker sites for subtyping, comprising the following four genomic regions: (a) speB Group A Streptococcus species-specific conserved sequences in the gene are used to confirm whether a sample is GAS positive. (b) emm Genes with emm1 Type-specific sequences are used to further confirm whether a patient is positive for GAS. emm1 type; (c) ssrA A single nucleotide polymorphism (SNP) site in the 5′ untranslated region of the gene, located in M1. UK The subtype contains mutant bases, in M1 global The subtype contains wild-type bases; (d) pstB A missense mutation site in the coding region of the gene that results in a histidine → asparagine substitution (H123N), this site is located in M1 UK The subtype contains mutant bases, in M1 global The subtype contains wild-type bases.

[0030] Wherein, only if the sample simultaneously satisfies: (i) speB Positive, (ii) emm1 Positive, (iii) ssrA SNP and pstB Only when all SNPs are mutant can it be determined to be M1. UK Subtype; only if the sample simultaneously satisfies: (i) speB Positive, (ii) emm1 Positive, (iii) ssrA SNP and pstB Only when all SNPs are wild-type can it be identified as M1. global Subtype; all other cases (including any SNP being wild-type) are not considered M1. UK Or M1 global This effectively avoids non- emm1 Background misjudgment and the risk of false positives from single SNPs.

[0031] Based on the detection results of the above four sites, the following hierarchical interpretation rules are adopted: Figure 1 As shown: (1) If the sample speB If negative, it is determined to be non-group A streptococcus; (2) If the sample speB Positive but emm1 If negative, it is considered non-negative. emm1 Type GAS; (3) If the sample speB Positive and emm1 If positive, further typing is performed based on SNP status: (a) when ssrA SNP and pstB When all SNPs are mutant, it is determined to be M1. UK Subtype; (b) when ssrA SNP and pstB When all SNPs are wild-type, it is classified as M1. global Subtype; (c) when ssrA and pstB When the SNP states are inconsistent (i.e., one is mutant and the other is wild-type), it is classified as other. emm1 Derivative subtype, not belonging to M1 UK Or M1 global .

[0032] The above-mentioned hierarchical interpretation mechanism can effectively avoid non-standard interpretations. emm1 Background misjudgment and the risk of false positives from a single SNP ensure M1 UK The specificity and reliability of the identification.

[0033] In the second embodiment, a method for identifying Group A Streptococcus species and M1 is provided. UK With M1 global A primer-probe composition for subtyping, the composition comprising four sets of specific primers and fluorescent probes, respectively targeting the four regions (a)–(d) above; wherein, for the two SNP sites (c) and (d), a variety of allele-specific recognition strategies can be adopted, including: (i) designing mismatched ARMS primers at the 3′ end of the primers; or (ii) using chemically modified allele-specific probes such as LNA and MGB.

[0034] Experimental evaluations have shown that all the above strategies can effectively distinguish between wild-type and mutant alleles. Specifically, the ARMS primer protocol has undergone complete quadruple qPCR performance validation (including sensitivity, specificity, stability, and clinical sample testing); the LNA and MGB probe protocols have been optimized under reaction conditions (such as Mg2+). After adjusting the concentration, annealing temperature, PCR buffer concentration, etc., it was also preliminarily confirmed that it has quadruple amplification compatibility and can be used as an alternative technical route.

[0035] The nucleotide sequences of the four sets of specific primers and fluorescent probes have been functionally verified and are numbered sequentially as SEQ ID NO.1 to SEQ ID NO.24, as shown in Table 1.

[0036] Table 1:

[0037] In the sequences shown in Table 1, the bases after "+" indicate locked nucleic acid (LNA) modified bases, such as "+T" indicating locked nucleic acid modification of base T; the underlined bases represent labeled SNP sites.

[0038] It should be noted that when there is a discrepancy between the provided sequence list and the sequence shown in Table 1, the sequence in Table 1 shall prevail.

[0039] The functional assignments of each sequence are as follows: SEQ ID NO.1~3: speB Forward primers, reverse primers, and VIC-labeled probes; SEQ ID NO.4~6: emm1 Forward primers, reverse primers, ROX-labeled probes; against ssrA SNP sites and pstB For SNP sites, this invention provides three optional allele-specific detection combinations: (1) Combinations of ARMS primers (SEQ ID NO. 7~9, 10~12), including ssrA , pstB ARMS forward primers, reverse primers, and probes; (2) Combinations of LNA-modified probes (SEQ ID NO.13~15, 16~18), including ssrA , pstB Conventional primer pairs and LNA chemically modified probes that cross SNP sites; (3) Combinations of MGB-modified probes (SEQ ID NO.19~21, 22~24), including ssrA , pstB Conventional primer pairs and short, highly specific probes that cross SNP sites and have MGB-NFQ linked at the 3′ end.

[0040] Preferably, this invention employs the ARMS primer scheme to construct a quadruple fluorescent PCR system, which is low in cost, simple in design, and provides balanced amplification efficiency and stable signal in multiplex reactions. All performance data, including sensitivity, stability, specificity, and clinical validation data, described in this invention are obtained based on the ARMS primer scheme.

[0041] Each probe is labeled with a different fluorescent reporter group (selected from FAM, VIC, ROX, Cy5) at its 5′ end and a quencher group (selected from BHQ1, BHQ2 or MGB-NFQ) at its 3′ end to achieve four-channel signal separation.

[0042] In the third embodiment, a method for identifying Group A Streptococcus species and M1 is provided. UK With M1 global A subtype genotyping detection kit, the kit comprising the primer-probe composition.

[0043] Furthermore, the kit is in ready-to-use premix form, containing an optimized concentration of primer and probe composition, hot-start Taq DNA polymerase, dNTPs, and Mg. UNG enzyme and PCR buffer; through systematic concentration gradient experiments, ensure that the amplification efficiency of each target in the quadruple amplification reaction is balanced (amplification efficiency 90–110%, R² > 0.99), and there is no significant inhibition or fluorescence crosstalk.

[0044] The detection sensitivity of the kit was determined by gradient dilution experiments, and the limit of detection (LoD) was 500 copies / mL (95% positive detection rate).

[0045] In terms of stability, the kit was verified by accelerated stability test at 37°C. It can be stably stored for more than 14 days under accelerated conditions without significant performance degradation (Ct value shift ≤1.0).

[0046] In addition, this kit is effective against non-group A streptococci (such as group B streptococci and Staphylococcus aureus) and other common streptococci. emm Type GAS (such as) emm12 , emm89 There was no cross-reactivity, and the intra-batch and inter-batch repeatability was good (Ct value coefficient of variation CV < 5%). It is suitable for various clinical sample types such as throat swabs, blood, cerebrospinal fluid, pus and bacterial cultures.

[0047] In the fourth embodiment, the application of the molecular marker site combination, primer-probe composition, or detection kit in the clinical diagnosis of group A streptococcal infection is provided.

[0048] In the fifth embodiment, its application in highly toxic M1 is provided. UK Applications in clonal active screening, tracing the source of invasive GAS (iGAS) outbreaks, hospital infection control, and the construction of a national pathogen surveillance network.

[0049] The specific operational procedures for the above applications include: extracting DNA from the sample to be tested (pharyngeal swab, blood, cerebrospinal fluid, pus, or culture), adding it to the premixed reagent kit, and running a single-tube quadruple amplification program in a standard real-time fluorescence PCR instrument; and performing hierarchical logical interpretation based on the four-channel fluorescence signals. First layer: speB Positive result → Confirmed as GAS; Second layer: speB and emm1 Double positive → Confirmed emm1 Type GAS; Third layer: In emm1 On the basis of a positive result, ssrA and pstB Double mutation → identified as M1 UK Otherwise, it is M1. global or others emm1 Derivative subtypes.

[0050] This closed-loop interpretation mechanism fundamentally eliminates the systematic misjudgment caused by "skipping species / type verification and directly typing".

[0051] Three types of quality control are set up simultaneously during the testing process: 1) Positive control: derived from M1 confirmed by whole-genome sequencing. UK Genomic DNA of clinical isolates of group A streptococci, during detection speB , emm1 , ssrA SNP and pstB All four targets of the SNP generated specific amplification signals; 2) Negative control: derived from M1 confirmed by whole-genome sequencing. global Genomic DNA of Group A Streptococcus strains, ssrA and pstB All loci were wild-type. The primer-probe combination used in this invention was designed to target mutant alleles; therefore, in this control, ssrA and pstB The target should not generate an amplification signal (i.e., the amplification curve shows no exponential growth or Ct > 40). This control is used to verify the specificity of the detection system for mutant sequences, i.e., wild-type templates will not be amplified and will not generate mutant signals; 3) Blank control: Water without nuclease, and all four targets should show no amplification signal.

[0052] It should be noted that the interpretation logic of this invention does not rely on ssrA or pstB The channel does not directly detect the "wild type" signal, but rather makes a judgment based on the presence or absence of the "mutant signal".

[0053] Example 1: Primer and probe design and construction of a quadruple qPCR system

[0054] Provided for identification of Group A Streptococcus species and M1 UK With M1 global The subtype-specific primer-probe composition contains four sets of specific primers and fluorescent probes, each targeting a specific subtype. speB , emm1 , ssrA SNPs and pstB The four SNP sites and four molecular marker sites were identified through whole-genome alignment and evolutionary analysis, and their genomic coordinates and reference sequence information are shown in Table 2. The nucleotide sequences of all primers and probes have been determined and assigned SEQ ID NO.1 to SEQ ID NO.24 (Table 1 above). For ease of reference, their functional classification and fluorescent labeling information are summarized in Table 3.

[0055] Table 2: Genomic coordinates and reference sequence information of four molecular marker sites

[0056] Table 3: Fluorescent labeling using three sets of primers and probes

[0057] Based on the above three primer-probe combinations, the following three methods can be used for detection: (a) ARMS-PCR method This method achieves specific amplification of mutant alleles by introducing a mismatch at the 3′ end or other position of one primer, while the other primer and probe are both located in a conserved sequence shared by wild type and mutant.

[0058] ① ssrA SNP combination: ARMS forward primer (SEQ ID NO.7) + conventional reverse primer (SEQ ID NO.8) + Cy5 labeled probe (SEQ ID NO.9); ② pstB SNP combination: ARMS forward primer (SEQ ID NO.10) + conventional reverse primer (SEQ ID NO.11) + FAM labeled probe (SEQ ID NO.12).

[0059] The final concentrations of primers and probes were determined through system optimization as follows: speB : 0.3 μM each of forward and reverse primers, 0.2 μM of VIC-labeled probe; emm1 : 0.3 μM each of forward and reverse primers, 0.15 μM of ROX-labeled probe; ssrASNP: 0.4 μM each of ARMS forward and reverse primers, and 0.15 μM of Cy5-labeled probe; pstB SNP: 0.4 μM each of ARMS forward and reverse primers, and 0.2 μM of FAM-labeled probe.

[0060] The PCR reaction system includes: the four sets of primers and probes mentioned above, 5 μL of template DNA, and hot-start Taq DNA polymerase, dNTPs, and Mg2+. UNG enzyme and PCR buffer. The amplification program was as follows: 95°C for 2 min (pre-denaturation), followed by 45 cycles (95°C for 5 s, 55°C for 30 s).

[0061] like Figure 2 As shown, in the single-tube reaction, all four targets exhibited typical S-shaped amplification curves, with good fluorescence signal separation and no cross-interference, indicating that the system has excellent multiplex compatibility. All performance data, including amplification efficiency, detection sensitivity, acceleration stability, cross-reaction verification, and clinical sample testing of the primer-probe set, were obtained based on this ARMS-PCR protocol.

[0062] (ii) LNA-modified probe method

[0063] As a second feasible technical approach for allele-specific identification, this invention also uses conventional primers to amplify all alleles, and LNA-modified probes cross SNP sites to achieve genotyping. LNA (Locked Nucleic Acid) modifies the nucleotides located at or near the corresponding SNP site in the probe, enhancing hybridization stability and mismatch identification ability through locked sugar rings.

[0064] ssrA SNP combination: conventional forward primer (SEQ ID NO.13) + conventional reverse primer (SEQ ID NO.14) + Cy5-labeled LNA probe (SEQ ID NO.15, A / T / C / G preceded by + indicates that the base is modified by LNA, underlined to mark SNP site); pstB SNP combination: conventional forward primer (SEQ ID NO.16) + conventional reverse primer (SEQ ID NO.17) + FAM-labeled LNA probe (SEQ ID NO.18, where A / T / C / G are preceded by + to indicate that the bases are modified by LNA, and the underlined SNP site).

[0065] Singleton qPCR assays showed that this protocol was effective against M1. UK (Double mutation) and M1 globalThe (double wild) template has a ΔCt > 15, demonstrating reliable SNP genotyping capability. Furthermore, a preliminary quadruple compatibility test was conducted on the LNA scheme in a single-tube reaction: after employing conventional system optimization strategies, Mg... With the concentration adjusted to 2.75 mM and the PCR buffer increased to 1.25×, four targets could be simultaneously amplified in a single tube reaction, and the four fluorescence signals were clearly separated without crosstalk. Figure 3 This indicates that the scheme is feasible for use in the quadruple detection system of this invention.

[0066] (III) MGB-modified probe method

[0067] As a third feasible technical approach for allele-specific identification, this invention also uses conventional primers to amplify all alleles, and uses short-chain probes modified with MGB to cross SNP sites for genotyping. The modification method usually involves replacing the quenching group at the 3′ end of the probe with MGB-NFQ (Minor Groove Binder-Non-Fluorescent Quencher), which shortens the probe length from 18~32nt to 12~18nt without reducing the probe Tm value, thus significantly improving the probe's specificity for SNP site identification.

[0068] ssrA SNP combination: conventional forward primer (SEQ ID NO.19) + conventional reverse primer (SEQ ID NO.20) + Cy5-labeled MGB probe (SEQ ID NO.21, underlined SNP site); pstB SNP combination: conventional forward primer (SEQ ID NO.22) + conventional reverse primer (SEQ ID NO.23) + FAM-labeled MGB probe (SEQ ID NO.24, underlined SNP site).

[0069] Singleton qPCR assays showed that this protocol was effective against M1. UK (Double mutation) and M1 global The (double wild) template has a ΔCt > 8, indicating reliable SNP genotyping ability. Similarly, in Mg... When the concentration is adjusted to 3.25 mM, the MGB protocol can also achieve simultaneous amplification of four targets, with clear separation of the four fluorescence signals and no crosstalk (see amplification curves). Figure 4 This confirms that the MGB-modified probe method can be used as an implementation scheme for simultaneous amplification in a single-tube reaction, indicating that this scheme is feasible for the quadruple detection system of this invention.

[0070] Although the LNA and MGB schemes have not undergone the same full-scale performance verification as the ARMS scheme (such as LoD, acceleration stability, large-sample cross-reactivity, etc.), they are both mature technologies in the field, and this invention has provided complete sequences and preliminary multi-compatibility data. Those skilled in the art can use conventional methods (such as fine-tuning annealing temperature, Mg...) The concentrations of primers and probes, the composition of the PCR buffer, etc., were successfully integrated into the multiplex detection of this invention. Therefore, all three schemes mentioned above are considered equivalent embodiments of this invention and fall within the scope of protection of the claims. Additionally, while PNA (peptide nucleic acid) is theoretically feasible, its high synthesis cost and poor compatibility with conventional qPCR systems have prevented practical testing in this invention, and therefore it is not considered a primary implementation scheme; however, its potential application in specific scenarios is not excluded.

[0071] Example 2: Verification of amplification efficiency in a quadruple qPCR system

[0072] In Example 1, the method was screened and preliminarily confirmed through a quadruple qPCR system to be suitable for the identification of Group A Streptococcus species and M1. UK / M1 global Four sets of specific primers and probes for genotyping were used. Based on this, the present invention further optimized the quadruple qPCR system and developed a detection kit using primer-probe compositions (ARMS protocol, SEQ ID NO:1-12). The optimizations included final primer concentrations (0.1-0.8 μM gradient testing), probe concentrations (0.1-0.4 μM gradient), and Mg... Concentration (2.0–5.0 mM), annealing / extension temperature (55 / 57 / 59℃), and PCR buffer components and concentrations, etc.

[0073] Through orthogonal experiments and Ct value stability evaluation, the optimal reaction system was finally determined to be: 1×PCR MasterMix, 3.5mM Mg 0.4μM SpeB Forward primer, 0.4 μM SpeB Reverse primer, 0.2 μM SpeB Fluorescent probe (VIC), 0.4 μM emm1 Forward primer, 0.4 μM emm1 Reverse primer, 0.2 μM emm1 Fluorescent probe (ROX), 0.4μM ssrA ARMS forward primer, 0.4 μM ssrA Reverse primer, 0.2 μM ssrA Fluorescent probe (Cy5), 0.4 μM pstB ARMS forward primer, 0.4 μM pstB Reverse primer, 0.2 μM pstBFluorescent probe (FAM); the optimal annealing temperature is 55°C, which is the optimal amplification program of Example 1.

[0074] This embodiment evaluates the quadruple qPCR amplification performance of the kit (ARMS primer scheme, SEQ ID NO. 1–12) described in this invention through an amplification efficiency verification experiment, as detailed below: Quantitative M1 UK Genomic DNA was serially diluted to 1×10⁻⁶ using TE buffer. 8 copies / mL, 1×10 7 copies / mL, 1×10 6 copies / mL, 1×10 5 copies / mL, 1×10 4 Copy / mL, with a total of 5 levels. Quadruple fluorescent PCR was performed using the kit of this invention according to the instructions. The amplification procedure was the same as in Example 1, and the results are as follows: Figure 5 As shown. Figure 5 In Figure a, the amplification curve is shown, and in Figure b, the linear fitting curve is shown. The results meet the performance requirements of the MIQE guidelines for multiplex qPCR systems, indicating that the quadruple qPCR detection system of this invention is highly efficient and stable, and suitable for subsequent M1 assays. UK / M1 global Fractal determination.

[0075] Each time the kit of this invention is used for testing, the positive control (M1) provided with the kit must also be tested simultaneously. UK Type GAS genomic DNA, all four channels were positive), negative control (M1) global DNA from the GAS standard strain, only speB and emm1 The positive control (positive channel) and blank control (water without nuclease, no amplification signal) wells are used to confirm the validity of the experiment. The amplification curves of the three controls in this experiment are shown below. Figure 6 As shown. Figure 6 In the diagram, a represents the positive control (M1). UK Type GAS genomic DNA, all four channels were positive), b is the negative control (M1) global DNA from the GAS standard strain, only speB and emm1 (Channel positive), c is blank control (no nuclease water, no amplification signal).

[0076] The Ct values ​​at each detection level were statistically analyzed, and a standard curve of Ct value versus the logarithm of the initial template copy number (Lg(concentration)) was plotted. The amplification efficiency (E) and linear correlation coefficient (R²) of each target were calculated. The results are shown in Table 4.

[0077] Table 4: Statistics of Amplification Efficiency Validation Results

[0078] The results show that within the quadruple system, all targets ( SpeB , emm1 , ssrA and pstB The primer-probe combination and kit designed in this invention exhibited excellent linearity (R² ≥ 0.99) within the test range, and the calculated amplification efficiency (E) ranged from 90% to 110%, meeting the requirements of a high-efficiency multiplex qPCR system. These data confirm that the primer-probe combination and kit designed in this invention have good amplification efficiency and stability, and are suitable for accurate detection and genotyping within a wide dynamic range from low to high.

[0079] Example 3: Determination of Detection Sensitivity (LoD)

[0080] This embodiment describes the use of the reagent kit for M1. UK The limit of detection (LoD) for group A streptococci was determined using the following method: quantitative M1... UK Genomic DNA reference samples were serially diluted. The reference samples were diluted with TE buffer to a final concentration of 2 × 10⁻⁶. 3 80 reactions were performed using ARMS primer protocols (Tables 1 and 3, SEQ ID NO. 1–12) according to the manufacturer's instructions. The amplification procedure was the same as in Example 1. Figure 7 As shown, Figure 7 The data show the amplification results for template concentrations of 2000 copies / mL (a), 1000 copies / mL (b), 500 copies / mL (c), and 200 copies / mL (d), and the 95% limit of detection (LoD) was determined to be 500 copies / mL.

[0081] The positive detection rate at each concentration level was calculated, and the lowest concentration with a positive detection rate of 95% was taken as the LoD of the kit of the present invention. The results are shown in Table 5.

[0082] Table 5: Summary of Sensitivity (LoD) Measurement Results

[0083] The results showed that the kit using ARMS primers was effective against M1. UK The LoD of group A streptococci is 500 copies / mL, which meets the detection requirements of low-load clinical samples, demonstrating that the primer-probe composition and kit of this invention have excellent detection sensitivity.

[0084] Example 4: Accelerated Stability Test

[0085] This embodiment investigates the stability and shelf life of the kit using ARMS primers through accelerated degradation assays. Three independent production batches of the kit (batch numbers: IC251101, IC251102, IC251103) were stored in a 37°C incubator, and samples were taken for testing at four time points: 0 days, 7 days, 14 days, and 21 days.

[0086] Medium concentration (1×1) was used in each test. (copy / mL) and low concentration (2×10) 3 (Copies / mL) Two levels M1 UK Genomic DNA samples were tested in triplicate at each time point (4 time points), each reagent batch (3 reagent batches), and each sample (2 samples), for a total of 72 reactions. The detection method was the same as in Example 1, and the amplification results are as follows. Figure 8 As shown. Figure 8 a, 8b, 8c, and 8d respectively show that after 0, 7, 14, and 21 days of storage, the CV of both medium-concentration and low-concentration samples was less than 5%, and the Ct values ​​of the four targets did not change for more than one cycle.

[0087] The Ct values ​​of the four targets were recorded and compared with the baseline value at day 0. The Ct value offset and coefficient of variation (CV) were calculated. The results are shown in Table 6: all four targets were stably amplified at all time points, with the maximum Ct value offset not exceeding one cycle. The CV range for medium-concentration samples was 0.05%-0.96%, and the CV range for low-concentration samples was 0.19%-1.90%. No false negative or false positive results were observed. This indicates that the kit of the present invention can be stably stored at 37°C for at least 14 days without significant performance degradation, meeting the stability requirements for transportation and short-term room temperature storage. Based on the Arrhenius Equation, it is estimated that the long-term stability of the kit containing ARMS primers reaches 12 months when stored at -20°C, meeting the shelf-life stability requirements of in vitro diagnostic reagents.

[0088] Table 6: Results of Accelerated Stability Test at 37℃

[0089] Note: (i) Ct mean refers to the average Ct value of 3 replicates (single batch) or 9 replicates (three batches) at each time point; (ii) ΔCt is defined as the average Ct value of each time point minus the average Ct value of 0 days (offset); (iii) CV refers to the coefficient of variation of Ct value of 3 replicates (single batch) or 9 replicates (three batches) at each time point.

[0090] Example 5: Specificity and Clinical Sample Validation

[0091] To fully evaluate the specificity of this invention, this embodiment verifies it from two levels: (i) Using bioinformatics methods, the selected molecular marker site combinations ( speB , emm1 , ssrA SNP, pstB A systematic analysis of the distribution of SNPs in the global GAS genome was conducted to verify their theoretical specificity. (ii) Using a wet experimental method, a quadruple fluorescent PCR system constructed with the ARMS primer scheme (SEQ ID NO.1~12) described in Example 1 was used to detect a series of samples to evaluate the specificity and cross-reactivity of the primer probe composition and the detection kit.

[0092] (a) Bioinformatics analysis and verification

[0093] This invention compares and analyzes 2606 high-quality GAS whole genomes (from the NCBI RefSeq Genome Database) published up to November 2025, focusing on the target status of the following four types of strains: (i)M1 UK type( emm1 Type and ssrA and pstB Both SNP sites are mutant). (ii) M1 global type( emm1 Type and ssrA and pstB Both SNP sites are wild-type). (iii) Other emm1 Derivative subtypes (such as M1) 13SNPs M1 19SNPs M1 23SNPs wait); (iv) Non- emm1 Type GAS (including) emm12 , emm89 , emm6 , emm3 wait).

[0094] The analysis results are shown in Table 7: speB The gene is highly conserved in all GAS strains (target sequence identity >99%), but is missing or has a large number of variations in non-GAS streptococci (identity <81%), and cannot be effectively amplified and identified by the primers and probes of this invention. It can be used as a reliable marker for identifying GAS species. Targeted emm1 Specific sequences only exist in emm1 Among the GAS strains, other emm Type GAS strains and non-GAS strains have no homologous sequences; ssrA SNP: The mutation is present in M1 UK and some emm1 Derivative subtype (M1) 23SNPs In strain ), this suggests that the site is not M1. UK Unique, but this site is related to M1 UK It is closely related to the high toxicity phenotype and has important value for pedigree identification; pstB SNP: In the current database (as of November 2025), the mutation exists only in M1. UK In China, not yet in other countries emm1 Derivative subtypes or non- emm1 Detected in the strain.

[0095] Table 7: Distribution Statistics of Four Target Sites in the Global GAS WGS Database

[0096] Note: ¹Includes M1 13SNPs M1 19SNPs M1 23SNPs wait emm1 Derivative lineage; "Double SNP mutation" refers to simultaneously satisfying ssrA SNP mutations and pstB SNP mutations; Data source: NCBI RefSeq Genome Database (as of November 2025).

[0097] (ii) Clinical sample validation

[0098] To evaluate the detection efficacy of the kit (ARMS primer scheme, SEQ ID NO.1~12) in real clinical scenarios and to verify the kit's specificity and cross-reactivity, this embodiment tested and verified a total of 94 clinical samples collected and confirmed by common laboratory methods such as culture identification or whole genome sequencing (WGS). These samples included 42 oropharyngeal swabs, 22 nasopharyngeal swabs, 17 bronchoalveolar lavage fluid samples, 6 sputum samples, 3 pus samples, 2 vaginal secretions samples, 1 anal swab, and 1 pleural effusion sample.

[0099] (1) Test Sample

[0100] The GAS sample consisted of 72 cases, including 3 cases of M1UK type, 23 cases of M1global type, 38 cases of emm12 type, 5 cases of emm28 type, 1 case of emm22 type, 1 case of emm75 type, and 1 case of emm89 type. The 22 non-GAS samples included one case each of Streptococcus agalactiae, Streptococcus stomatologicus, Streptococcus pneumoniae, Streptococcus blood, Streptococcus pharyngitis, Streptococcus suis type II, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Bordetella pertussis, Bordetella bronchiseptica, Acinetobacter baumannii, Aspergillus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Legionella, Candida albicans, and Pneumocystis jirovecii.

[0101] Specific information is shown in Table 8.

[0102] Table 8: Test Sample Information Table

[0103]

[0104]

[0105] (2) Detection method

[0106] Genomic DNA from all test samples was extracted using a validated magnetic bead-based nucleic acid extraction kit. Detection was performed using the detection kit of this invention (containing the primers and probes shown in SEQ ID NO. 1-12): 20 μL of ready-to-use premixed PCR reaction solution was added to a PCR reaction tube, followed by 5 μL of nucleic acid sample. After thorough mixing and brief centrifugation, amplification was performed according to the procedure described in Example 1.

[0107] (3) Results statistics

[0108] The test results are shown in Table 9, and the specific findings are as follows: 1) M1 UK sample: speB , emm1 , ssrA and pstB All four targets were positive; 2) M1 global sample: speB and emm1 The target was positive, and ssrA and pstB The target was negative; 3) Non- emm1 GAS Sample: speB The target was positive. emm1、ssrA and pstB All targets were negative; 4) Non-GAS samples: All four targets were negative.

[0109] Table 9: Summary of Specificity Validation Results of the Reagent Kit of the Present Invention

[0110]

[0111]

[0112] (III) Conclusion

[0113] This embodiment demonstrates, through a dual approach of bioinformatics analysis and wet laboratory testing (clinical sample validation), that the " speB + emm1 + ssrA SNP+ pstB The quadruple target combination of "SNP" is unique in the current global GAS genome database and sample testing, corresponding only to M1. UK Subtype. This design not only conforms to the internationally recognized M1 UK The definition of molecular characteristics, through a two-site cross-verification mechanism, fundamentally avoids two types of misjudgments: 1) non- emm1 The strain was misidentified as M1 due to its two SNPs being naturally wild-type. global ;2) Carrying only a single SNP emm1 Derivative subtypes (such as M1) 23SNPs It was misclassified as M1 UK This provides a high-confidence classification basis for clinical diagnosis and public health monitoring.

[0114] Example 6: Inter-batch precision verification

[0115] This embodiment selects a medium concentration (10) 4 (copy / mL) and low concentrations (close to LoD, 2×10) 3 (Copies / mL) Two levels M1 UK The batch-to-batch precision of the kit is assessed using genomic DNA samples of strains, which is used to evaluate batch-to-batch differences in the kit.

[0116] Samples at the two levels were tested using three independently manufactured batches of the kit (batch numbers: IN251101, IN251102, IN251103), with three replicates for each level. The positive rate of each target and the coefficient of variation (CV) of the Ct value for each channel were calculated for a total of 18 replicates across the three batches. The results (see Table 10) showed that the positive detection rate of all four targets in the 18 replicates was 100%, and the inter-batch CV of the Ct value for all four channels was less than 5%, indicating that the manufacturing process of the kit of this invention is stable and its performance is reliable, meeting the inter-batch precision requirements of in vitro diagnostic reagents.

[0117] Simultaneously, during the experiment, the three batches of reagent kits tested their respective corresponding control standards, including the positive control (M1). UK DNA, all four channels were positive), negative control (M1) global DNA, speB and emm1 The channel is positive, and ssrA and pstB The results of the four control groups (no amplification signal in any of the four channels) and the blank control (no nuclease water, no amplification signal in any of the four channels) were all in line with expectations, further verifying the robustness of the detection system.

[0118] Table 10: Summary of inter-batch precision test results

[0119] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A primer-probe composition, characterized in that, Including for detection speB The first primer and probe set for the gene is used for detection. emm Primer-probe set two with type 1 specific sequence, used for detection pstB and ssrA Primer and probe set three for gene SNP sites; The primer-probe set includes primers with sequences as shown in SEQ ID NO. 1-2, and probes as shown in SEQ ID NO. 3; The primer-probe set two includes primers with sequences as shown in SEQ ID NO.4-5, and probes as shown in SEQ ID NO.6; The primer-probe set three is selected from any one of the following: a) Primers with nucleotide sequences as shown in SEQ ID NO.7-8, 10-11, and probes as shown in SEQ ID NO.9, 12; b) Primers with nucleotide sequences as shown in SEQ ID NO. 13-14, 16-17, and probes as shown in SEQ ID NO. 15, 18, wherein the probes are modified with LNA; c) Primers with nucleotide sequences as shown in SEQ ID NO.19-20, 22-23, and probes as shown in SEQ ID NO.21, 24, wherein the probes are modified with MGB.

2. The primer-probe composition according to claim 1, characterized in that, Each probe in the primer-probe composition is labeled with a fluorescent group, and different probes are labeled with different fluorescent groups.

3. The primer-probe composition according to claim 1, characterized in that, The LNA modification method of the probe shown in SEQ ID NO.15 is: modification of the 8th, 10th, 12th and 14th bases starting from the 5' end; and / or, the LNA modification method of the probe shown in SEQ ID NO.18 is: modification of the 8th, 11th and 15th bases starting from the 5' end; And / or, the 3' end of the probes shown in SEQ ID NO. 21, 24 is modified with NFQ-MGB.

4. The application of the primer-probe composition according to any one of claims 1-3, characterized in that, The application is for preparing detection products for at least one of the following purposes: 1) Used for identification of Group A Streptococcus species; 2) Used for emm 1. Type identification; 3) Used for M1 UK Subtype identification; 4) Used for M1 global Subtype identification.

5. The application according to claim 4, characterized in that, The testing product is a testing or diagnostic kit, chip, system, or device.

6. A test kit, characterized in that, Includes the primer-probe composition according to any one of claims 1-3.

7. The detection kit according to claim 6, characterized in that, The detection kit also includes Taq DNA polymerase, dNTPs, and Mg. At least one of UNG enzyme and PCR buffer.

8. The detection kit according to claim 6, characterized in that, The test kit also includes nucleic acid extraction and / or purification reagents.

9. Identification of a group A streptococcal species and M1 UK With M1 global The subtype typing detection method is for non-diagnostic purposes and is characterized by: include: 1) Nucleic acid extraction is performed on the sample; 2) The extracted nucleic acid is amplified using the detection kit described in any one of claims 6-8, and the amplification signal is collected; 3) Based on speB Gene, emm Under the premise of a positive type 1 specific sequence, according to ssrA and pstB The subtype is determined by the amplification signal of the gene SNP site.

10. The detection method according to claim 9, characterized in that, The samples were selected from throat swabs, blood, cerebrospinal fluid, pus, or cultures.

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