A primer probe combination, a method for differentiating different subtypes of microorganisms, and a kit

Abstract

The application belongs to the field of biology and discloses a primer probe combination for detecting an SNP site in a target nucleic acid sequence, which comprises a first inner primer, a first outer primer, a second inner primer, a second outer primer and at least one probe; the first inner primer, the first outer primer, the second inner primer and the second outer primer are used for amplifying a nucleic acid sequence with a single loop structure; and the probe is used for specifically combining with a nucleic acid region where the SNP site is located. The multiplex detection of pathogens is realized by removing the loop inner primer and adding the linear inner primer, the unique primer design strategy provides greater flexibility, thereby facilitating the flexible introduction of a single nucleotide polymorphism (SNP) recognition probe. Meanwhile, the application also discloses a method for distinguishing different types of microorganisms and a kit.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a primer-probe combination, a method for distinguishing different types of microorganisms, and a kit. Background Technology

[0002] Single nucleotide polymorphisms (SNPs) are associated with the risk of various diseases, including cancer, genetic disorders, and infectious diseases. Therefore, SNPs are considered key biomarkers for molecular diagnostics, especially for pathogens with high mutation rates. Traditional SNP detection technologies are highly accurate, some even capable of identifying previously unknown SNP sites. However, these technologies are time-consuming or rely on complex, expensive equipment and specialized operators, limiting their application in point-of-care testing (POCT).

[0003] LAMP is widely used in point-of-care testing (POCT) because it requires only a constant temperature environment and is rapid, providing results within an hour. Some LAMP methods modify one of the loop primers into a fluorescent probe. This probe specifically binds to the SNP site, is then cleaved by enzymes to generate a signal, enabling SNP detection. Therefore, this fluorescent probe, derived from the loop primer, is located in the same region as the SNP site. However, since LAMP primer sets contain four to six primers, the area available for designing SNP detection probes is too short, significantly limiting the design of SNP-targeting probes.

[0004] Although some LAMP-based POCT detection technologies have been established for the rapid detection of SNPs, most of these methods are used for the detection of a single target, and multiple mutation detection is difficult to achieve due to the complex primer set of LAMP.

[0005] Therefore, developing an advanced isothermal amplification method that can flexibly combine SNP-specific probes for multiple mutation detection is of great research significance and application value. Summary of the Invention

[0006] Based on this, the first objective of the present invention is to provide a primer-probe combination for detecting SNP sites in target nucleic acid sequences. We define this primer-probe combination as the smart single-loop mediated isothermal amplification (ssLAMP) method, which performs multiple typing of pathogens by removing the circular inner primer and adding a linear inner primer. This unique primer design strategy provides greater flexibility, thereby facilitating the flexible introduction of single nucleotide polymorphism (SNP) recognition probes.

[0007] In addition, the present invention also discloses a method for distinguishing different types of microorganisms and a reagent kit.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] A primer-probe combination for detecting SNP sites in a target nucleic acid sequence, wherein a single strand of the target nucleic acid sequence has a first nucleic acid region, a second nucleic acid region, a third nucleic acid region, a fourth nucleic acid region, and a fifth nucleic acid region in sequence; and an SNP site is located between the third and fourth nucleic acid regions.

[0010] The primer-probe combination includes a first inner primer, a first outer primer, a second inner primer, a second outer primer, and at least one probe;

[0011] The 3' end of the first inner primer is a sequence complementary to the second nucleic acid region, and the 5' end is a sequence identical to the third nucleic acid region;

[0012] The first outer primer is complementary to the first nucleic acid region;

[0013] The sequences of the second inner primer and the fourth nucleic acid region are identical;

[0014] The second outer primer and the fifth nucleic acid region have the same sequence;

[0015] The first inner primer, the first outer primer, the second inner primer, and the second outer primer are used to amplify nucleic acid sequences with a single circular structure;

[0016] The probe is used to specifically bind to the nucleic acid region where the SNP site is located.

[0017] The innovation of this invention lies in:

[0018] 1. This invention changes the characteristic of traditional LAMP detection methods that amplify fragments that are double-looped. The fragments of this invention are single-looped at one end and straight-chain at the other end (i.e., the region extending towards the fifth nucleic acid region). As long as the third and fourth nucleic acid regions are designed on both sides of the SNP site during the primer design process, the detection of a single SNP site can be satisfied.

[0019] 2. This invention effectively overcomes the limitation of traditional LAMP methods, which can only detect a maximum of one SNP site. Since the straight chain ends do not need to form a loop, it can accommodate the detection of multiple SNP sites. Theoretically, it is not limited by the number of SNP sites.

[0020] 3. Traditional methods such as PCR and NGS involve high temperatures and long processing times, making them unsuitable for on-site testing.

[0021] In the primer-probe combination described above for detecting SNP sites in a target nucleic acid sequence, the single strand of the target nucleic acid sequence also has a sixth nucleic acid region located between the second and third nucleic acid regions, and the primer-probe combination also includes a circular primer with the same sequence as the sixth nucleic acid region.

[0022] In the primer-probe combination described above for detecting SNP sites in target nucleic acid sequences, when there are multiple SNP sites between the third and fourth nucleic acid regions, the probes are multiple corresponding probes, and the fluorescent groups carried by each probe emit different colors.

[0023] In the primer-probe combination described above for detecting SNP sites in target nucleic acid sequences, the target nucleic acid sequence is a sequence fragment of a microorganism, an animal, or a plant.

[0024] In some application scenarios of this invention, the microorganism is a virus or bacteria;

[0025] Viruses such as porcine pseudorabies virus, novel coronavirus, avian influenza virus, infectious bursal disease virus, Newcastle disease virus, African swine fever virus, and porcine epidemic diarrhea virus;

[0026] Bacteria such as Salmonella, Escherichia coli, Campylobacter, Haemophilus parasuis, Staphylococcus, Streptococcus suis, and Lebsiella pneumoniae;

[0027] Animals include, but are not limited to, mammals and oviparous animals; mammals include humans.

[0028] Plants can include trees, herbaceous plants, algae, etc.

[0029] Meanwhile, this invention also discloses a method for distinguishing different types of microorganisms, comprising the following steps:

[0030] Step 1: Identify the SNP sites of microorganisms with different subtypes;

[0031] Step 2: Design any of the primer-probe combinations described above for the SNP site;

[0032] Step 3: Use primer-probe combination to treat microorganisms to determine their typing or to distinguish whether the sample contains microorganisms that match the primer-probe combination.

[0033] In the above-mentioned method for distinguishing different types of microorganisms, if the number of microorganisms to be classified exceeds 3, then based on the differences in SNP sites of different types of microorganisms, the different types of microorganisms are divided into at least two groups of microorganisms; the microorganisms include a first microorganism and a second microorganism; the first microorganism has at least one identical SNP site, and the second microorganism does not have the same SNP site.

[0034] Primer-probe combinations are designed for this SNP site and applied to microorganisms to distinguish whether the microorganism belongs to the first microbiome or the second microbiome, or to determine whether the first microbiome exists in the sample.

[0035] In the above-mentioned methods for distinguishing different types of microorganisms, one or more primer-probe combinations are designed to distinguish different types of microorganisms in the first microbiome by targeting the different SNP sites present in different types of microorganisms.

[0036] To distinguish between different subtypes of microorganisms in the second microbiome, one or more primer-probe combinations are designed to target the different SNP sites present in the subtypes of microorganisms.

[0037] In addition, the present invention also discloses a kit containing the primer-probe combination as described above.

[0038] In the above-mentioned kit, the amplification temperature is 60-70℃ and the amplification time is 20-60 min.

[0039] The kit also includes 10×Bst Reaction Buffer, magnesium chloride solution, betaine solution, dNTPs solution, nuclease-free pure water and Bst WarmStart DNA polymerase, which constitute the basic components of the kit.

[0040] If the kit is used to detect the presence of a certain segment of a target nucleic acid sequence, SYTO9 fluorescent dye is used as an indicator of the reaction result;

[0041] If the kit is used to detect whether a specific SNP site is present in a segment of the target nucleic acid sequence, then RNase H2 enzyme is used to cut the probe to generate a fluorescent signal to determine the reaction result.

[0042] The beneficial effects of this invention are as follows:

[0043] 1. Traditional SNP detection techniques often employ sequencing and high-resolution melting curves, which are time-consuming and require specialized laboratories and personnel. Using isothermal nucleic acid amplification technology for rapid SNP detection significantly reduces the time required to differentiate high-mutation-rate pathogens through SNPs.

[0044] 2. Based on LAMP technology, primers were optimized and designed to establish intelligent single-loop mediated isothermal amplification technology (ss-LAMP), which provides a larger designable area for fluorescent probes and makes it easier to achieve single-tube multiplex detection mode with compatibility of multiple primer sets in a single tube compared to LAMP.

[0045] 3. Combining optimized DNA-RNA polymer probes with RNase H2 enzyme-activated hydrolysis ensures the specificity of the SNP reaction, making SNP site detection more accurate.

[0046] 4. Establishing a multi-mutation site typing and identification system based on SNP sites provides a new option for the detection of pathogens with high mutation rates and has broader application prospects. Attached Figure Description

[0047] Figure 1 This is a diagram showing the relationship between the target nucleic acid sequence and primers in Example 1 of the present invention;

[0048] Figure 2A Schematic diagram of ssLAMP amplification system with different inner primer region lengths;

[0049] Figure 2B This is a graph showing the effect of the presence or absence of external primer sets on the ssLAMP amplification system.

[0050] Figure 2C This is a graph showing the effect of the presence or absence of accelerating primers on the ssLAMP amplification system.

[0051] Figure 2D To accelerate the use of primers, the concentration of the test chromatogram was optimized;

[0052] Figure 2E This is an agarose gel electrophoresis analysis of the ssLAMP amplification products;

[0053] Figure 2F This is a graph showing the sequencing results of the ssLAMP amplification products.

[0054] Figure 3A A comparison graph of single-loop mediated isothermal amplification (ssLAMP) with loop mediated isothermal amplification (LAMP) and Smart Amplification Method Version 2 (SAMP 2).

[0055] Figure 3B The graph shows the sensitivity test results of single-stranded loop-mediated isothermal amplification (ssLAMP) and loop-mediated isothermal amplification (LAMP) under high GC content (gE gene) plasmid templates, as well as the linear regression graph between the plasmid indicator dilution factor and the time to reach the threshold (TT value);

[0056] Figure 3C The graph shows the sensitivity test results of single-stranded loop-mediated isothermal amplification (ssLAMP) and loop-mediated isothermal amplification (LAMP) under a plasmid template with low GC content (invA gene), as well as the linear regression graph between the plasmid indicator dilution factor and the time to reach the threshold (TT value).

[0057] Figure 4A A schematic diagram showing the locus information of the ins214EPE, N211del, L212I, Q493R, N658S, and F486V genes;

[0058] Figure 4B and Figure 4CThis is a schematic diagram of the color development principle in Example 3;

[0059] Figure 5 Figure showing the specificity results of multiplex ssLAMP on plasmids of five SARS-CoV-2 Omicron lineages;

[0060] Figures 6A to 6E Figure showing the single-plasm sensitivity assessment results for five SARS-CoV-2 Omicron lineages;

[0061] Figure 7A This is a graph showing the detection sensitivity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system II for the BA.1 plasmid;

[0062] Figure 7B This is a graph showing the detection sensitivity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system II for BA.3 plasmid;

[0063] Figure 7C This is a graph showing the detection sensitivity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system III for the BA.4 plasmid;

[0064] Figure 7D This is a graph showing the detection sensitivity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system III for BA.5;

[0065] Figure 7E This is a graph showing the detection specificity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system II for different plasmids;

[0066] Figure 7F This is a graph showing the detection specificity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system III for different plasmids.

[0067] Figure 8A This is a fluorescence visualization result of the detection sensitivity test of plasmids BA.1, BA.2, BA.3, BA.4, and BA.5 using the multiple single-loop mediated isothermal amplification (ssLAMP) detection method.

[0068] Figure 8B This is a fluorescence visualization result of the detection specificity test of plasmids BA.1, BA.2, BA.3, BA.4, and BA.5 using the multiple single-loop mediated isothermal amplification (ssLAMP) detection method. Detailed Implementation

[0069] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0070] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0071] Example 1

[0072] A primer-probe combination for detecting SNP sites in a target nucleic acid sequence, wherein a single strand of the target nucleic acid sequence has a first nucleic acid region, a second nucleic acid region, a third nucleic acid region, a fourth nucleic acid region, and a fifth nucleic acid region in sequence; and an SNP site is located between the third and fourth nucleic acid regions.

[0073] The primer-probe combination includes a first inner primer, a first outer primer, a second inner primer, a second outer primer, and at least one probe;

[0074] The 3' end of the first inner primer is a sequence complementary to the second nucleic acid region, and the 5' end is a sequence identical to the third nucleic acid region;

[0075] The first outer primer is complementary to the first nucleic acid region;

[0076] The sequences of the second inner primer and the fourth nucleic acid region are identical;

[0077] The second outer primer and the fifth nucleic acid region have the same sequence;

[0078] The first inner primer, the first outer primer, the second inner primer, and the second outer primer are used to amplify nucleic acid sequences with a single circular structure;

[0079] The probe is used to specifically bind to the nucleic acid region where the SNP site is located.

[0080] For the sake of simplicity in describing the present invention, see references. Figure 1 In this embodiment, the first nucleic acid region, the second nucleic acid region, the third nucleic acid region, the fourth nucleic acid region, and the fifth nucleic acid region are divided into F3C, F2C, F1C, B2, and B3 according to conventional division methods in the art.

[0081] The corresponding first inner primer, first outer primer, second inner primer, and second outer primer are FIP primer, F3 primer, B2 primer, and B3 primer, respectively;

[0082] The FIP primer has a sequence complementary to F2C at the 3' end and the same sequence as F1C at the 5' end, which is used to form a loop;

[0083] The F3 primer is complementary to the F3C primer;

[0084] The B2 primer and the B2 sequence are identical;

[0085] The B3 primer and the B3 sequence are identical;

[0086] In addition, Figure 1 There is also a sixth nucleic acid region, namely LF; the primer corresponding to this region is the circular primer LF primer;

[0087] The LAMP assay is characterized by the ease with which double-stranded nucleic acid sequences dissociate at around 63°C. This allows the FIP primer to penetrate the double-stranded DNA template and initiate the reaction; the presence of the LF primer accelerates this dissociation. Subsequently, the single-stranded DNA generated by FIP primer extension is replaced by F3 primer extension, providing templates for the LF, B2, and B3 primers. The two single-stranded DNA fragments generated by LF and B2 primer extensions are replaced during B2 and B3 primer extensions, forming short DNA fragments. These fragments undergo double-stranded DNA respiration at around 60°C, reaching a dynamic equilibrium between double-stranded and single-stranded structures. At this point, the single-stranded DNA primers generated by LF primer extension can hybridize with the FIP primers to form short double-stranded DNA. The LF primers bind during single-stranded DNA respiration and generate new double-stranded DNA amplicones. These single-stranded DNA fragments become new templates and cycle in the reaction (Cycle I). On the other hand, the single-stranded DNA generated by B2 primer extension forms a single-circular double-stranded DNA structure, and the FIP and B2 primers can easily hybridize to generate new single-stranded DNA. With the help of LF primers, the same single-loop DNA double-stranded structure is formed, thereby initiating another round of cyclic reaction (cycle II).

[0088] The above steps can be used to quickly initiate the amplification reaction and obtain a nucleic acid sequence with a single circular structure.

[0089] For each mutation site, probes containing ribonucleotide modifications can be designed to distinguish between wild-type and mutant alleles;

[0090] The probe is paired with Rnase H2 enzyme; based on the characteristic that Bst DNA polymerase lacks 3'→5' exonuclease activity, Rnase H2 enzyme is introduced into the ssLAMP reaction to cleave the probe.

[0091] The probe is modified to contain ribonucleotides, a 5′ fluorescent group, a 3′ quencher group, and a C3Spacer group. Different probes contain different fluorescent groups, each with its own corresponding excitation wavelength range. For example, the Quasar 670 fluorescent group uses 620 nm excitation light in this embodiment; the FAM fluorescent group uses 500 nm excitation light in this embodiment. These specific wavelengths of light can be obtained by using a combination of LEDs and filters within the corresponding wavelength range.

[0092] 3' C3 spacer modification blocks BST DNA polymerase-mediated elongation, preventing the probe from acting as a primer for elongation amplification during template-directed DNA synthesis. If the ribonucleotides on the probe perfectly match the target gene sequence during the LAMP reaction, RNase H2 will be activated and cleave the probe. This causes the surface plasmon resonance between the fluorophore and quencher to disappear, releasing a fluorescent signal. Conversely, if the probe does not match the target gene, it remains intact and does not produce a fluorescent signal. Only when the modified ribonucleotides in the probe perfectly match the template will the probe be cleaved and produce a fluorescent signal, thus enabling SNP detection.

[0093] The above is a description of the basic working principle of this invention; the following specific examples will further explain the above principle.

[0094] Example 2

[0095] In this embodiment, the amplification efficiency, specificity, and sensitivity of the ssLAMP method of the present invention are only used to verify the detection, and the detection of SNP sites is not involved. Therefore, this embodiment does not involve the use of probes.

[0096] Detection of the gE gene (GenBank: KT936468.1) of pseudorabies virus (PRV)

[0097] First, a primer set for the ssLAMP method was designed using the gE gene of pseudorabies virus (PRV) as the target gene (Table 1). Isothermal amplification reactions were performed by designing LIRs at different positions.

[0098] The kit for detecting the gE gene of pseudorabies virus (PRV) consists of the following components: 1×Bst reaction buffer, SYTO9 dye, 1.4 mM dNTPs, 6 U Bst WarmStart DNA polymerase, and primer set.

[0099] The concentrations of ssLAMP primers were optimized to be 1.6 μM FIP, 1.6 μM LIR (i.e., primer B2 mentioned above), 0.8 μM LF, 0.2 μM F3, and 0.2 μM B3.

[0100] LAMP: The optimal primer concentrations are 1.6 μM FIP or BIP, 0.8 μM LF or LB, and 0.2 μM F3 or B3.

[0101] The reaction amplification procedure for this kit is as follows: the reaction is performed on a Roche Light Cycler 96 real-time detection system (Roche, Switzerland) and Applied Biosystems. TMThe reaction was performed on a 7500 real-time PCR system (Thermo Fisher Scientific, USA). The reaction consisted of 60 cycles at 63°C for 1 minute each, with fluorescence signals measured at the end of each cycle.

[0102] The results showed that primer sets with inner primer regions ranging from 60 to 140 bp in length could efficiently amplify ( ) within 40 minutes. Figure 2A );

[0103] Furthermore, the necessity of the outer primers (F3 and B3) was assessed, and the results showed that the absence of outer primers in the reaction system reduced nucleic acid amplification efficiency. Figure 2B ).

[0104] To accelerate the ssLAMP reaction, the accelerating effect of LF primers was investigated. Based on the ssLAMP principle, it was hypothesized that LF invasion of dsDNA might accelerate reaction initiation. Experimental results showed that adding LF primers significantly shortened the threshold time (TT value) of ssLAMP. Figure 2C This study confirmed the crucial role of LF in accelerating the initiation of the ssLAMP reaction. By testing the effect of different concentrations of LF primers on reaction stability, it was found that no nonspecific amplification occurred even at an LF concentration of 1.6 μM, indicating that the ssLAMP system containing LF primers possesses stability. Figure 2D ).

[0105] Specificity verification of the reaction: The isothermal amplification products were analyzed by 3% agarose gel electrophoresis. Figure 2E ), and sequenced the reaction products. Figure 2F This verifies that the amplified product sequence obtained by this method is consistent with the expected sequence.

[0106] Sensitivity assessment: We also assessed the sensitivity of this method by preparing 10-fold serial dilutions (10^6, 10^5, 10^4, 10^3, 10^2, 10, and 1 copies) of plasmid DNA containing the PRV gE gene and the Salmonella invA gene (GenBank: 1254419) (the primer and probe set specifically designed for the invA gene is shown in Table 1; the kit composition and amplification procedure are the same as above). These results were compared with the conventional LAMP method. Figures 3A to 3C As can be seen, this method has good sensitivity, and a detection rate of 10 copies can be achieved within 30 cycles.

[0107] Figure 2A Schematic diagram of ssLAMP amplification system with different inner primer region lengths;

[0108] Figure 2B This is a graph showing the effect of the presence or absence of external primer sets on the ssLAMP amplification system.

[0109] Figure 2CThis is a graph showing the effect of the presence or absence of accelerating primers on the ssLAMP amplification system.

[0110] Figure 2D To accelerate the use of primers, the concentration of the test chromatogram was optimized;

[0111] Figure 2E This is an agarose gel electrophoresis analysis of the ssLAMP amplification products;

[0112] Figure 2F This is a graph showing the sequencing results of the ssLAMP amplification products.

[0113] Figure 3A A comparison graph of single-loop mediated isothermal amplification (ssLAMP) with loop mediated isothermal amplification (LAMP) and Smart Amplification Method Version 2 (SAMP 2).

[0114] Figure 3B The graph shows the sensitivity test results of single-stranded loop-mediated isothermal amplification (ssLAMP) and loop-mediated isothermal amplification (LAMP) under high GC content (gE gene) plasmid templates, as well as the linear regression graph between plasmid indicator dilution factor and threshold time (TT value);

[0115] Figure 3C The graph shows the sensitivity test results of single-stranded loop-mediated isothermal amplification (ssLAMP) and loop-mediated isothermal amplification (LAMP) under a plasmid template with low GC content (invA gene), as well as the linear regression graph between the plasmid indicator dilution factor and the threshold time (TT value).

[0116] Table 1 Primer set list

[0117]

[0118] Example 3

[0119] Establishment of a multi-analysis system

[0120] This embodiment verifies the practicality of this method in a multiple mutation site detection system. Using the SARS-CoV-2 Omicron variant as a model and the spike protein mutation sites of the SARS-CoV-2 Omicron variant (BA.1, BA.2, BA.3, BA.4, and BA.5) as targets, specific probes were designed (BA.1 probe recognizes ins214EPE; BA.1 / 3 probes recognize N211del and L212I; BA.1 / 2 / 3 probes recognize Q493R; BA.4 probe recognizes N658S; BA.4 / 5 probes recognize F486V). When attempting to design LAMP primers containing these probes using online software, it was found that effective primer combinations could not be generated if the probes were located in the loop primer region. Therefore, this embodiment redesigned the primers based on the ssLAMP principle. Detailed primer and probe information is shown in Table 2.

[0121] Locus information for the genes ins214EPE, N211del, L212I, Q493R, N658S, and F486V is available. Figure 4A ;

[0122] Its color rendering principle can be referenced. Figure 4B and Figure 4C ;

[0123] The reaction system is similar to that of the dye method, except that the SYTO9 dye is removed as the signal reporter group and replaced with the corresponding enzyme blocking probe. Additionally, RnaseH2 enzyme is added. The reaction procedure is the same as that of the dye method, as follows:

[0124] 1. Probe method reaction system: 1×Bst reaction buffer, 1.4mM dNTPs, 6U Bst WarmStart DNA polymerase, 6U RNase H2 enzyme, primer set and probe. The primer set and probe for each system are shown in Table 2.

[0125] 2. Probe method reaction procedure: The reaction consists of 60 cycles at 63°C, each lasting 1 minute, with the fluorescence signal measured at the end of each cycle.

[0126] Single-plasm specificity validation: First, the analytical specificity of plasmids against five SARS-CoV-2 Omicron lineages was tested. The results showed that multiplex ssLAMP had good specificity. Figure 5 ).

[0127] Single-particle sensitivity assessment: The analytical sensitivity for five SARS-CoV-2 Omicron lineages was subsequently tested, with a detection limit of 10 copies / μL and good linearity. Figures 6A to 6E ).

[0128] Multiplex system validation: Primers and probes were assembled into three multiplex reaction systems (primers and probes are shown in Table 2):

[0129] System I: Group BA.1 / 2 / 3 (FAM-labeled probes) were used to detect BA.1, BA.2, and BA.3;

[0130] System II: Group BA.1 (QUASAR 670 labeled probe) and group BA.1 / 3 (FAM labeled probe) distinguish BA.1 and BA.3;

[0131] System III: Group BA.4 (FAM-labeled probes) and group BA.4 / 5 (QUASAR 670-labeled probes) distinguish BA.4 and BA.5;

[0132] Combination Figure 4B and Figure 4C Further analysis was conducted on systems I through III as described above:

[0133] First, BA1 / 2 / 3 and BA4 / 5 are distinguished by System I (primer and probe set BA1 / 2 / 3). BA1 / 2 / 3 is represented by green fluorescence, while BA4 / 5 or BA4 / 5 is represented by no color development.

[0134] Secondly, BA1 / 3 are distinguished by system II (primer and probe set BA1 / 3). Red and green indicate BA1, green indicates BA3, and no color indicates BA2.

[0135] Finally, BA4 / 5 was distinguished using System III (primer-probe set BA4 / 5 and primer-probe set BA4).

[0136] Multiplex ssLAMP typing strategies were designed based on the mutation sites of different variants. Specificity and sensitivity were evaluated by constructing plasmid templates. The results showed that the detection limit of multiplex ssLAMP was 100 copies / μL. Specificity verification in systems II and III indicated that ssLAMP could accurately distinguish different plasmid templates, while traditional LAMP showed nonspecific signals.

[0137] Figure 7A This is a graph showing the detection sensitivity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system II for the BA.1 plasmid;

[0138] Figure 7B This is a graph showing the detection sensitivity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system II for BA.3 plasmid;

[0139] Figure 7CThis is a graph showing the detection sensitivity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system III for the BA.4 plasmid;

[0140] Figure 7D This is a graph showing the detection sensitivity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system III for BA.5;

[0141] Figure 7E This is a graph showing the detection specificity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system II for different plasmids;

[0142] Figure 7F This is a graph showing the detection specificity test results of the multiple single-loop mediated isothermal amplification (ssLAMP) detection method system III for different plasmids.

[0143] Figure 8A This is a fluorescence visualization result of the detection sensitivity test of plasmids BA.1, BA.2, BA.3, BA.4, and BA.5 using the multiple single-loop mediated isothermal amplification (ssLAMP) detection method.

[0144] Figure 8B This is a fluorescence visualization result of the detection specificity test of plasmids BA.1, BA.2, BA.3, BA.4, and BA.5 using the multiple single-loop mediated isothermal amplification (ssLAMP) detection method.

[0145] Table 2. List of primer and probe sets

[0146]

[0147]

[0148] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.

Claims

1. A primer-probe combination for detecting SNP sites in a target nucleic acid sequence, characterized in that, The target nucleic acid sequence has a first nucleic acid region, a second nucleic acid region, a third nucleic acid region, a fourth nucleic acid region, and a fifth nucleic acid region sequentially on one single strand; there is an SNP site between the third nucleic acid region and the fourth nucleic acid region; The primer-probe combination includes a first inner primer, a first outer primer, a second inner primer, a second outer primer, and at least one probe; The 3' end of the first inner primer is a sequence complementary to the second nucleic acid region, and the 5' end is a sequence identical to the third nucleic acid region; The first outer primer is complementary to the first nucleic acid region; The sequences of the second inner primer and the fourth nucleic acid region are identical; The second outer primer and the fifth nucleic acid region have the same sequence; The first inner primer, the first outer primer, the second inner primer, and the second outer primer are used to amplify nucleic acid sequences with a single circular structure; The probe is used to specifically bind to the nucleic acid region where the SNP site is located; The target nucleic acid sequence also has a sixth nucleic acid region located between the second and third nucleic acid regions on a single strand, and the primer-probe combination also includes a circular primer with the same sequence as the sixth nucleic acid region.

2. The primer-probe combination for detecting SNP sites in a target nucleic acid sequence according to claim 1, characterized in that, When there are multiple SNP sites between the third and fourth nucleic acid regions, the probes are multiple corresponding probes, and the emission wavelengths of the fluorescent groups carried by each probe are different.

3. The primer-probe combination for detecting SNP sites in a target nucleic acid sequence according to claim 1, characterized in that, The target nucleic acid sequence is a sequence fragment from a microorganism, an animal, or a plant.

4. A method for differentiating microbial types for non-disease diagnostic purposes, characterized in that, Includes the following steps: Step 1: Identify the SNP sites of microorganisms with different subtypes; Step 2: Design primer-probe combinations as described in any one of claims 1 to 3 for the SNP sites; Step 3: Use primer-probe combination to treat microorganisms to determine their typing or to distinguish whether the sample contains microorganisms that match the primer-probe combination.

5. The method for differentiating microbial types for non-disease diagnostic purposes according to claim 4, characterized in that, If the number of microorganisms to be genotyped exceeds 3, then based on the differences in SNP sites of different genotypes, the microorganisms of different genotypes are divided into at least two microbiomes; the microbiome includes a first microbiome and a second microbiome; the first microbiome has at least one identical SNP site, and the second microbiome does not have that SNP site. Primer-probe combinations are designed for this SNP site and applied to microorganisms to distinguish whether the microorganism belongs to the first microbiome or the second microbiome, or to determine whether the first microbiome exists in the sample.

6. The method for differentiating microbial types for non-disease diagnostic purposes according to claim 5, characterized in that, To distinguish different subtypes of microorganisms in the first microbiome by designing one or more primer-probe combinations targeting the different SNP sites present in the subtypes of microorganisms in the first microbiome; To distinguish between different subtypes of microorganisms in the second microbiome, one or more primer-probe combinations are designed to target the different SNP sites present in the subtypes of microorganisms.

7. A reagent kit, characterized in that, Contains the primer-probe combination as described in any one of claims 1 to 3.

8. The reagent kit according to claim 7, characterized in that, The amplification temperature is 60~70℃ and the amplification time is 20~60min.

9. The reagent kit according to claim 7, characterized in that, It also includes 10× Bst Reaction Buffer, magnesium chloride solution, betaine solution, dNTPs solution, nuclease-free pure water and Bst WarmStart DNA polymerase, which form the basic components of the kit; If the kit is used to detect the presence of a certain segment of a target nucleic acid sequence, SYTO9 fluorescent dye is used as an indicator of the reaction result; If the kit is used to detect whether a specific SNP site is present in a segment of the target nucleic acid sequence, then RNase H2 enzyme is used to cut the probe to generate a fluorescent signal to determine the reaction result.

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