Primer probe combination, method for distinguishing different types of microorganisms and kit

Abstract

The invention belongs to the field of biology, and discloses a primer probe combination for detecting SNP (Single Nucleotide Polymorphism) sites in a target nucleic acid sequence, the primer probe combination 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 monocyclic structure; the probe is used for being specifically combined with a nucleic acid area where an SNP site is located. By removing the annular inner primers and adding the linear inner primers to perform multiple typing detection on pathogens, the unique primer design strategy provides greater flexibility, thereby facilitating flexible introduction of a single nucleotide polymorphism (SNP) recognition probe. Meanwhile, the invention also discloses a method for distinguishing different types of microorganisms and a kit.

Description

Technical Field

[0001] The present invention belongs to the field of biology, and particularly relates to a primer-probe combination, a method for differentiating different genotypes of microorganisms, and a kit. Background Art

[0002] Single nucleotide polymorphisms (SNPs) are associated with various disease risks including cancer, genetic diseases, and infectious diseases. Therefore, SNPs are considered key biomarkers for molecular diagnosis, especially for pathogens with high mutation rates. Traditional SNP detection techniques are highly accurate in detecting SNPs, and some can even discover unknown SNP loci. However, these techniques are very time-consuming or rely on complex, expensive instruments and professional operators, which limit their application in point-of-care testing (POCT).

[0003] LAMP has been widely used in POCT detection because its reaction only requires a constant temperature reaction environment and the reaction is rapid, and results can be obtained within one hour. In some LAMP methods, one of the loop primers of its primers is modified into a fluorescent probe, and the fluorescent probe specifically binds to the SNP locus, and thus is cleaved by an enzyme to generate a signal value, realizing the recognition and detection of SNPs. Therefore, this fluorescent probe modified from the loop primer is in the same region as the SNP locus. However, since the primer set of LAMP contains four to six primers, the region available for designing SNP detection probes is too short, which greatly limits the design of SNP-targeted probes.

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

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

[0006] Based on this, the first object of the present invention is to provide a primer-probe combination for detecting SNP loci in a target nucleic acid sequence. We define this primer-probe combination as the smart single-loop-mediated isothermal amplification (ssLAMP) method. By removing the circular inner primer and adding a linear inner primer for multiplex genotyping detection of pathogens, this unique primer design strategy provides greater flexibility, thus facilitating the flexible introduction of single nucleotide polymorphism (SNP) recognition probes.

[0007] Meanwhile, the present invention also discloses a method for differentiating different genotypes of microorganisms and a kit.

[0008] To achieve the above invention object, the present invention adopts the following technical solutions:

[0009] A primer-probe combination for detecting SNP sites in a target nucleic acid sequence. One single strand of the target nucleic acid sequence sequentially 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. There is an SNP site between the third nucleic acid region and the fourth nucleic acid region.

[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 second inner primer has the same sequence as the fourth nucleic acid region.

[0014] The second outer primer has the same sequence as the fifth nucleic acid region.

[0015] The first inner primer, the first outer primer, the second inner primer, and the second outer primer are used to amplify a nucleic acid sequence with a single-loop structure.

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

[0017] The innovation of the present invention lies in:

[0018] 1. It changes the characteristic that the fragment amplified by the traditional LAMP detection method has a double-loop structure. One end of the fragment of the present invention is a single loop and the other end is a straight chain (i.e., the region extending in the direction of the fifth nucleic acid region). As long as during the primer design process, according to the SNP site, the third nucleic acid region and the fourth nucleic acid region are designed on both sides of the SNP site, the detection of a single SNP site can be satisfied.

[0019] 2. The present invention effectively overcomes the defect that the traditional LAMP method can detect at most one SNP site. Since the straight-chain end does not need to form a loop, the detection of multiple SNP sites can be accommodated. Theoretically speaking, it is not limited by the number of SNP sites.

[0020] 3. Traditional methods such as PCR and NGS have high temperatures and long times, and are not suitable for on-site detection.

[0021] In the above-mentioned primer-probe combination for detecting SNP sites in a target nucleic acid sequence, there is also a sixth nucleic acid region located between the second nucleic acid region and the third nucleic acid region on the single strand of the target nucleic acid sequence, and the primer-probe combination further includes a loop primer having the same sequence as the sixth nucleic acid region.

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

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

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

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

[0026] Bacteria such as Salmonella, Escherichia coli, Campylobacter, Haemophilus parasuis, Staphylococcus, Streptococcus suis, Klebsiella pneumoniae, etc.;

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

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

[0029] Meanwhile, the present invention also discloses a method for distinguishing different genotypes of a microorganism, including the following steps:

[0030] Step 1: Determine the SNP sites of microorganisms with different genotypes;

[0031] Step 2: Design a primer-probe combination as described above for the SNP site;

[0032] Step 3: Use the primer-probe combination to act on the microorganism to determine the genotype of the microorganism, or to distinguish whether the sample contains a microorganism that matches the primer-probe combination.

[0033] In the method for distinguishing different genotypes of the microorganism described above, if the number of microorganisms to be genotyped exceeds 3; then according to the differences in the SNP sites of microorganisms with different genotypes, the microorganisms with different genotypes are divided into at least two groups of microorganisms; the groups of microorganisms include a first group of microorganisms and a second group of microorganisms; the first group of microorganisms has at least one identical SNP site, and the second group of microorganisms does not have this SNP site;

[0034] Design a primer-probe combination for this SNP site and act on the microorganism to distinguish whether the microorganism belongs to the first group of microorganisms or the second group of microorganisms, or to determine whether the first group of microorganisms exists in the sample.

[0035] In the above method for distinguishing different genotypes of microorganisms, for the different SNP sites existing in the microorganisms of different genotypes in the first microbiome, one or more sets of primer-probe combinations are designed to distinguish different genotypes in the first microbiome;

[0036] For the different SNP sites existing in the microorganisms of different genotypes in the second microbiome, one or more sets of primer-probe combinations are designed to distinguish different genotypes in the second microbiome.

[0037] Meanwhile, the present invention also discloses a kit containing any one of the above-mentioned primer-probe combinations.

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

[0039] In the above kit, it also includes 10×Bst Reaction Buffer, magnesium chloride solution, betaine solution, dNTPs solution, nuclease-free pure water and Bst WarmStart DNA polymerase as the basic components of the kit;

[0040] If the kit is used to detect whether a certain sequence exists in the 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 exists in a certain sequence of the target nucleic acid sequence, RNase H2 enzyme is used to cut the probe to generate a fluorescent signal to judge the reaction result.

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

[0043] 1. Traditional SNP site detection technologies mostly use sequencing methods, high-resolution melting curves, etc., which are time-consuming and rely on professional laboratories and professional personnel for operation. Using nucleic acid isothermal amplification technology for rapid SNP detection greatly shortens the time for distinguishing highly mutable pathogens through SNPs.

[0044] 2. Based on the LAMP technology, primers are optimized and designed to establish an intelligent single-loop mediated isothermal amplification technology (ss-LAMP), providing a larger designable area for fluorescent probes and making it easier to achieve a single-tube multiplex detection mode with multiple primer sets compatible in a single tube compared to LAMP.

[0045] 3. Combining the optimized DNA-RNA polymer probe and the hydrolysis reaction activated by Rnase H2 enzyme ensures the specificity of the SNP reaction, making the detection of SNP sites more accurate.

[0046] 4. Establish a multiplex mutation site genotyping and identification system based on SNP sites, which provides a new option for the detection of high-mutation-rate pathogen genotyping and has a broader application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0048] Figure 2A It is a test diagram of the ssLAMP amplification system with different lengths of internal primer regions;

[0049] Figure 2B It is a test diagram of the influence of the presence or absence of external primer groups on the ssLAMP amplification system;

[0051] Figure 2C It is a test diagram of the influence of the presence or absence of acceleration primers on the ssLAMP amplification system;

[0052] Figure 2D It is a test diagram for optimizing the usage concentration of acceleration primers;

[0053] Figure 2E It is an agarose gel electrophoresis analysis diagram of ssLAMP amplification products;

[0054] Figure 2F It is a sequencing result diagram of ssLAMP amplification products;

[0055] Figure 3A It is a comparison diagram of single-strand loop-mediated isothermal amplification (ssLAMP) with loop-mediated isothermal amplification (LAMP) and smart amplification method version 2 (SAMP 2);

[0056] Figure 3B It is a sensitivity test result diagram of single-strand loop-mediated isothermal amplification (ssLAMP) and loop-mediated isothermal amplification (LAMP) with a high GC content (gE gene) plasmid template, and a linear regression diagram between the plasmid indication dilution factor and the time to reach the threshold (TT value);

[0057] Figure 3C It is a sensitivity test result diagram of single-strand loop-mediated isothermal amplification (ssLAMP) and loop-mediated isothermal amplification (LAMP) with a low GC content (invA gene) plasmid template, and a linear regression diagram between the plasmid indication dilution factor and the time to reach the threshold (TT value);

[0058] Figure 4A It is a schematic diagram of the site information of the ins214EPE, N211del, L212I, Q493R, N658S, F486V genes;

[0059] Figure 4B and Figure 4CIt is the color development principle diagram of Example 3;

[0060] Figure 5 It is the multiplex ssLAMP specificity result diagram of plasmids of 5 SARS-CoV-2 Omicron lineages;

[0061] Figures 6A to 6E It is the singleplex sensitivity evaluation result diagram of plasmids of 5 SARS-CoV-2 Omicron lineages;

[0062] Figure 7A It is the detection sensitivity test result diagram of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method system Ⅱ for the BA.1 plasmid;

[0063] Figure 7B It is the detection sensitivity test result diagram of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method system Ⅱ for the BA.3 plasmid;

[0064] Figure 7C It is the detection sensitivity test result diagram of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method system Ⅲ for the BA.4 plasmid;

[0065] Figure 7D It is the detection sensitivity test result diagram of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method system Ⅲ for BA.5;

[0066] Figure 7E It is the detection specificity test result diagram of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method system Ⅱ for different plasmids;

[0067] Figure 7F It is the detection specificity test result diagram of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method system Ⅲ for different plasmids.

[0068] Figure 8A It is the fluorescence visualization result diagram of the detection sensitivity test of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method for plasmids BA.1, BA.2, BA.3, BA.4 and BA.5.

[0069] Figure 8B It is the fluorescence visualization result diagram of the detection specificity test of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method for plasmids BA.1, BA.2, BA.3, BA.4 and BA.5. Specific implementation manners

[0070] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0071] For those without specific experimental steps or conditions indicated in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments without the manufacturer indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0072] Example 1

[0073] A primer-probe combination for detecting SNP sites in a target nucleic acid sequence. One single strand of the target nucleic acid sequence successively 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; there is an SNP site between the third nucleic acid region and the fourth nucleic acid region.

[0074] 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.

[0075] 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.

[0076] The first outer primer is complementary to the first nucleic acid region.

[0077] The second inner primer has the same sequence as the fourth nucleic acid region.

[0078] The second outer primer has the same sequence as the fifth nucleic acid region.

[0079] The first inner primer, the first outer primer, the second inner primer, and the second outer primer are used to amplify a nucleic acid sequence with a single-loop structure.

[0080] The probe is used to specifically bind to the nucleic acid region where the SNP site is located.

[0081] For the sake of simplicity in describing the present invention, with reference to Figure 1 , in this example, 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 the conventional division method in this field.

[0082] The corresponding first inner primer, first outer primer, second inner primer, and second outer primer are respectively the FIP primer, the F3 primer, the B2 primer, and the B3 primer.

[0083] The 3'-end of the FIP primer is a sequence complementary to F2C and the 5'-end is a sequence identical to F1C, for forming a loop.

[0084] The F3 primer is complementary to F3C.

[0085] The B2 primer has the same sequence as B2.

[0086] The B3 primer is identical to the B3 sequence;

[0087] In addition, in Figure 1 there is also a region which is the sixth nucleic acid region, namely LF; the primer corresponding to this region is the loop primer LF primer;

[0088] The characteristic of the LAMP detection method is that at about 63 °C, double-stranded nucleic acid sequences are relatively easy to dissociate. The FIP primer invades the double-stranded DNA template and initiates the reaction. In particular, in the presence of the LF primer, the dissociation rate will be accelerated; subsequently, the single-stranded DNA generated by the extension of the FIP primer is replaced by the extension of the F3 primer, providing a template for the LF primer, B2 primer and B3 primer. The two single-stranded DNAs generated by the extension of the LF primer and B2 primer are replaced during the extension of the B2 primer and B3 primer, forming approximately short DNA fragments. These fragments undergo double-stranded DNA breathing at about 60 °C, reaching a dynamic equilibrium between double-stranded and single-stranded structures. At this time, the single-stranded DNA primer formed by the extension of the LF primer can hybridize with the FIP primer to form a short double-stranded DNA. The LF primer binds and generates a new double-stranded DNA amplicon during the single-stranded DNA breathing process. These single-stranded DNAs become new templates and cycle (Cycle I). On the other hand, the single-stranded DNA formed by the extension of the B2 primer forms a single-loop DNA double-stranded structure. The FIP primer and B2 primer can easily hybridize to generate new single-stranded DNA. With the help of the LF primer, the same single-loop DNA double-stranded structure is formed, thus initiating another round of cycling reaction (Cycle II).

[0089] Through the above operations, the amplification reaction can be quickly initiated to obtain a nucleic acid sequence with a single-loop structure.

[0090] For each mutation site, probes containing ribonucleotide modifications can be designed to distinguish wild-type and mutant alleles;

[0091] The enzyme used in conjunction with the probe is Rnase H2; based on the characteristic that Bst DNA polymerase lacks 3'→5' exonuclease activity, Rnase H2 enzyme is introduced to cleave the probe in the ssLAMP reaction;

[0092] The probe is modified and contains ribonucleotides, a fluorescent group at the 5′ end, a quenching group at the 3′ end and a C3Spacer group. Different probes contain different fluorescent groups, and each fluorescent group has its own corresponding excitation light in a certain 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 the combined use of LED lights and filters in the corresponding wavelength ranges.

[0093] 3'-end C3 Spacer modification can block the extension mediated by BST DNA polymerase and prevent the probe from being extended and amplified as a primer during template-directed DNA synthesis. If the ribonucleotides on the probe are completely complementary to the target gene sequence during the LAMP reaction, the RNase H2 enzyme will be activated and cleave the probe. This will result in the disappearance of surface plasmon resonance between the fluorophore and the quencher group, thus releasing a fluorescent signal. Conversely, if the probe does not match the target gene, the probe will remain intact and no fluorescent signal will be generated. Only when the modified ribonucleotides in the probe are completely complementary to the template, the probe will be cleaved and a fluorescent signal will be generated, thus achieving SNP detection.

[0094] The above is the description of the basic principle of the work of the present invention; the above principle will be further explained through specific cases below.

[0095] Example 2

[0096] In this example, it is only used to verify the amplification efficiency, specificity, sensitivity, etc. of the ssLAMP method of the present invention, and does not involve the detection of SNP sites. Therefore, this example does not involve the use of probes.

[0097] Detection of the gE gene (GenBank: KT936468.1) of Pseudorabies virus (PRV)

[0098] First, the gE gene of Pseudorabies virus (PRV) was used as the target gene, and a primer set for the ssLAMP method was designed (Table 1). Isothermal amplification reactions were carried out by designing LIRs at different positions;

[0099] The related substances of the kit for detecting the gE gene of Pseudorabies virus (PRV) consisted of: 1×Bst reaction buffer, SYTO9 dye, 1.4 mM dNTPs, 6 U Bst WarmStart DNA polymerase, and the primer set.

[0100] The concentration of the ssLAMP primers was optimized to 1.6 μM FIP, 1.6 μM LIR (i.e., the above-mentioned B2 primer), 0.8 μM LF, 0.2 μM F3, and 0.2 μM B3.

[0101] LAMP: The optimal concentration of the primers is 1.6 μM FIP or BIP, 0.8 μM LF or LB, and 0.2 μM F3 or B3.

[0102] The reaction amplification procedure of this kit was as follows: The reaction was carried out on a Roche Light Cycler 96 real-time detection system (Roche, Switzerland) and Applied Biosystems TMPerformed on a 7500 Real-Time PCR System (Thermo Fisher Scientific, USA). The reaction included 60 cycles at 63°C for 1 minute each, and the fluorescence signal was measured at the end of each cycle.

[0103] The results showed that primer sets with inner primer region lengths of 60 to 140 bp could efficiently amplify within 40 minutes ( Figure 2A );

[0104] In addition, the necessity of outer primers (F3 and B3) was also evaluated. The results showed that the lack of outer primers in the reaction system would reduce the nucleic acid amplification efficiency ( Figure 2B ).

[0105] To accelerate the ssLAMP reaction, the accelerating effect of LF primers was explored. According to the ssLAMP principle, it was speculated that the invasion of LF into dsDNA might accelerate the reaction initiation. The experimental results showed that the addition of LF primers could significantly shorten the threshold time (TT value) of ssLAMP ( Figure 2C ), confirming the key role of LF in accelerating the ssLAMP reaction initiation. By testing the effect of different concentrations of LF primers on the reaction stability, it was found that there was no non-specific amplification when the LF concentration reached 1.6 μM, indicating the stability of the ssLAMP system containing LF primers ( Figure 2D ).

[0106] Verification of reaction specificity: The isothermal amplification products were analyzed by 3% agarose gel electrophoresis ( Figure 2E ), and the reaction products were sequenced ( Figure 2F ), verifying that the product sequence amplified by this method was consistent with the expectation.

[0107] Assessment of reaction sensitivity: Meanwhile, we evaluated the sensitivity of this method. Ten-fold serial dilutions (10^6, 10^5, 10^4, 10^3, 10^2, 10, and 1 copy) of plasmid DNA containing PRV gE gene and Salmonella invA gene (GenBank: 1254419) (the primer-probe sets designed specifically for the invA gene are shown in Table 1, and the kit composition and amplification procedure are the same as above) were prepared and compared with the traditional LAMP method ( Figures 3A to 3C ). It can be seen that this method has good sensitivity and can reach a detection rate of 10 copies within 30 cycles.

[0108] Figure 2A Test diagram for ssLAMP amplification systems with different inner primer region lengths;

[0109] Figure 2B Test diagram for the influence of the presence or absence of outer primer sets on the ssLAMP amplification system;

[0110] Figure 2CTest chart for the influence of the presence or absence of an acceleration primer pair on the ssLAMP amplification system;

[0111] Figure 2D Test chart for optimizing the usage concentration of the acceleration primer;

[0112] Figure 2E Agarose gel electrophoresis analysis chart of ssLAMP amplification products;

[0113] Figure 2F Sequencing result chart of ssLAMP amplification products;

[0114] Figure 3A Comparison chart of single-stranded loop-mediated isothermal amplification (ssLAMP) with loop-mediated isothermal amplification (LAMP) and smart amplification method version 2 (SAMP 2);

[0115] Figure 3B Sensitivity test result chart of single-stranded loop-mediated isothermal amplification (ssLAMP) and loop-mediated isothermal amplification (LAMP) with a plasmid template of high GC content (gE gene), and the linear regression chart between the plasmid indication dilution factor and the threshold time (TT value);

[0116] Figure 3C Sensitivity test result chart of single-stranded loop-mediated isothermal amplification (ssLAMP) and loop-mediated isothermal amplification (LAMP) with a plasmid template of low GC content (invA gene), and the linear regression chart between the plasmid indication dilution factor and the threshold time (TT value).

[0117] Table 1 List of primer sets

[0118]

[0119] Example 3

[0120] Establishment of a multiplex analysis system

[0121] This example verified 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 (the BA.1 probe recognizes ins214EPE; the BA.1 / 3 probe recognizes N211del and L212I; the BA.1 / 2 / 3 probe recognizes Q493R; the BA.4 probe recognizes N658S; the BA.4 / 5 probe recognizes F486V). When attempting to design LAMP primers containing these probes using online software, it was found that no effective primer combinations could be generated if the probes were located in the loop primer region. Therefore, in this example, the primers were redesigned according to the ssLAMP principle. The detailed primer and probe information is shown in Table 2.

[0122] The locus information of the ins214EPE, N211del, L212I, Q493R, N658S, and F486V genes can be seen Figure 4A ;

[0123] Its color development principle can be referred to Figure 4B and Figure 4C ;

[0124] The reaction system is similar to the dye method. Remove the SYTO9 dye as the signal reporting group and replace it with the corresponding enzyme-blocking probe, and add RnaseH2 enzyme. The reaction program is the same as the dye method as follows:

[0125] 1. Probe method reaction system: 1×Bst reaction buffer, 1.4 mM dNTPs, and 6 U Bst WarmStart DNA polymerase, 6 U RnaseH2 enzyme, primer set, and probe. The primer set and probe for each system are shown in Table 2.

[0126] 2. Probe method reaction program: The reaction includes 60 cycles at 63 °C for 1 minute each, and the fluorescence signal is measured at the end of each cycle.

[0127] Single-specificity verification: First, the analytical specificity for plasmids of 5 SARS-CoV-2 Omicron lineages was tested, and the results showed good multiple ssLAMP specificity ( Figure 5 ).

[0128] Single-sensitivity assessment: Subsequently, the analytical sensitivity for plasmids of 5 SARS-CoV-2 Omicron lineages was tested. The detection limit was 10 copies / μL, and there was a good linear relationship ( Figures 6A to 6E ).

[0129] Multiplex system verification: The primers and probes were assembled into three multiplex reaction systems (the primers and probes are shown in Table 2):

[0130] System I: Detection of BA.1, BA.2, and BA.3 by group BA.1 / 2 / 3 (probe labeled with FAM);

[0131] System II: Differentiation between BA.1 and BA.3 by group BA.1 (probe labeled with QUASAR 670) and group BA.1 / 3 (probe labeled with FAM);

[0132] System III: Differentiation between BA.4 and BA.5 by group BA.4 (probe labeled with FAM) and group BA.4 / 5 (probe labeled with QUASAR 670);

[0133] Combined Figure 4B and Figure 4C and further analysis was performed on the above Systems I to III:

[0134] First, BA1 / 2 / 3 and BA4 / 5 were distinguished by System I (primer-probe set BA1 / 2 / 3). Green fluorescence indicates BA1 / 2 / 3, and no color development indicates BA4 / 5 or the absence of the target gene and target SNP sites;

[0135] Secondly, BA1 / 3 was distinguished by System II (primer-probe set BA1 / 3). Red and green fluorescence indicates BA1, green fluorescence indicates BA3, and no color development indicates BA2;

[0136] Finally, BA4 / 5 was distinguished by System III (primer-probe set BA4 / 5 and primer-probe set BA4).

[0137] According to the mutation sites of different variants, a multiplex ssLAMP genotyping strategy was designed, and the specificity and sensitivity were evaluated by constructing plasmid templates. The results showed that the detection limit of multiplex ssLAMP was 100 copies / μL. The verification of reaction specificity in Systems II and III indicated that ssLAMP could accurately distinguish different plasmid templates, while non-specific signals appeared in traditional LAMP;

[0138] Figure 7A It is the test result graph of the detection sensitivity of System II of the multiplex single-strand loop-mediated isothermal amplification (ssLAMP) detection method for the BA.1 plasmid;

[0139] Figure 7B It is the test result graph of the detection sensitivity of System II of the multiplex single-strand loop-mediated isothermal amplification (ssLAMP) detection method for the BA.3 plasmid;

[0140] Figure 7CIt is a graph showing the test results of the detection sensitivity of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method system III for the BA.4 plasmid;

[0141] Figure 7D It is a graph showing the test results of the detection sensitivity of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method system III for BA.5;

[0142] Figure 7E It is a graph showing the test results of the detection specificity of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method system II for different plasmids;

[0143] Figure 7F It is a graph showing the test results of the detection specificity of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method system III for different plasmids.

[0144] Figure 8A It is a graph showing the fluorescence visualization results of the detection sensitivity of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method for plasmids BA.1, BA.2, BA.3, BA.4, and BA.5.

[0145] Figure 8B It is a graph showing the fluorescence visualization results of the detection specificity of the multiplex single-loop mediated isothermal amplification (ssLAMP) detection method for plasmids BA.1, BA.2, BA.3, BA.4, and BA.5.

[0146] Table 2 List of primer-probe sets

[0147]

[0148]

[0149] The embodiments presented herein are merely selected embodiments according to all possible combinations of embodiments. The appended claims should not be limited by the embodiments illustrating the invention. Variations within some numerical ranges used in the claims, including sub-ranges within them, 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, One single strand of the target nucleic acid sequence successively 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; there is a 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 second inner primer has the same sequence as the fourth nucleic acid region; The second outer primer has the same sequence as the fifth nucleic acid region; The first inner primer, the first outer primer, the second inner primer, and the second outer primer are used to amplify a nucleic acid sequence with a single-loop structure; The probe is used to specifically bind to the nucleic acid region where the SNP site is located.

2. The primer-probe combination for detecting SNP sites in a target nucleic acid sequence according to claim 1, wherein There is also a sixth nucleic acid region between the second nucleic acid region and the third nucleic acid region on the single strand of the target nucleic acid sequence, and the primer-probe combination further includes a loop primer having the same sequence as the sixth nucleic acid region.

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

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

5. A method for differentiating different genotypes of a microorganism, characterized in that, It includes the following steps: Step 1: Determine the SNP sites of microorganisms of different genotypes; Step 2: Design a primer-probe combination as described in any one of claims 1 to 3 for the SNP sites; Step 3: Use the primer-probe combination to act on the microorganism to determine the genotype of the microorganism, or to distinguish whether the sample contains a microorganism that matches the primer-probe combination.

6. The method for distinguishing different subtypes of microorganisms according to claim 5, wherein If the number of microorganisms to be genotyped exceeds 3; then according to the differences in the SNP sites of microorganisms of different genotypes, the microorganisms of different genotypes are divided into at least two groups of microorganism groups; the microorganism groups include a first microorganism group and a second microorganism group; the first microorganism group has at least one identical SNP site, and the second microorganism group does not have this SNP site; Design a primer-probe combination for this SNP site and act on the microorganism to distinguish whether the microorganism belongs to the first microorganism group or the second microorganism group, or to determine whether the first microorganism group exists in the sample.

7. The method for differentiating different subtypes of microorganisms according to claim 6, characterized in that, Design one or more groups of primer-probe combinations for the different SNP sites existing in the microorganisms of different genotypes in the first microorganism group to distinguish the different genotypes in the first microorganism group; Design one or more groups of primer-probe combinations for the different SNP sites existing in the microorganisms of different genotypes in the second microorganism group to distinguish the different genotypes in the second microorganism group.

8. A kit, characterized in that, It contains a primer-probe combination as described in any one of claims 1 to 4.

9. The kit according to claim 8, wherein Its amplification temperature is 60-70°C and the amplification time is 20-60 min.

10. The kit according to claim 8, wherein It further includes the basic components of a kit consisting of 10×Bst Reaction Buffer, magnesium chloride solution, betaine solution, dNTPs solution, nuclease-free pure water, and Bst WarmStart DNA polymerase; If the kit is used to detect the presence of a certain sequence in the 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 contained in a certain sequence of the target nucleic acid sequence, RNase H2 enzyme is used to cleave the probe to generate a fluorescent signal to judge the reaction result.

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