CN116497098A - SNP detection kit and detection method based on fluorescent marker primer - Google Patents

Abstract

The invention relates to a SNP detection kit based on fluorescent labeled primers, comprising: forward and reverse primers for each SNP detection of a class of animals, and two ET-tagged fluorescent primers; the 5' ends of the two ET marked fluorescent primer sequences are respectively connected with a fluorescent group, and the middle of the sequences are respectively connected with a quenching group; the sequence structures of the two ET-labeled fluorescent primers are shown as SEQ_1 and SEQ_2 respectively. The present invention utilizes efficient integration of allele-specific PCR with universal Energy Transfer (ET) fluorescent tagged primers by PCR amplification of genomic DNA in the presence of these several primers, wherein the allele-specific primers include a Tail-containing forward Primer (Tail Primer) and a Reverse Primer (Reverse Primer), and two universal ET fluorescent tagged primers (Reporter primers). The detection process is carried out by fluorescent quantitative PCR amplification and the green and red fluorescent signals of an amplification product system are evaluated, thereby achieving the identification and screening of homozygous and heterozygous individuals.

Description

SNP detection kit and detection method based on fluorescent marker primer

Technical Field

The invention relates to the technical field of SNP detection, in particular to a SNP detection kit and a detection method based on fluorescent marker primers.

Background

Single Nucleotide Polymorphism (SNP) mainly refers to a polymorphism of a DNA sequence at the genomic level caused by a base change of a single nucleotide, and is one of the most common heritable variations. The number of SNP loci can reach more than millions in a genome, and the sequences at the upstream and downstream of each locus are different, so that different SNP locus detection needs to design specific primers and probes for the sequences at the upstream and downstream of each locus, and the cost of the fluorescent probes is far higher than that of the primers. In general, the requirements of SNP detection are determined according to specific projects, so that the different requirements of different clients are greatly different, and the detection cost is very high because the SNP detection is often customized according to the requirements of the clients. Meanwhile, the difference of primer sequences at the upstream and downstream of SNP loci is limited, and the annealing temperature for carrying out amplification reaction is possibly different, so that single-tube detection generally only relates to one SNP locus, and the detection material and operation cost are further increased. In the prior art, the mouse strain identification generally needs to detect dozens of SNP loci simultaneously, and the primer probe design of the sequence specificity is needed for different SNP loci, so that the SNP detection of the mouse population with low cost and high flux is difficult to realize under the condition of high diversity requirement.

Specifically, the detection means commonly used at present areSNP genotyping techniques that use the 5' nuclease activity of Taq polymerase to generate a fluorescent signal during PCR. For each SNP, the detection method uses two different probes (i.e., for the wild type and variant type, respectively) directed only to the sequence of the SNP site, the two probes being complementary to the DNA sequence containing a particular base in the SNP site, respectively. The technology utilizes FRET technology, and both ends of wild type and variant allele probe sequences are respectively connected with a 5 'report fluorescent group and a 3' quenching group. When the probe is intact, fluorescence is inhibited because the quenching groups are located within close physical ranges of the quenching groups. In the PCR annealing step, the probe hybridizes to the target SNP site. During the PCR extension, the Taq polymerase is usedThe 5' nuclease activity results in the quenching group and the quenching group being released, resulting in an increase in the characteristic fluorescence of the quenching group. Exonuclease activity occurs only on perfectly hybridized probes, as probes containing mismatched bases are not recognized by Taq polymerase. At the end of the PCR reaction, the fluorescent signal of both quenching groups will be measured. The ratio of the signals will be indicative of the genotype of the sample. The current Taqman probe method generally aims at SNP identification by using a pair of specific probes of the site, and for a plurality of SNP sites to be detected, a plurality of groups of Taqman probes are required to be designed for each site, and the synthesis cost of the probes is far higher than that of primers, so that the sample detection cost is extremely high. FIG. 1 shows->SNP genotyping technology detection principle. Wherein D1 is exemplified by a red fluorescent group Dye1, D2 is exemplified by a green fluorescent group Dye2, and Q is exemplified by a quenching group.

Disclosure of Invention

In view of the above, the present invention provides a SNP detection kit and detection method based on fluorescent labeled primers, which address the above problems of the prior art.

In order to achieve the technical purpose, the technical scheme adopted by the application is as follows:

in a first aspect, the present invention provides a SNP detection kit based on fluorescent-labeled primers, comprising: forward and reverse primers for each SNP detection of a class of animals, and two ET-tagged fluorescent primers; the 5' ends of the two ET marked fluorescent primer sequences are respectively connected with a fluorescent group, and the middle of the sequences are respectively connected with a quenching group; the sequence structures of the two ET-labeled fluorescent primers are shown as SEQ_1 and SEQ_2 respectively.

Furthermore, two forward primers for each SNP detection of a certain animal are provided, and the forward primers correspond to the wild type and the variant type of each SNP locus of the certain animal respectively.

Further, the tail sequences of the 5 'ends of the two forward primers are respectively identical to the 3' end sequences of the two ET-labeled fluorescent primers.

Further, the fluorescent groups of the two ET-labeled fluorescent primers are different and are FAM and Texas Red-X respectively; the quenching groups are the same and are all Dabcyl.

Further, in the ET-labeled fluorescent primer shown in SEQ_1, the Dabcyl group is located between positions 21 and 22 of the sequence; in the ET-tagged fluorescent primer shown in seq_2, the Dabcyl group is located between positions 19 and 20 of the sequence.

Further, the kit further comprises a diluent, a 2×qpcr reaction premix, gDNA, and purified water.

For each SNP locus of a certain animal, three PCR primers are used, wherein one reverse primer specific to the SNP locus and two forward primers specific to the SNP locus are respectively aimed at one of wild type or variant type in the SNP locus, and the 5 'end of each forward primer is provided with a universal tail sequence which is identical to the 3' end sequence of the fluorescent report primer. In addition, two ET marked fluorescent primers are added in the amplification system, the 5' end of each fluorescent report primer sequence is connected with a fluorescent group, and meanwhile, the middle of the sequence is connected with a quenching group, the quenching groups of the two fluorescent report primers are identical, and the fluorescent groups are different, so that the fluorescent signals are different. At the same time, the fluorescent reporter primer will be incorporated into the final amplification product during PCR amplification by annealing extension to the complementary strand of the amplification product of the forward primer, and in the process will result in a physical distance of the fluorescent group from the quenching group, resulting in fluorescence detectable by the instrument.

In a second aspect, the invention provides an application of the detection kit in SNP detection of a certain type of animal or identification of a certain type of animal variety.

In a third aspect, the invention provides a method for detecting an animal SNP based on a fluorescent-labeled primer, comprising the steps of:

step 1, dissolving and diluting a forward primer, a reverse primer and two ET marked fluorescent primers by adopting a diluent, and taking pure water as a negative control;

step 2, qPCR amplification is carried out;

and 3, carrying out data analysis according to qPCR amplification results.

Further, the data analysis in step 3 includes:

1) The reaction result of the specific SNP locus is calculated as follows:

result value= (result fluorescence value-initial fluorescence value) _{[ sample of strain ]]} - (result fluorescence value-initial fluorescence value) _{[ negative control ]]}

2) Based on the reaction result calculated for the specific SNP site, the SNP site base corresponding to the fluorescence with a stronger fluorescence value is used as the judgment result.

The invention discloses a mouse SNP detection method based on fluorescent marker primers, which utilizes effective integration of allele-specific PCR and universal Energy Transfer (ET) fluorescent marker primers, and genomic DNA is amplified by PCR under the existence of the primers, wherein the allele-specific primers comprise Tail-containing forward Primer (Tail Primer) and Reverse Primer (Reverse Primer) and two universal ET fluorescent marker primers (report Primer). Two fluorescently labeled primers are Green fluorescent (Green Primer) and red fluorescent (Green Primer). The detection process is carried out by fluorescent quantitative PCR amplification and the green and red fluorescent signals of an amplification product system are evaluated, thereby achieving the identification and screening of homozygous and heterozygous individuals.

The invention has the advantages that:

1. unlike the existing Taqman probe method, the fluorescent signal is generated not by hydrolysis of a sequence-specific probe by DNA polymerase, but by the sequence of two universal fluorescent primers at each position and the sequence of a site-specific SNP upstream amplification primer, annealing of an initial amplification product and secondary neck ring structure expansion of the fluorescent primers in the process of extension integration, so that the design of the sequence-specific fluorescent probe for each SNP site is not required; the fluorescent primers at different sites can be used universally, and the cost is lower;

2. the detection can be realized on a common domestic fluorescent quantitative PCR instrument without a matched fluorescent quantitative PCR amplification instrument. The experimental result can be processed and analyzed by simple mathematical operation on a common office computer such as Excel.

Drawings

FIG. 1 is a diagram of the prior artSNP genotyping technology detection schematic diagram.

FIG. 2 is a schematic diagram of the sequence structure of 2 ET-labeled fluorescent primers according to the invention.

FIG. 3 is a schematic diagram (red fluorophores) of the present invention for SNP detection in mice using five different primers.

FIG. 4 is a schematic diagram (green fluorophores) of the present invention for SNP detection in mice using five different primers.

FIG. 5 is a graph showing SNP detection results in C57BL/6J mice in example 1.

FIG. 6 is a graph showing SNP detection results in C57BL/6J mice of comparative example 1.

FIG. 7 is a graph showing SNP detection results in C57BL/6J mice of comparative example 2.

FIG. 8 is a graph showing SNP detection results in C57BL/6J mice in comparative example 3.

FIG. 9 is a graph showing SNP detection results in C57BL/6J mice in comparative example 3.

FIG. 10 is a graph showing SNP detection results in C57BL/6J mice in comparative example 3.

Detailed Description

In the description of the present invention, it is to be noted that the specific conditions are not specified in the examples, and the description is performed under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The invention provides a mouse SNP detection kit based on fluorescent marker primers, which adopts five PCR primers to carry out amplification reaction aiming at each mouse SNP detection: 2 double-tailed allele-specific primers, one reverse primer and two universal ET-tagged fluorescent primers. The tail sequences of the 5 'ends of the 2 double-tail allele-specific primers are respectively identical with the 3' end sequences of the two ET-labeled fluorescent primers. Of the two ET-labeled fluorescent primers, one was labeled with a green group and the other was labeled with a red group. Through signal amplification, one homozygous genotype produces only green fluorescence, the other homozygous genotype produces red fluorescence, and the heterozygous genotype mixes red and green fluorescence. The sequence structures of the two ET-labeled fluorescent primers are shown as SEQ_1 and SEQ_2 respectively. The sequence structure of the two ET-labeled fluorescent primers is also given in table 1.

TABLE 1

Two different tail sequence allele-specific primers are employed for each SNP site, each comprising a segment of the 3' -terminal allele-specific nucleotide complementary to the gene to be detected. In our technical design, the distance between the allele and the reverse primer is not critical, and it is possible to range from 1-160 bp. The two ET-tagged fluorescent 5' hairpin structures contain a fluorescent group and a quenching group. Before the reaction starts, the primer is in a hairpin-like structure, and the emitted fluorescent energy is transferred from the fluorescent group to a nearby quenching group to be dissipated in the form of heat.

When the reaction proceeds, the primer will linearize in the PCR product, thereby separating the fluorescent group from the quenching group, and the fluorescent signal is significantly enhanced. In the first cycle, the 3' -a or 3' -G allele-specific primer initiates a polymerase reaction, resulting in the synthesis of a fragment with either tail 1 or tail 2 (or both products of the heterozygous DNA sample) at the 5' end. The second cycle produces a complementary sequence comprising either tail 1 or tail 2. From cycle 3, green fluorescently labeled ET primers were synthesized on the A/tail 1 fragment, while red fluorescently labeled ET primers were synthesized on the G/tail 2 product. From cycle 4, the complementary strand is synthesized using the previously reacted product as a template, resulting in the hairpin structure of the ET tagged primer to punch and the ET primer to become part of a double stranded PCR product.

According to the mouse SNP detection technology based on the fluorescent marker primers, fluorescent molecules and quenching molecules of two ET marker fluorescent primer sequences are FAM/Dabcyl and Texas Red-X/Dabcyl combinations similar to qPCR molecular beacon fluorescent probes respectively, and the sequence structure is shown in figure 2.

When SNP identification is carried out, PCR amplification occurs in the reaction tube, the tail-containing primer aiming at the specific SNP locus can obtain complete sequence matching and realize amplification, and meanwhile, the sequence at the tail of the tail is complementary with the primer marked by fluorescence, so that a fluorescent signal group and a quenching group are separated from each other, and fluorescent signal accumulation is obtained along with product accumulation, thereby achieving the purpose of being monitored in real time. FIGS. 3 and 4 show schematic diagrams of the invention for SNP detection in mice using five different primers.

After the reaction is finished, the SNP locus is judged by judging the fluorescence color with the highest increment in the reaction system. Since 20-30 SNP standard loci of mice to be detected are provided, SNP identification of two mice can be completed in a conventional PCR tray theoretically, so that the purity of the strain of the mice is determined.

The invention will now be described in further detail with reference to the drawings and to specific examples, which are given by way of illustration and not limitation.

Example 1

In this example, the application of the mouse SNP detection kit of the present invention will be specifically described by taking C57BL/6J mouse SNP detection as an example.

1. Primer dissolution and dilution:

1) All primers were synthesized at 5 nmol; the corresponding forward primer and reverse primer sequences are shown in FIGS. 5-10;

2) The stock was dissolved in DNase, RNase Free water to 50. Mu.l of 100. Mu.M stock (Original Stock Con);

3) Diluting the original Stock solution of different primers according to the concentration (Stock Con) of the Stock solution; dilution ratios, etc. are shown in table 2;

2. the gDNA of the genome of the mouse and the 2 xqPCR reaction premix are required to be extracted and prepared separately, and are not included in the patent, but the quality of the extracted gDNA is ensured to be qualified, and the 2 xqPCR reaction premix can select reliable suppliers by self:

TABLE 2

Primer type	Original Stock Con	Stock Con	Dilution degree
				Tail 1 Primer	100μM	500nM	1:200
Tail 2 Primer	100μM	500nM	1:200
				Reverse Primer	100μM	5μM	1:20
FAM	100μM	5μM	1:20
				Texas Red-X	100μM	5μM	1:20

3. Reaction system and qPCR amplification procedure:

1) qPCR amplification system: fluorescent PCR amplification systems were formulated as in table 3:

TABLE 3 Table 3

Component (A)	Stock Con	Final concentration	Volume of
				2×qPCR Pre mix	1×	1×	10μl
Tail 1 Primer	500nM	25nM	1μl
				Tail 2 Primer	500nM	25nM	1μl
Reverse Primer	5μM	250nM	1μl
				Green Primer	5μM	250nM	1μl
Red Primer	5μM	250nM	1μl
				gDNA	10ng/μl gDNA	2ng/μl gDNA	4μl
H 2 O	——	——	1μl
				Total volume	——	——	20μl

2) qPCR amplification procedure, conditions are shown in table 4:

TABLE 4 Table 4

3) Data analysis was performed according to qPCR amplification results:

4. the calculation formula of the reaction result of the specific SNP locus comprises the following steps:

result value= (result fluorescence value-initial fluorescence value) _{[ sample of strain ]]} - (result fluorescence value-initial fluorescence value) _{[ negative control ]]} The preliminary result judging method comprises the following steps: for the SNP locus result of a strain, after calculation according to the formula, the fluorescent value of the reaction result detected by the locus is strongerThe SNP locus base corresponding to the fluorescence of (2) is used as the judgment result. The results are shown in FIGS. 5-10, and the primer sequences referred to in the figures are shown as SEQ_3-SEQ_74.

In summary, the detection technology of the present invention is different from the existing Taqman probe method in that the fluorescent signal is generated not by hydrolysis of the sequence-specific probe by DNA polymerase, but by the two universal fluorescent primers at each site and the sequence of the site-specific SNP upstream amplification primer, annealing of the initial amplification product and secondary neck ring structure expansion of the fluorescent primers during the extension integration process, so that it is not necessary to design the sequence-specific fluorescent probe for each SNP site. The existing Taqman probe rule requires designing a sequence-specific Taqman fluorescent probe for each SNP site.

In addition, the forward tail-containing primer sequences, the reverse sequence primer sequences, and the fluorescent primer sequences respectively marking green and red fluorescent groups and corresponding quenching groups of all alleles of the SNP loci to be detected; the existing Taqman probe rule is based on a common Taqman fluorescent quantitative PCR method, and the upstream amplification primer of the SNP locus is a common PCR amplification primer and does not contain a sequence section for other purposes.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (9)

1. A SNP detection kit based on fluorescent labeled primers is characterized in that: comprising the following steps: forward and reverse primers for each SNP detection of a class of animals, and two ET-tagged fluorescent primers; the 5' ends of the two ET marked fluorescent primer sequences are respectively connected with a fluorescent group, and the middle of the sequences are respectively connected with a quenching group; the sequence structures of the two ET-labeled fluorescent primers are shown as SEQ_1 and SEQ_2 respectively.

2. The SNP detection kit based on fluorescent labeled primers according to claim 1, wherein: the number of forward primers for each SNP detection of a certain animal is two, and the forward primers correspond to the wild type and the variant type of each SNP locus of the certain animal respectively.

3. The SNP detection kit based on fluorescent labeled primers according to claim 1, wherein: the tail sequences of the 5 'ends of the two forward primers are respectively identical to the 3' end sequences of the two ET-labeled fluorescent primers.

4. The SNP detection kit based on fluorescent labeled primers according to claim 1, wherein: the fluorescent groups of the two ET-labeled fluorescent primers are different and are FAM and Texas Red-X respectively; the quenching groups are the same and are all Dabcyl.

5. The SNP detection kit based on a fluorescent labeled primer according to claim 4, wherein: in the ET-labeled fluorescent primer shown in SEQ_1, the Dabcyl group is positioned between the 21 st and 22 nd positions of the sequence; in the ET-tagged fluorescent primer shown in seq_2, the Dabcyl group is located between positions 19 and 20 of the sequence.

6. The SNP detection kit based on fluorescent labeled primers according to claim 1, wherein: the kit also comprises a diluent, a 2×qpcr reaction premix, gDNA and purified water.

7. Use of the detection kit according to any one of claims 1 to 6 for SNP detection or identification of a class of animals.

8. A SNP detection method based on fluorescent labeled primers is characterized in that: the method comprises the following steps:

step 1, dissolving and diluting a forward primer, a reverse primer and two ET marked fluorescent primers by adopting a diluent, and taking pure water as a negative control;

step 2, qPCR amplification is carried out;

and 3, carrying out data analysis according to qPCR amplification results.

9. The method for detecting an animal SNP based on a fluorescent-labeled primer according to claim 8, wherein: the data analysis in the step 3 comprises the following steps:

1) The reaction result of the specific SNP locus is calculated as follows:

result value= (result fluorescence value-initial fluorescence value) _{[ sample of strain ]]} - (result fluorescence value-initial fluorescence value) _{[ negative control ]]}

2) Based on the reaction result calculated for the specific SNP site, the SNP site base corresponding to the fluorescence with a stronger fluorescence value is used as the judgment result.