CN113201593B

CN113201593B - Primer probe combination product for detecting ACTN3 genotype

Info

Publication number: CN113201593B
Application number: CN202110568459.1A
Authority: CN
Inventors: 李志强; 李星豪; 陈翀
Original assignee: Guangzhou Shengshi Zhonghao Sports Technology Co ltd
Current assignee: Guangzhou Shengshi Zhonghao Sports Technology Co ltd
Priority date: 2021-05-25
Filing date: 2021-05-25
Publication date: 2024-01-23
Anticipated expiration: 2041-05-25
Also published as: CN113201593A

Abstract

The invention relates to the field of molecular biology, in particular to a primer probe combination product for ACTN3 genotype detection. The primer probe combination product comprises an outer primer pair consisting of F3 and B3, an inner primer pair consisting of FIP and BIP, and a loop primer and a probe. The kit provided by the invention is used for detecting the ACTN3 genotype, has the characteristics of simplicity and rapidness in operation, provides an effective technical means for rapid detection of the ACTN3 genotype, and has important significance.

Description

Primer probe combination product for detecting ACTN3 genotype

Technical Field

The invention relates to the field of molecular biology, in particular to a primer probe combination product for ACTN3 genotype detection.

Background

The ACTN3 gene is a well-studied sports gene, and is considered to be a gene related to explosive sports such as sprinting and weightlifting for athletes in the olympic games and the top-grade sports, which are found by statistics of athletes, wherein the ACTN3 gene carries a much higher proportion than the high-level endurance athletes. The ACTN3 gene encodes alpha-actin (alpha-actinin-3), a protein that plays a structural role in skeletal muscle and plays a role in the process of muscle contraction.

rs1815739 is a common genetic variation on the gene, and a single base on the 16 th exon of the gene changes from C to T, so that 577 th amino acid is converted from arginine to a stop codon, and the synthesis of functional alpha-actinin-3 protein is prevented. Different genotypes differ significantly in athletic ability. The CC muscle performance is good, and the explosive force is strong, is fit for making the sprint person. CT type composite muscles, which generally have explosive force, may be suitable for sprint runners. The TT-type muscle has weak explosive force and strong endurance, and is suitable for long-distance runners.

Genotyping is carried out on rs1815739, so that the athlete can be guided to select and pull out better, the waste of resources is reduced, and the best performance is obtained. The rs1815739 is quickly typed, so that on one hand, the rapid selection of athletes is facilitated, the construction of sports industry in China is recommended, and on the other hand, the effect of better guiding movement can be achieved for common citizens, and the improvement of the health of the citizens is facilitated.

Conventional SNP typing techniques, including AS-PCR, taqman probe method, sanger sequencing, mass spectrometry, etc., none of which requires no complicated instrument, so that SNP typing must be performed in a conditional central laboratory, greatly limiting the application of the techniques. The isothermal amplification technology has lower requirements on equipment, but the conventional isothermal amplification technology has poor differentiating effect on single base and is not suitable for SNP detection.

In view of this, the present invention has been made.

Disclosure of Invention

The first aspect of the present invention relates to a primer probe combination product comprising an outer primer pair consisting of F3 and B3 and an inner primer pair consisting of FIP and BIP, as well as a loop primer and a probe;

the nucleotide sequences of F3, B3, FIP and BIP are shown in SEQ ID NO: 1-4;

the nucleotide sequence of the loop primer is shown in SEQ ID NO: 5-6;

the nucleotide sequence of the probe is shown in SEQ ID NO: 7-8, wherein 8 th and 9 th nucleotides at the 5' ends of the two probes are ribonucleotides.

The second aspect of the invention relates to a kit comprising a primer probe combination product as described above.

The third aspect of the invention relates to the use of a primer probe combination product as described above for the preparation of a reagent or kit for the genotyping of ACTN 3.

Compared with the prior art, the invention has the beneficial effects that:

1) The kit provided by the invention is used for detecting the ACTN3 genotype, has the characteristics of simplicity and rapidness in operation, provides an effective technical means for rapid detection of the ACTN3 genotype, and has important significance.

2) The kit provided by the invention adopts a ribonuclease HII report system to synchronously react with isothermal amplification, can realize effective amplification and detection of target genes at 55-65 ℃, and does not need temperature change or complex instruments. The reaction time is short, the reaction can be completed within 10-40min, and the detection result is accurate and specific.

3) In the method, ribonuclease HII has high specificity to the probe and the target sequence, and only the amplified product and the probe sequence are completely complementary to the probe can be cut off, so that a report signal is generated, the identification effect on single base change is better than that of other primer-based identification methods, and the specificity and accuracy of the result are greatly improved.

4) The reaction conditions required by detection are simple, the requirements on hardware are low, the cost is low, the method has no harm to the subjects, and the method is favorable for wide popularization of the technology.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of verification of different probes in one embodiment of the invention;

FIG. 2 shows the detection result of a real sample according to an embodiment of the present invention;

FIG. 3 shows the stability test results of a cartridge according to one embodiment of the present invention;

FIG. 4 shows the results reported using a colloidal gold immunochromatographic card according to an embodiment of the present invention.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The invention relates to a primer probe combination product, which comprises an outer primer pair consisting of F3 and B3, an inner primer pair consisting of FIP and BIP, and a loop primer and a probe;

the nucleotide sequences of F3, B3, FIP and BIP are shown in SEQ ID NO: 1-4;

the nucleotide sequence of the loop primer is shown in SEQ ID NO: 5-6;

the nucleotide sequence of the probe is shown in SEQ ID NO: 7-8, wherein the 8 th nucleotide and the 9 th nucleotide of the 5' end of each probe are ribonucleotides.

FIP is a primer generated in such a way that it has an F2 region complementary to the F2c region of the target sequence at the 3-terminus and a sequence identical to the F1c region of the target gene at the 5-terminus.

F3 is a primer generated in such a manner that it has an F3 region complementary to the F3c region of the target gene.

BIP is a primer generated in such a way that it has a B2 region complementary to the B2c region of a target sequence at the 3 'end and a sequence identical to the Blc region of a target gene at the 5' end.

B3 is a primer generated in such a manner that it has a B3 region which is complementary to the B3c region of the target gene.

When the primer set of the present invention is used, one or two kinds of circular primers (LF primers or LB primers) may be added in order to accelerate the nucleic acid amplification reaction. Such a circular primer is designed so as to anneal to a region between F1 and F2 or a region between B1 and B2, and then added to the LAMP reaction system. In this way, these primers bind to loop portions that are not used in the nucleic acid amplification process, so that the nucleic acid reaction is promoted using all loop portions as the origin, thereby accelerating the nucleic acid amplification reaction.

The primer probe combination product can be matched with ribonuclease HII to realize more sensitive and specific diagnosis of ACTN3 genotype.

Ribonuclease HII (RNase HII) is an endonuclease suitable for nicking the 5' -end of ribonucleic acid within double-stranded DNA to produce 5' -phosphate and 3' -hydroxyl ends. RNase HII specifically recognizes an RNA base in a DNA double strand and breaks a phosphodiester bond connecting the DNA bases in the 5 '-direction of the RNA base, resulting in a nick of the DNA double strand in the 5' -direction of the RNA base. In organisms, DNA repair is initiated by the cleavage, RNA bases in double-stranded DNA can be removed, and the accuracy of genetic information is ensured. The invention designs a single-stranded probe based on the characteristic of RNase HII on strict pairing requirement of a substrate, wherein RNA base is embedded in the probe, and the RNase HII is applied to a report system of an amplification system. Since RNase HII recognizes and cleaves only double strands, it does not act on a single-stranded probe. Under a proper temperature, when the single-stranded DNA complementarily paired with the probe exists in the environment, the probe can be hybridized with the single-stranded DNA, the probe is cut off at a specific position by RNase HII, the melting temperature of the cut-off probe is reduced, the single-stranded DNA can not be stably combined with the complementary single-stranded DNA and can be dissociated, and the complementary single-stranded DNA can be combined with a new probe to start a new cycle, so that signal expansion is realized. In the whole process, the input of the single-stranded DNA complementary to the probe is converted into the output of the probe breakage through the cleavage of RNase HII, the specificity of the generated product is ensured through the complementary pairing principle of bases, meanwhile, the amplification of signals is realized, and the detection result is obtained through detecting the breakage condition of the probe.

In one aspect, useful primers and probes include those that hybridize to SEQ ID NO: 1-8 has a nucleotide sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. Such primer and probe modifications are also contemplated, as well as the introduction of base substitutions, and can be prepared according to standard techniques.

The term "base substitute" is a structure that contains no bases and that is attached upstream and downstream of a nucleic acid strand to maintain the integrity of the probe as a whole, nor does it interfere with hybridization of the nucleic acid strand. For example, when polymorphic sites that do not need to be detected are present in the target sequence, degenerate analysis can be achieved by substituting bases at positions corresponding to the probes with base substitutes. Base substitutes are commonly used as deoxynucleotide spacers (dSpacer) or C3 Spacer (Spacer). The number of base substitutes may be 1, 2, 3, 4, 5, 6.

The term "% identity" in the context of two or more nucleotide sequences or amino acid sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example,% identity is the entire length of the coding region relative to the sequences to be compared.

For sequence comparison, typically one sequence is used as a reference sequence, and the test sequence is compared to that sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. The percent identity can be determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al, 1990, J Mol Biol 215:3, 403-410; altschul et al, 1997, nucleic Acids Res25:17, 3389-402).

The primer and probe modification may be performed by a known method. Modified versions of these primer and/or probe sequences can include, by way of non-limiting example, adding one or more nucleotides to the 5 'end, one or more nucleotides to the 3' end, one or more nucleotides to the 5 'and 3' ends, adding tails, shortening the sequence, extending the sequence, shifting the sequence several bases upstream and downstream, or any combination thereof.

Base modifications such as 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2 ' -deoxyadenosine, C-5 propynyl-deoxycytidine, C-5 propynyl-deoxyuridine, 2-amino-2 ' -deoxyadenosine-5 ' -triphosphate, 2, 6-diaminopurine (2-amino-dA), inverted dT, inverted dideoxy-T, hydroxymethyl dC, iso-dC, 5-methyl dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNA's) and include at least one mismatched base at one of the bases, or at least one of the bases is replaced with an RNA base, to effect, for example, an increase in nucleic acid interactions at the 3' end of the mutant-specific primer to increase Tm. The modified probe should retain the ability to distinguish between the mutation site to be detected and the wild-type site.

Furthermore, with the exception of the specifically emphasized sites, the probes generally consist of DNA bases, as is generally understood by those skilled in the art. Similarly, the primer is composed of DNA bases.

In some embodiments, the RNA base of the probe corresponds to a SNP site base.

In some embodiments, the RNA base of the probe corresponds to the base adjacent to the SNP site.

In some embodiments, blocking of the 3 'end may also be achieved by adding a nucleic acid sequence to the 3' end of the probe that is unrelated to the target sequence.

In some embodiments, the arm modification is selected from any of ethylene glycol, C9 arm (Spacer 9), C18 arm (Spacer 18), dideoxy arm [1',2' -Dideoxyribose (dSpacer) ], C3 arm (C3 Spacer).

In some embodiments, the spacer modification is selected from the group consisting of a C3 spacer.

In some embodiments, the DNA bases of the probe on both sides of the ribonucleotide are labeled with a fluorescent group and a quenching group, respectively.

In some embodiments, the fluorophore of the probe is independently selected from any of AMCA, pacific Blue, atto 425, BODIPY FL, FAM, alexa Fluor 488, TET, JOE, yakima Yellow, VIC, HEX, quasar 570, cy3, NED, TAMRA, ROX, aqua phoor 593, texas Red, atto 590, cy5, quasar 670, cy5.5, and Cy 5.5.

In some embodiments, the quenching group of the probe is independently selected from any of BHQ1, BHQ2, BHQ3, dabcyl, eclipse, and MGB.

In some embodiments, the fluorescent groups with the same or similar excitation/emission wavelengths are labeled for different allelic forms of the probes, and different probes are respectively amplified and detected by a physical interval mode such as a branched pipe.

In some embodiments, different allelic probes are labeled with fluorescent groups of different excitation/emission wavelengths, and the different probes react in the same liquid system, distinguishing the genotype of the sample by the difference in fluorescent signals.

In some embodiments, the DNA bases of the probe on both sides of the ribonucleotide are each labeled with a molecule for interaction with a protein, said molecule being selected from any one of biotin, digoxin and FITC.

The invention also relates to a kit comprising the primer probe combination product.

The term "kit" refers to any article of manufacture (e.g., package or container) comprising at least one device, which may further comprise instructions, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.

In some embodiments, the kit further comprises at least one of the following ingredients:

ribonuclease HII, sample lysate, control, reagents required by isothermal amplification reaction and immunochromatography detection card.

Depending on the reaction temperature, different RNase HII may be used. In some embodiments, the reaction temperature is 50℃to 70 ℃, preferably 55℃to 65 ℃, using a thermophilic, e.gThermus thermophilus、Pyrococcus abyssiRNase HII from source.

In some embodiments, the reagents required for the isothermal amplification reaction comprise at least one of the following components:

DNA polymerase, dNTPs and Mg with strand displacement capability ²⁺ 、Na ⁺ 、K ⁺ Buffer component, dithiothreitol.

In some embodiments, the DNA polymerase is Bst DNA polymerase.

The enzymes used in the present invention may be wild-type enzymes or mutant enzymes (typically with enhanced enzymatic activity or other preferred properties, such as better thermostability) that are sequence optimized.

The components are preferably realized in lyophilized form, for example in the form of one or more so-called lyophilized beads. Lyophilization beads are generally understood to mean lyophilisates which are pressed into spheres after manufacture, after which the substance is usually present as a powder. Thus, the components required for a isothermal reaction, in particular the DNA polymerase, the nucleic acid component and the reaction buffer component, can be provided, for example, in lyophilized form. In this way, the isothermal reaction process can be started directly in a very user-friendly manner by adding the sample to be quantified and optionally other desired components. In particular, the provision of a lyophilized form is very advantageous for automated applications. In some embodiments, the reagents required for the isothermal amplification reaction are lyophilized reagents.

In some embodiments, the reporter system of the immunochromatographic detection card is selected from any one of colloidal gold, latex, colloidal iron, labeled fluorescent molecules, and fluorescent quantum dots.

In some embodiments, a single immunochromatographic assay card detects for a single allele.

In some embodiments, a single immunochromatographic assay card detects for two or more allelic forms.

According to yet another aspect of the invention, the invention also relates to the use of a primer probe combination product as described above for the preparation of a diagnostic reagent or kit for detecting the ACTN3 genotype.

The invention also relates to the use of a primer probe combination product as described above, or a kit as described above, for detecting ACTN3 genotype.

In some embodiments, the temperature detected is 50 ℃ to 70 ℃, preferably 55 ℃ to 65 ℃, and optionally 60 ℃.

The temperature detected may be provided by a water bath.

In some embodiments, the time of detection is 10-60 minutes; for example 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 50 minutes; preferably 30 minutes.

Such use may be used to detect ACTN3 genotype.

The sample used to detect ACTN3 genotype is preferably an oral specimen (e.g., oral swab, etc.) of the subject, and the noninvasive sampling is effective in protecting the subject.

Embodiments of the present invention will be described in detail below with reference to examples.

Example 1 establishment of a method for detecting ACTN3 genotype by fluorescence method

And (3) performing conservation analysis on the sequence around the SNP locus rs1815739 of the ACTN3 gene, and performing primer and probe design aiming at rs 1815739. The primers and probes were designed as follows:

group A:

primer F3:5'-TGTTGCCTGTGGTAAGTGG-3' (SEQ ID NO: 1)

Primer B3:5'-GGGCTGAGGGTGATGTAGG-3' (SEQ ID NO: 2)

Primer FIP:5'-TGCCTTGAACTGATCGTGCGCGGGACACCAGCTGACACT-3' (SEQ ID NO: 3)

Primer BIP:5'-CCATCATGGGCATCCAGGGTATTGGTGGAGCAGGGCC-3' (SEQ ID NO: 4)

Primer LF:5'-AGGCTCTGGGGACGACA-3' (SEQ ID NO: 5)

Primer LB:5'-GAGATCCAGAAGATCTGCCAGA-3' (SEQ ID NO: 6)

Primer probe C:5'-CGCTC [ dT-BHQ1] C [ rG ] GT [ dC-FAM ] AGCC-3' (SEQ ID NO: 7)

Primer probe T:5'-TCGCTC [ dT-BHQ1] C [ rA ] GT [ dC-FAM ] AGCCT-3' (SEQ ID NO: 8)

Primer probe C (17 base): 5'-TCGCTC [ dT-BHQ1] C [ rG ] GT [ dC-FAM ] AGCCT-3'

Primer probe C (13 base): 5'-GCTC [ dT-BHQ1] C [ rG ] GT [ dC-FAM ] AGC-3'

Primer probe T (19 base): 5'-CTCGCTC [ dT-BHQ1] C [ rA ] GT [ dC-FAM ] AGCCTC-3'

Primer probe T (15 base): 5'-CGCTC [ dT-BHQ1] C [ rA ] GT [ dC-FAM ] AGCC-3 ]'

The primer group is matched with probes with different lengths, and respectively carries out the amplification of C, T allelic plasmids and blank (NF-H ₂ O) detecting.

The results are shown in FIG. 1. The different probes have no non-specific reaction to the blank group detection. For probes of different lengths, long probes will result in reduced allelic discrimination and short probes will have lower positive signals. In the subsequent examples of the present invention, the reaction system was prepared using primers and a medium-length probe (C-15 base, T-17 base).

Establishment of detection method

The invention constructs a kit for detecting ACTN3 genotype based on a isothermal amplification system and a ribonuclease HII report system, which comprises a sample lysate, the isothermal amplification system and a ribonuclease HII report system reaction module, wherein the sample lysate contains a Tris-HCL buffer system, naOH, SDS, EDTA, guanidine isothiocyanate, tween80 and triton; the optimal allocation ratio of the reaction system in the reaction module of the isothermal amplification system and ribonuclease HII report system is shown in table 1, and the optimal allocation ratio comprises the fluorescent labeled probe and the primer; the control was a plasmid containing the ACTN3 target gene, and the C, T control nucleotides at position rs1815739 were C and T, respectively.

TABLE 1 reaction System ratio of reaction modules

Sequence number	Component (A)	Concentration of
			1	Tris-HCl（pH 8.8）	20 mM
2	Ammonium sulfate	10 mM
			3	Potassium chloride	50 mM
4	Magnesium sulfate	2 mM
			5	Tween 20	0.10%
6	Group A primer F3	0.2 μM
			7	Group A primer B3	0.2 μM
8	Group A primer FIP	1.6 μM
			9	Group A primer BIP	1.6 μM
10	Group A primer LF	0.4 μM
			11	Group A primer LB	0.4 μM
12	Group A primer probe	0.1 μM
			13	Bst polymerase	16 U

The reaction conditions of the reaction system are as follows: reacting for 10-60min at 50-65 ℃.

The optimal reaction conditions are as follows: the reaction was carried out at 63℃for 30min.

The detection flow of the kit is as follows:

step one, sample processing. Shaking and uniformly mixing 15 mu L of sample lysate and 10 mu L C control/T control/sample to be detected, and standing at room temperature for 5 min;

step two, adding all the products of the step one into a reaction module of a constant-temperature amplification system and a ribonuclease HII report system, covering a tube cover, vibrating and centrifuging, and immediately detecting; the reaction procedure is: the fluorescent signal is collected at 63 ℃ for 1 minute and 30 cycles, and the detection can be completed within 30 minutes;

and step three, judging the result.

(1) C control: the typical amplification curve appears in the probe system C, and the fluorescence value of the end point is higher than or equal to the fluorescence value of the starting point by more than 3 times; no amplification curve appears in the T probe system, or the end point fluorescence value is 3 times lower than the starting point fluorescence value; the two points are met as effective results;

(2) t control: typical amplification curves appear in the T probe system, and the fluorescence value of the end point is higher than or equal to that of the starting point by more than 3 times; no amplification curve appears in the probe system, or the end point fluorescence value is 3 times lower than the starting point fluorescence value; the two points are met as effective results;

(3) the sample to be tested:

a. if the end point fluorescence value in the single probe system is more than or equal to 3 times of the start point fluorescence value, judging that the single probe system is homozygote of the same type;

b. if the end point fluorescence values in the two probe systems are higher than or equal to the start point fluorescence value by more than 3 times, judging the two probe systems as heterozygotes;

c. if the end point fluorescence values in the two probe systems are lower than 3 times of the starting point fluorescence values, judging that the two probe systems are abnormal, and re-detecting or re-sampling and detecting.

Example 2 analysis of real samples by ACTN3 genotyping fluorescence detection kit

The detection effect of the kit on the real sample was examined by using 4 cases of oral swabs (CC type 1 case, TT type 1 case, CT type 2 case) with known genotypes.

The specific operation is as follows:

4 cases of oral swabs were taken and tested against C, T, and the results were recorded.

The results are shown in FIG. 2. C. The T control detection result is normal. The detection results of the 4 samples are identical to the known genotypes. The kit is shown to be accurate and effective in detecting a real oral swab sample.

Example 3 stability test of ACTN3 genotype fluorescence detection kit

The liquid reagent needs to be stored at low temperature and can not be repeatedly frozen and thawed. The kit is characterized in that a fluorescent reaction module comprising a constant-temperature amplification system and a ribonuclease HII reporting system is dried into a powdery reagent in vacuum, the freeze-dried powdery reagent can be stored at normal temperature, the cost of cold chain transportation and low-temperature storage is saved, and the operation is simpler. The stability of the ACTN3 genotype fluorescence detection kit was verified in this example.

The specific operation is as follows:

eight-tube containing lyophilized reagents were sealed in aluminum foil bags containing a desiccant and stored in a 37 ℃ incubator. Freeze-dried reagents were taken and tested at 0 day, 30 day, 90 day, 180 day, respectively.

Taking C control and T control for detection respectively, and recording the results.

The results are shown in FIG. 3. The reagent freeze-dried powder of the reaction module stored for 0 day, 30 days, 90 days and 180 days is tested respectively, and the detection results of the reagent in the kit of the invention after freeze-drying are all in line with each other in 0 day, 30 days, 90 days and 180 days. The reagent in the kit of the invention can be stably stored for at least 3 months at 37 ℃ after freeze-drying.

Example 4 establishment of a method for detecting ACTN3 genotyping colloidal gold assay kit

Compared with detection equipment for judging through fluorescence, the immunochromatography card with the color change judging through naked eyes, such as colloidal gold, latex and the like, omits a module for fluorescence excitation and reading, has lower cost and is more suitable for a detection scene outside a laboratory.

In the embodiment, a detection kit using a colloidal gold immunochromatographic card as a reporting system is constructed.

The kit configuration of this example was substantially identical to that of example 1, except that: (1) The probe modification is adjusted from fluorescent labeling to labeling that can be recognized by an immunochromatographic card; (2) Based on the composition of the kit in the example 1, a colloidal gold immunochromatographic card for detecting an amplification product is added.

Probe sequences used in this example:

primer probe C:5 '-biotin-CGCTCTCTC [ rG ] GTCAGCC-digoxin-3' (SEQ ID NO: 7)

Primer probe T:5'-biotin-TCGCTCTC [ rA ] GTCAGCCT-digoxin-3' (SEQ ID NO: 8)

The colloidal gold immunochromatographic card used was divided into 3 regions: sample pad, NC membrane, absorbent pad.

The sample pad contains nano gold particles marked with digoxin antibody. The NC is respectively provided with a quality control line (C line) and a detection line (T line) in sequence, the C line is fixed with streptavidin, and the T line is fixed with a goat anti-mouse secondary antibody, so that the digoxin antibody of the gold standard can be identified.

When no allelic form consistent with the probe exists in the sample, the probe remains intact, a gold labeled digoxin antibody-probe-streptavidin complex is formed, all the nano gold particles are captured by the C line, the C line develops color, and the T line does not develop color. When the allelic form consistent with the probe exists in the sample, the probe is partially/completely cut off, the formation of the gold labeled digoxin antibody-probe-streptavidin complex is reduced or impossible, the nano gold particles cannot be completely captured by the C line, the nano gold particles which cannot be captured continue to flow and then are captured by the T line, the C line is colored or not colored, and the T line is colored.

The detection flow is as follows:

step one, sample processing. Shaking and uniformly mixing 30 mu L of sample lysate and 20 mu L of control/sample to be detected, and standing at room temperature for 5 min;

step two, adding 25 mu L of the product obtained in the step one into a C, T reaction module, covering a tube cover, vibrating and centrifuging, and immediately reacting; the reaction procedure is: 30 minutes at 63 ℃;

step three, respectively adding the products of each tube of the step two to sample pads of different colloidal gold chromatographic cards, waiting for 5 min, and observing the results;

and step three, judging the result.

(1) C control: c, detecting card of system loading: developing by a T line; t system sample detection card: the color of the C line is developed, and the T line is not developed;

(2) t control: c, detecting card of system loading: the color of the C line is developed, and the T line is not developed; t system sample detection card: developing by a T line;

(3) the sample to be tested:

a. if the T line of the detection card of the single probe system is developed, judging that the single probe system is homozygote of the allelic type;

b. if the T lines of the detection cards of the two probe systems are developed, judging that the two probe systems are heterozygotes;

c. if the C, T line in any system does not develop color, or the T line in both systems does not develop color, judging that the system is abnormal, and re-detecting or re-sampling and detecting.

The detection results are shown in figure 4, and the detection results of the colloidal gold immunochromatographic card are identical with the known genotypes. Indicating the validity of the system.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as 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.

SEQUENCE LISTING

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Claims

1. The kit for detecting the rs1815739 site nucleotide of the ACTN3 gene comprises a primer probe combination product, a sample lysate, a control, a constant-temperature amplification system and a ribonuclease HII report system reaction module;

the primer probe combination product comprises an outer primer pair consisting of F3 and B3, an inner primer pair consisting of FIP and BIP, and a loop primer and a probe;

the nucleotide sequences of F3, B3, FIP and BIP are shown in SEQ ID NO: 1-4;

the nucleotide sequence of the loop primer is shown as SEQ ID NO: 5-6;

the nucleotide sequence of the probe is shown as SEQ ID NO: 7-8, wherein 8 th and 9 th nucleotides of the 5' ends of the two probes are ribonucleotides;

the sample lysate contains a Tris-HCL buffer system, naOH, SDS, EDTA, guanidine isothiocyanate, tween80 and triton;

the reagent required by the isothermal amplification reaction comprises the following components: ribonuclease HII, bst DNA polymerase, dNTPs, mg ²⁺ 、Na ⁺ 、K ⁺ Buffer component, dithiothreitol;

the isothermal amplification system and a fluorescence reaction module of the ribonuclease HII report system are dried into powdery reagents in vacuum;

the reaction system of the reaction module comprises the following components:

Tris-HCl pH 8.8 20 mM； Ammonium sulfate 10 mM； Potassium chloride 50 mM； Magnesium sulfate 2 mM； Tween 20 0.10%； Group A primer F3 0.2 µM； Group A primer B3 0.2 µM； Group A primer FIP 1.6 µM； Group A primer BIP 1.6 µM； Group A primer LF 0.4 µM； Group A primer LB 0.4 µM； Group A primer probe 0.1 µM； Bst polymerase 16 U

。