CN114606310A - Detection method and kit for frame shift mutation of human DEFB126 gene - Google Patents
Detection method and kit for frame shift mutation of human DEFB126 gene Download PDFInfo
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Abstract
The invention relates to a detection kit and a method for detecting the genotype of a frame shift mutation site rs11467417 and rs11467497 of a human beta defensin 126 gene (DEFB126), wherein the kit comprises (1) specific primers designed according to sequences on two sides of the rs11467417 site; (2) specific primers designed according to sequences on two sides of the locus rs 11467497; (3) a specific probe designed according to the adjacent sequence of the rs11467417 locus; (4) probes respectively designed according to the adjacent sequence of the locus rs11467497 and the sequences of the locus before and after mutation; (5) and (3) PCR reaction reagents. The kit and the detection method can accurately detect 6 genotypes of the DEFB126 genes (rs11467417 and rs11467497) by utilizing one PCR reaction and three probes.
Description
Technical Field
The invention relates to a detection method and a kit for frame shift mutation of a human DEFB126 gene, which are used for detecting genotypes of frame shift mutation sites rs11467417 and rs11467497 of a human beta defensin 126 gene (DEFB 126).
Background
beta-Defensins (DEFB) are an important natural immune molecule, and 39 DEFB genes are co-generated in human beings and are mainly expressed in epididymis tissues.
Human DEFB126 is a cysteine-rich secreted glycoprotein that is secreted by the major epithelial cells of the epididymis, covering the entire surface of the sperm. Unlike other DEFB family members, DEFB126 protein is a highly glycosylated modified short peptide with multiple O-link oligosaccharide modification sites at the C-terminus, and it has been found that sialic acid residues or acidic sperm antigens adsorb to the surface of the DEFB protein during its transit through the epididymis, thus enriching the surface with negative charges and facilitating its transit through the similarly enriched cervical mucus of women when the sperm enters the reproductive tract. Theodere L et al demonstrated that DEFB126 is critical to penetration and efficient movement of sperm in cervical mucus before and after ovulation in cynomolgus monkeys. After DEFB126 protein is removed from the sperm, the number of sperm entering and passing through the cervical mucus is significantly reduced. The addition of soluble DEFB126 protein to the sperm surface fully restores the ability of the sperm to cross cervical mucus.
In 2011, Tollner and the like firstly find that two nucleotides (CC /) deletion mutation (SNP number is rs11467417) exist in a coding region of a DEFB126 gene of a human, which causes a problem of protein generation, namely a homozygote mutation (abbreviated as 2DD) type male has high and low DEFB126 protein expression level on sperms, and even no DEFB126 protein expression can be detected on sperms of some patients. The 2DD frameshift mutation is also a high-frequency mutation, and about 20% of men in China (data from 509 cases of newly-married men in Anhui province in China) are homozygous mutations of 2DD type, and the natural fertility of the mutations is remarkably lower than that of the two other genotypes, namely 2WW type (wild type) or 2WD type (heterozygous mutant type).
There is another 4 nucleotide frameshift mutation (CAAA /) 151bp upstream of locus rs11467417 (SNP encoded as rs11467497, FIG. 2). Homozygous mutant type (referred to as 4DD) men do not express DEFB126 protein on sperms, are more serious DEFB126 gene expression defects, do not find the mutation in fertile men, and only appear in infertile and infertile donated males. However, the frequency of this mutation is not high, and the incidence of type 4DD in infertile men is about 0.1-0.3%.
The inventor of the application researches two mutations of DEFB126 gene (defensin beta 126) of 303 cases of Chinese male sperms and the capability of penetrating HA, and finds that: DEFB126 rs11467417 homozygous mutant donor sperm (2DD, n-42) showed a reduced ability to penetrate HA compared to male sperm of the other two genotypes (2WW, n-29 and 2WD, n-232) with a significant difference (P-0.015); the penetration rate of the cervical mucus of DEFB126 rs11467497 homozygote mutant donor sperm (4DD, n ═ 1) is 0%.
It should be noted that the reduction in cervical mucus penetration of sperm may not be the only reason for the reduced fertility of 2DD or 4DD homozygous mutations. DEFB126 also helps to protect sperm from recognition by the female immune system, promoting attachment of sperm to the oviduct epithelium. Perhaps, DEFB126 gene 2DD or 4DD is responsible for infertility by affecting various aspects of sperm function.
From the above, it can be seen that it is of great significance to develop a kit and a detection method for simultaneously detecting the genotypes of the frame shift mutation sites rs11467417 and rs11467497 of the human beta defensin 126 gene (DEFB 126).
Disclosure of Invention
The invention aims to provide a method and a kit for detecting the genotype of the frame shift mutation sites rs11467417 and rs11467497 of a human beta defensin 126 gene (DEFB 126).
The human DEFB126 gene frameshift mutation detection kit comprises:
(1) specific primers designed according to sequences on two sides of the locus rs 11467417;
(2) specific primers designed according to sequences on two sides of the locus rs 11467497;
(3) a specific probe designed according to a neighboring sequence of the rs11467417 locus;
(4) probes respectively designed according to the adjacent sequence of the locus rs11467497 and the sequences of the locus before and after mutation;
(5) PCR reaction reagent;
optionally (6) a positive control;
optionally (7) a negative control,
wherein, the specific probe designed according to the adjacent sequence of the rs11467417 locus, the probes respectively designed according to the adjacent sequence of the rs11467497 locus and the locus sequences before and after mutation are respectively marked with different fluorescent luminous groups, and the kit utilizes a PCR reaction to detect a plurality of genotypes of the frame shift mutation loci rs11467417 and rs 11467497.
The PCR reaction reagent may include Taq polymerase, UNG, dNTP for PCR amplification. In each unit of kit, the Taq polymerase may be 1-3 units, e.g., 1 or 2 units; the UNG enzyme may be 1-3 units, e.g., 1 or 2 units; the dNTP may be 0.2-0.6mM, e.g., 0.4 mM.
The concentration of each primer or probe may be 0.02 to 1. mu.M, further 0.1 to 0.4. mu.M, for example 0.2. mu.M or 0.3. mu.M or 0.4. mu.M.
In one embodiment, the sequences at both sides of the wild-type rs11467417 site (the sequences at both ends of the upstream and downstream of rs11467417) are shown as sequence 1 in the sequence table, and the sequences at both sides of the mutant-type rs11467417 site (the sequences at both ends of the upstream and downstream of rs11467417) are shown as sequence 2 in the sequence table.
In general, the rs11467417 site includes the wild type rs11467417 site and the mutant rs11467417 site.
In one embodiment, the sequences flanking the wild type rs11467497 site (sequences at two end parts of the upstream and downstream of rs11467497) are shown as sequence 3 in the sequence table, and the sequences flanking the mutant rs11467497 site (sequences at two end parts of the upstream and downstream of rs11467497) are shown as sequence 4 in the sequence table.
In one embodiment, the positive control is a mixture of two-site heterozygote U-containing DNA (Rs11467417PCR product sequence and synthetic sequence with Rs11467497PCR product sequence with T replaced in whole or in part with U). In one embodiment, the sequence of the Rs11467417PCR product is shown as the sequence 5 in the sequence table, and the sequence of the Rs11467497PCR product is shown as the sequence 6 in the sequence table. Preferably, the mixture further comprises (e.g. 8, 9, 10 or 11 units) UNG enzyme inhibitor UGI.
In one embodiment, the base sequences of the specific primers designed according to the sequences flanking the rs11467417 locus are, for example, sequence 7(5 'primer) and sequence 8 (3' primer) in the sequence listing and shown in fig. 1.
In one embodiment, the base sequences of the specific primers designed according to the sequences flanking the rs11467497 site are shown as sequence 9(5 'primer) and sequence 10 (3' primer) in the sequence listing and in fig. 2.
In one embodiment, the base sequence of the specific probe designed based on the sequence adjacent to the rs11467417 site is shown as sequence 11 in the sequence table and fig. 3, wherein the upper case sequence is a gene-specific sequence and the lower case sequence is an additional sequence that helps to form a stem-loop.
In one embodiment, the sequences of the probes designed based on the sequence adjacent to the rs11467497 site and the site sequences before and after mutation are shown as sequences 12 and 13 in the sequence listing and fig. 4 and 5, wherein the upper case sequence is a gene-specific sequence and the lower case sequence is an additional sequence that helps to form a stem loop.
In one embodiment, a specific probe designed according to the sequence adjacent to the rs11467417 site labels a VIC fluorophore; the sequence probe before rs11467497 mutation is labeled with CY5 fluorophore, and the sequence probe after rs11467497 mutation is labeled with FAM fluorophore.
In one embodiment, the negative control is water or TE buffer.
According to a second aspect of the present invention, there is provided a method for detecting a frame shift mutation in the human DEFB126 gene, the method comprising the steps of:
(1) extracting a DNA sample;
(2) amplifying the DNA sample to be detected, the optional negative control and the optional positive control by using specific primers designed according to the sequences on two sides of the locus rs11467417 and specific primers designed according to the sequences on two sides of the locus rs11467497 respectively;
(3) labeling with specific probe designed based on the adjacent sequence of rs11467417 site and probes designed based on the adjacent sequence of rs11467497 site and site sequences before and after mutation, wherein the three probes are respectively different fluorescent labels (fluorescent dyes);
(4) detecting by using the probe according to the corresponding relation between the mutation site and the fluorescence channel;
(5) and (4) carrying out typing according to the dissolution temperature of the melting curve.
Regarding step (5), since the probe is designed according to the wild-type sequence of rs11467417, for example, the wild-type sequence has a higher Tm, the wild-type sequence is detected at the high temperature Tm of the same channel, the mutant type sequence is detected at the low temperature Tm, and the mixed type sequence is detected at both the high temperature Tm and the low temperature Tm; for rs11467497 gene, probes are designed according to wild type and mutant type, respectively, and a wild type probe is a wild type probe when a peak is detected at the wild type Tm, a mutant type probe when a peak is detected at the mutant type Tm, and a mixed type probe when peaks are detected at the wild type Tm and the mutant type Tm.
In one embodiment, a specific probe designed according to the sequence adjacent to the rs11467417 site labels a VIC fluorophore; the sequence probe before rs11467497 mutation is labeled with CY5 fluorophore, and the sequence probe after rs11467497 mutation is labeled with FAM fluorophore, and the results are judged according to the following criteria.
Note: w, means the wild (wild) type allele; d, means deletion (deletion) mutant allele.
In this application, "optional" means the presence or absence of a subsequent component.
THE ADVANTAGES OF THE PRESENT INVENTION
The kit and the detection method can accurately detect 6 genotypes of DEFB126 genes (rs11467417 and rs 11467497). The genotype of 2 sites of human DEFB126 gene and reproduction related genes is simultaneously detected by a fluorescence PCR reaction. The product adopts a molecular beacon probe, and the genotype can be accurately and conveniently judged according to the characteristic melting temperature of the probe. The technology overcomes the defects of the Taqman technology of monopoly technology for detecting gene mutation. The Taqman technique requires 2 probes to achieve genotyping of a site. There are two methods of typing. Typing is carried out according to the difference of Ct values of the two probes, and typing is carried out according to the cluster statistical analysis of the fluorescence values of the end points of the 2 probes. Both of these methods have significant drawbacks for use in diagnostic reagents. According to the difference type of the Ct values of the two probes, the accurate Ct value is depended on, and the Ct value changes along with the change of the sample concentration, so that the product judgment standard is not easy to form. By means of end point fluorescence value typing of 2 probes, reliable cluster analysis can be carried out only by detecting a plurality of samples once, and application scenes of products are limited. The product is classified by the characteristic melting temperature of the probe, which is the physical characteristic value of the probe, does not change along with the change of the concentration of the sample, does not need to set a threshold value, and does not need to refer to the results of other samples. The product typing judgment standard is simple and clear, and the result is accurate. The product uses positive control, and avoids typing error caused by positive control pollution. The kit and the detection method can simultaneously and accurately detect 6 genotypes of the DEFB126 genes (rs11467417 and rs11467497) by utilizing one PCR reaction and three probes.
Drawings
FIG. 1 shows the base sequence of a specific primer designed based on the sequence flanking site rs 11467417.
FIG. 2 shows the base sequence of a specific primer designed based on the sequence flanking site rs 11467497.
FIG. 3 shows the base sequence of a specific probe designed based on the adjacent sequence of site rs 11467417.
FIGS. 4 and 5 show the sequences of probes designed based on the sequence adjacent to the rs11467497 site and the site sequences before and after mutation, respectively.
FIG. 6 shows the melting temperature of the melting curve of the negative control.
FIG. 7 shows the melting temperature of the melting curve of the positive control.
FIGS. 8-15 show the melting temperature and genotyping for the melting curves of samples P1-P8, respectively.
Detailed Description
The invention is further illustrated by the following specific examples.
Example 1
Designing specific primers (specific primer sequences) according to sequences at two sides of the locus rs11467417 and rs 11467497; designing a specific probe (the specific probe sequence is matched with a wild type) according to the adjacent sequence (containing sites) of the rs11467417 site, and labeling a VIC fluorescent group on the probe; probes (respectively designed probe sequences) are respectively designed according to the adjacent sequence of the rs11467497 site and the site sequences before and after mutation, the sequence probe before mutation marks CY5 fluorescent group, and the sequence probe after mutation marks FAM fluorescent group. And simultaneously amplifying sequence segments where the 2 frameshift mutation sites are located by a multichannel real-time fluorescence PCR method, performing melting curve analysis on the amplified segments, reading the Tm value of each fluorescence channel, and finally judging the genotype of each frameshift mutation site according to the Tm value of each channel.
TABLE 1 kit composition
Before unsealing, the kit is stored at the temperature below-15 ℃ and the validity period is 1 year (tentative); sealing, and storing at 2-10 deg.C for 10 days; repeated freezing and thawing are avoided. The kit is suitable for use in, for example, a SLAN-96S PCR instrument; is suitable for 48-hole PCR reaction plates, 96-hole PCR reaction plates and 8-connecting tubes.
The PCR assay was performed in divided zones.
1. Extraction of DNA
The kit does not contain DNA extraction and purification components, and can adopt a commercial DNA extraction reagent to prepare a DNA sample according to the operation of a specification. Clinical blood samples were taken.
PCR amplification and melting
2.1 preparation of reaction solution
According to the total number of samples (containing DNA samples to be detected, negative control and positive control) detected each time, according to the table 2, taking PCR Master Mix and PP Mix from the kit, and preparing reaction liquid with calculated amount for later use.
TABLE 2 reaction solution composition for each reaction
2.2 sample application
Taking the DNA sample to be detected and the negative control of the kit, adding a PCR plate in 1 hole each, and adding 2 mu L of the DNA sample and the negative control of the kit. The positive control was not added at all.
2.3 addition of reaction solution
Add 18. mu.L of the prepared reaction solution to each well.
2.4 plus Positive control
Taking 2 mu L of positive control of the kit, and adding a positive control hole corresponding to the PCR plate.
2.5 closing or sealing tubes
A PCR reaction plate is sealed by a special membrane; 8 connecting pipes and sealing by a pipe cover.
2.6 centrifugation
After the PCR plate was closed, gently patted several times. The plate was inserted into a centrifuge and centrifuged at low speed for 10 s.
8 connecting pipes, flicking the pipes one by one, inserting the flicked pipes into a centrifugal groove, and centrifuging at a low speed for 10 s.
2.7 setting PCR instrument
According to the specification of the PCR instrument, a software setting interface is started, the PCR instrument is set according to the parameters listed in Table 3, the fluorescence channels listed in Table 4 are selected, and the peak height threshold is set to be 5.00.
TABLE 3 PCR amplification and melting procedure parameters
TABLE 4 correspondence of mutation sites to fluorescent channels
2.8 run
And (4) putting the prepared PCR plate into a sample bin of the PCR instrument, closing a bin door, and clicking to operate.
3. Analysis of results
And opening a data file according to the operation of a software instruction of an applicable machine type, and analyzing and calculating a melting curve peak diagram and a Tm value of the sample to be detected and the negative control and the positive control.
The melting curve peak plot for each sample should be reviewed manually before reporting the test results. When abnormal conditions such as peak inversion, baseline fluctuation or drifting and the like appear in a melting curve peak diagram, the detection value of the melting curve peak reported by software can be influenced and is manually interpreted; if necessary, the samples of the corresponding reaction wells should be retested.
[ Positive judgment value ]
TABLE 5 typing judgment values
Note: w, means the wild (wild) type allele; d, means deletion (deletion) mutant allele.
The following are the positive control, the negative control, and the melting temperature of each sample and genotyping based on the melting temperature. In the following table, the known genotypes are the genotypes that have been determined by sequencing.
Table 6 shows the melting temperatures of the melting curves of the negative controls.
TABLE 6
Table 7 shows the melting temperatures of the melting curves of the positive controls.
TABLE 7
Table 8 shows the melting temperature of the melting curve of sample 1 and the genotyping based on the melting temperature.
TABLE 8
Table 9 shows the melting temperature of the melting curve of sample 2 and the genotyping based on the melting temperature.
TABLE 9
Table 10 shows the melting temperature of the melting curve of sample 3 and the genotyping based on the melting temperature.
Watch 10
Table 11 shows the melting temperature of the melting curve of sample 4 and the genotyping based on the melting temperature.
TABLE 11
Table 12 shows the melting temperature of the melting curve of sample 5 and the genotyping based on the melting temperature.
TABLE 12
Table 13 shows the melting temperature of the melting curve of sample 6 and the genotyping based on the melting temperature.
Watch 13
Table 14 shows the melting temperature of the melting curve of sample 7 and the genotyping based on the melting temperature.
TABLE 14
Table 15 shows the melting temperature of the melting curve of sample 8 and the genotyping based on the melting temperature.
Watch 15
As can be seen from the above, the determination method according to the present invention is completely consistent with the genotyping determined by sequencing.
Sequence listing
<110> institute of biomedical technology in Shanghai city
Shanghai Bingxiang Biotech Co., Ltd
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Claims (10)
1. A human DEFB126 gene frameshift mutation detection kit for detecting the genotype of frameshift mutation sites rs11467417, rs11467497 of human beta defensin 126 gene (DEFB126), comprising:
(1) specific primers designed according to sequences on two sides of the locus rs 11467417;
(2) specific primers designed according to sequences on two sides of the locus rs 11467497;
(3) a specific probe designed according to the adjacent sequence of the rs11467417 locus;
(4) probes respectively designed according to the adjacent sequence of the locus rs11467497 and the sequences of the locus before and after mutation;
(5) PCR reaction reagent;
optionally (6) a positive control;
optionally (7) a negative control,
wherein, the specific probe designed according to the adjacent sequence of the rs11467417 locus, the probes respectively designed according to the adjacent sequence of the rs11467497 locus and the locus sequences before and after mutation are respectively marked with different fluorescent luminous groups, and the kit utilizes a PCR reaction to detect a plurality of genotypes of the frame shift mutation loci rs11467417 and rs 11467497.
2. The human DEFB126 gene frameshift mutation detection kit of claim 1, wherein the PCR reaction reagents comprise Taq polymerase, UNG, dNTP for PCR amplification;
preferably, in each unit of the kit, Taq polymerase is 1-3 units, e.g., 1 unit, UNG enzyme is 1-3 units, e.g., 1 unit, dNTP is 0.2-0.6mM, e.g., 0.4mM, primer concentration is 0.02-1. mu.M, further 0.1-0.4. mu.M, e.g., 0.2. mu.M.
3. The human DEFB126 gene frame shift mutation detection kit of claim 1, wherein the sequences at both sides of the wild rs11467417 site (the sequences at both ends of the upstream and downstream of rs11467417) are shown as sequence 1 in the sequence table, and the sequences at both sides of the mutant rs11467417 site (the sequences at both ends of the upstream and downstream of rs11467417) are shown as sequence 2 in the sequence table.
4. The human DEFB126 gene frame shift mutation detection kit of any one of claims 1-3, wherein the sequences flanking the wild type rs11467497 site (sequences at both ends of the upstream and downstream of rs11467497) are shown as sequence 3 in the sequence listing, and the sequences flanking the mutant rs11467497 site (sequences at both ends of the upstream and downstream of rs11467497) are shown as sequence 4 in the sequence listing.
5. The DEFB126 gene frameshift mutation detection kit of any of claims 1-4, wherein the positive control is a DNA heterozygote containing U at two sites, i.e., a mixture of Rs11467417PCR product sequence and synthetic sequence of Rs11467497PCR product sequence with T replaced in whole or in part by U; preferably, it also contains (e.g. 10 units of) the UNG enzyme inhibitor UGI;
preferably, the Rs11467417PCR product sequence is shown as sequence 5 in the sequence table, and the Rs11467497PCR product sequence is shown as sequence 6 in the sequence table.
6. The human DEFB126 gene frameshift mutation detection kit of any one of claims 1-5, wherein the base sequence of the specific primers designed according to the sequences at both sides of the rs11467417 locus are shown as sequence 7 (end primer No. 5) and sequence 8 (end primer No. 3) in the sequence table; and/or
The method is characterized in that the base sequence of the specific primers designed according to the sequences on both sides of the rs11467497 locus is shown as a sequence 9 (a No. 5 end primer) and a sequence 10 (a No. 3 end primer) in the sequence table.
7. The human DEFB126 gene frame shift mutation detection kit of any one of claims 1-6, wherein the base sequence of the specific probe designed according to the adjacent sequence of rs11467417 locus is shown as sequence 11 in the sequence table; and/or the sequences of probes respectively designed according to the adjacent sequence of the locus rs11467497 and the locus sequences before and after mutation are shown as sequences 12 and 13 in the sequence table.
8. The human DEFB126 gene frameshift mutation detection kit of any one of claims 1-7, wherein a specific probe designed according to the sequence adjacent to the rs11467417 site is used for labeling a VIC fluorophore; the sequence probe before rs11467497 mutation is labeled with CY5 fluorophore, and the sequence probe after rs11467497 mutation is labeled with FAM fluorophore.
9. The detection method of the frame shift mutation of the human DEFB126 gene comprises the following steps:
(1) extracting a DNA sample;
(2) amplifying the DNA sample to be detected, the optional negative control and the optional positive control by using specific primers designed according to the sequences on two sides of the locus rs11467417 and specific primers designed according to the sequences on two sides of the locus rs11467497 respectively;
(3) labeling with specific probes designed according to the adjacent sequence of the locus rs11467417 and probes respectively designed according to the adjacent sequence of the locus rs11467497 and the locus sequences before and after mutation, wherein the three specific probes respectively adopt different fluorescent dyes;
(4) detecting by using the probe according to the corresponding relation between the mutation site and the fluorescence channel;
(5) typing was performed based on the melting temperature of the melting curve.
10. The method for detecting the frameshift mutation of human DEFB126 gene as claimed in claim 9, wherein a specific probe designed according to the sequence adjacent to the rs11467417 site is used to mark a VIC fluorophore; the rs11467497 pre-mutation sequence probe is labeled with CY5 fluorophore, and the rs11467497 post-mutation sequence probe is labeled with FAM fluorophore;
and judging the result according to the following criteria:
wherein, W means the wild type allele; d, means deletion mutant allele.
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