CN114317694A - Probe, primer and kit for detecting ACE2 gene polymorphism - Google Patents
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Abstract
The invention provides a probe for detecting ACE2 gene polymorphism, wherein the sequence of the probe is shown in SEQ ID No. 1, or both ends of the sequence of the SEQ ID No. 1 are modified by groups. The invention also provides a primer for detecting the ACE2 gene polymorphism, and the sequence of the primer is shown as SEQ ID NO. 2 and SEQ ID NO. 3. The probe and the primer for detecting the ACE2 gene polymorphism have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and the like when the ACE2 gene polymorphism is detected.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a probe, a primer and a kit for detecting ACE2 gene polymorphism.
Background
The ACE2 (angiotensin I converting enzyme 2) gene encodes a protein belonging to the angiotensin converting enzyme family of dipeptidyl carboxypeptidase and having a homology with human angiotensin 1 converting enzyme. This secreted protein catalyzes the cleavage of angiotensin I to angiotensin 1-9 and angiotensin II to the vasodilator angiotensin 1-7. Organ and cell specific expression of this gene suggests that it may play a role in regulating cardiovascular and renal function as well as fertility. Furthermore, the encoded protein is a functional receptor for the spike glycoprotein of the human coronavirus SARS and HCoV-NL 63.
The mutation detection method commonly applied at present is a direct DNA sequencing method, and the PCR product is directly subjected to DNA sequence analysis, so that mutation sites can be defined, but the defects of time and labor waste, high cost, inapplicability to detection of a large number of samples and the like exist. Therefore, it is necessary to develop a method suitable for the detection of large-scale samples.
Disclosure of Invention
The invention aims to provide a probe for detecting ACE2 gene polymorphism, wherein the sequence of the probe is shown in SEQ ID No. 1, or groups are modified at two ends of the sequence of the SEQ ID No. 1;
wherein the modifying group is: 5' modifying groups are fluorescent groups, such as CY 5; the 3' modifying group is a quenching group, such as BHQ 2;
specifically, the probe for detecting the polymorphism of the ACE2 gene provided by the invention is as follows: 5 '-CY 5-CCGAATTAGTAGCNTACCTGGTTCGG-BHQ 2-3';
wherein N is a variant base, specifically C > T;
the probe comprises a loop sequence and a stem sequence, wherein the loop sequence is 6-21bp (5 ' -CCGAATTAGTAGCNTACCTGGTTCGG-3 '), two reverse complementary stem sequences are arranged on two sides of the loop sequence, and the stem sequence is (5 ' -CCGAA … … TTCGG-3).
The design principle of the probe is as follows: according to the ACE2 gene polymorphism rs2285666, a probe and a primer are designed, and the probe is required to be in a stem-loop state when a DNA template does not exist at an annealing temperature. The quenching group adopted by the probe is BHQ2, the quenching space range is very small, and the fluorescence can be well quenched only when the molecular beacon is in a stem-loop structure by matching with a fluorescent group with short emission wavelength, such as CY 5. When the loop sequence is complementary with the target DNA sequence, the optimal PCR primer is determined to be 40-45 bp in size, and the length of the PCR product is 100-150 bp.
The invention also provides a primer for detecting the polymorphism of the ACE2 gene, and the sequence of the primer is shown as SEQ ID NO. 2 (primer 1) and SEQ ID NO. 3 (primer 2):
primer 15 '-AGGTGACACTATAGAATATGAAACACACATATCTGCAATCA-3'
Primer 25 '-GCAAGCCCTCACGTAGCGAACTGGAAAAGTTTGTAACCCAGA-3'.
The invention also provides a kit for detecting the polymorphism of the ACE2 gene, which comprises the probe and/or the primer.
The kit further comprises Taq enzyme, dNTP, and/or Mg2+And instructions for use.
The invention also provides a method for detecting the polymorphism of the ACE2 gene, which comprises the following steps:
(1) collecting a sample and extracting DNA;
(2) carrying out fluorescent quantitative PCR reaction by using the probe and the primer;
(3) analyzing the result by using the matched software of the PCR instrument, defining proper base lines and threshold values according to an amplification curve, and displaying different genotypes based on the difference of Tm values displayed by formed hybridization peaks;
wherein the Tm value is 58 ℃ for the wild type, and the Tm value is 50 ℃ for the mutant type.
Wherein the total amount of PCR reaction is 15ul (including PCR Mix 7.5ul, forward primer solution 0.5ul and reverse primer solution 0.5ul, probe 0.1ul, sample DNA 2ul, sterilized double distilled water 4.4 ul); carrying out reaction on a fluorescent quantitative PCR instrument, wherein the PCR reaction condition is pre-denaturation at 92-97 ℃ for 5-15 minutes; denaturation at 92-97 deg.C for 10-30 s, annealing at 57-65 deg.C for 10-30 s, extension at 70-75 deg.C for 10-30 s, and 40-50 cycles; extension at 72 ℃ for 10 min; denaturation at 92-97 deg.C for 1 min, renaturation at 40 deg.C for 1 min, and real-time monitoring of fluorescence signal at 45-80 deg.C, and recording 5 times at 1 deg.C per time.
The invention detects the polymorphism of the ACE2 gene, can realize the rapid screening of the polymorphism of the ACE2 gene, and assists clinical adjustment of the dosage of a patient according to the genotype of the patient, so as to improve the curative effect and reduce the occurrence of adverse reactions.
The invention utilizes a fluorescent probe capable of specifically identifying a nucleic acid sequence, releases fluorescent dye through conformational change after hybridization with a target sequence, and judges a typing result according to peak patterns at different temperatures generated after hybridization. Under the condition that no target DNA exists, the fluorescent group and the quenching group can be stably combined together, and no fluorescent signal can be detected; when the target DNA exists, the structure of the fluorescence labeling probe is damaged, and the fluorescent group and the quenching group are separated from each other, so that the fluorescence signal can be detected. Compared with other genetic typing techniques, the method is simple to operate, can finish 96 cases of detection within 2-3 hours, has the advantages of high sensitivity, good specificity, rapidness, high-flux detection and the like, obtains a detection result by directly detecting a fluorescent signal in a PCR process, has clear genotyping, does not need PCR post-treatment or electrophoresis detection, and can realize real closed-tube operation.
Drawings
FIG. 1, three peak types (CC type, CT type, TT type) at rs2285666 site.
FIG. 2 shows the results of precision experiments (two peaks are CT type, and one peak is CC type).
Detailed Description
Example 1 Probe and primer design
The invention designs a probe and a primer sequence aiming at an ACE2 SNP locus. The specific principle is that the fluorescent probe and a target sequence are hybridized to release fluorescent dye through conformational change, and a genotyping result is judged according to peak graphs and Tm values of different temperatures generated after hybridization. Under the condition that no target DNA exists, the fluorescent group and the quenching group can be stably combined together, and no fluorescent signal can be detected; when the target DNA exists, the structure of the fluorescence labeling probe is damaged, and the fluorescent group and the quenching group are separated from each other, so that the fluorescence signal can be detected.
Designing a probe and a primer, and realizing that the probe is in a stem-loop state when the template does not exist at the annealing temperature, the probe comprises a loop sequence and stem sequences with two sides in reverse complementarity, the total length is 30bp, wherein the loop sequence is 6-21bp (5 '-TTAGTAGCNTACCTGG-3'), and the stem sequences are formed by 5 bases at two ends; and the stem sequences at the two ends are just complementary; the adopted quenching group is BHQ2, and is matched with a fluorescent group CY5 with short emission wavelength. The loop sequence is complementary to the target DNA sequence, and the PCR primer is determined to be 38-40 bp in size.
The primer and probe sequences were as follows:
primer 15 '-AGGTGACACTATAGAATATGAAACACACATATCTGCAATCA-3';
primer 25 '-GCAAGCCCTCACGTAGCGAACTGGAAAAGTTTGTAACCCAGA-3';
probe 5 '-CY 5-CCGAATTAGTAGCNTACCTGGTTCGG-BHQ 2-3'.
Wherein N is a variant base, specifically G > C;
the above-mentioned probe and primer were synthesized by Biotechnology engineering (Shanghai) Ltd.
Example 2 detection of different genotype standards
1. Wild type standard plasmid and mutant standard plasmid containing target gene rs2285666 site are constructed and prepared by using plasmid ACE2 (plasmid source, and synthesis of plasmid containing target gene is synthesized by Biotechnology engineering (Shanghai) GmbH). Determining the accuracy of the sequence by sanger sequencing, wherein the genotype of the wild type standard quality particle rs2285666 is CC; the rs2285666 genotype of the mutant standard plasmid is TT. Standard plasmid DNA concentrations were normalized to 10 ng/ul.
2. The probe and primer in example 1 were used.
3. And (3) PCR reaction system:
1) sequentially adding 7.5ul of PCR Mix, 0.5 uM of primer 1 solution, 0.5 uM of primer 2 solution and 0.1 uM of probe into each PCR reaction hole, then respectively adding 2ul of wild type standard plasmid DNA, mutant type standard plasmid DNA and mixed type DNA (the wild type standard plasmid and the mutant type standard plasmid are mixed according to a ratio of 1: 1) into 3 different PCR reaction holes, and supplementing 15ul of sterilized redistilled water;
2) carrying out reaction on a fluorescent quantitative PCR instrument, wherein the PCR reaction condition is pre-denaturation at 95 ℃ for 5 minutes; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 45 cycles; extension at 72 ℃ for 10 min; denaturation at 95 ℃ for 1 min, renaturation at 40 ℃ for 1 min, and real-time monitoring of fluorescence signals at a melting temperature of 45-80 ℃ with 5 recordings at 1 ℃ per temperature rise.
4. The results of analysis by SLAN software equipped with a PCR instrument revealed different genotypes based on the difference in Tm values exhibited by the formed hybridization peaks. The peak at rs2285666 locus appeared at 58 ℃ and was CC genotype, the peak appeared at 50 ℃ and was TT genotype, and the peaks at both positions were CT genotype (FIG. 1).
Example 3 test method Performance analysis experiment
1. Precision experiment
Wild type standard plasmid DNA and mixed type DNA (rs 2285666 plasmid source, and synthesis of plasmid containing target gene synthesized by Biotechnology engineering (Shanghai) GmbH, wild type standard plasmid and mutant type standard plasmid are mixed at a ratio of 1: 1) are taken, each part is detected for 3 times a day for 5 days continuously, the amplification reaction program adopts the method in example 2, and the result is shown in figure 2. The fluorescent PCR amplification reaction has good repeatability (the coincidence rate is more than 95 percent, and the Ct value variation coefficient CV of the detection result is less than 5 percent).
2. Coincidence rate experiment
DNA samples of 20 healthy volunteers in Shanghai regions are selected, the method of the embodiment 2 is applied to carry out rs2285666 locus detection, meanwhile, the sanger sequencing method is applied to carry out verification, the consistency of the detection results of the two methods is compared, and the result shows that the coincidence rate of the typing result of the embodiment 2 and the sanger sequencing method is 100% in percentage of consistency degree and excellent in consistency.
3. Limit of detection experiment
A portion of mixed DNA (wild type standard plasmid and mutant type standard plasmid mixed at a ratio of 1: 1) with known concentration was diluted to four concentrations of 10ng/ul, 5ng/ul, 2ng/ul and 1ng/ul, and each concentration was assayed in 3 tubes in parallel. The results show that the corresponding genotypes can be detected when the sample concentration is 10ng/ul, 5ng/ul, 2ng/ul and 1ng/ul, namely the lowest detectable concentration is 1 ng/ul.
Example 4 detection of DNA in samples of oral epithelial cells
1. Extracting the genome DNA of oral epithelial cells of 100 healthy volunteers in Shanghai region by a silica gel adsorption method, detecting the concentration and purity of the DNA by an electrophoresis gel imaging method, and marking the concentration of the DNA of a sample to be detected to 10 ng/ul.
2. The detection method comprises the following steps: sequentially adding 7.5ul of PCR Mix, 0.5 uM of forward primer solution, 0.5 uM of reverse primer solution and 0.1 uM of probe into each PCR reaction hole, simultaneously detecting a weak positive control (genotype is CT), a negative control (genotype is CC) and a sample to be detected, adding 2ul of DNA into each reaction hole, and supplementing 15ul of sterilized redistilled water; carrying out reaction on a fluorescent quantitative PCR detector, wherein the PCR reaction condition is pre-denaturation at 95 ℃ for 5 minutes; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 45 cycles; extension at 72 ℃ for 10 min; denaturation at 95 ℃ for 1 min, renaturation at 40 ℃ for 1 min, and real-time monitoring of fluorescence signals at a melting temperature of 45-80 ℃ with 5 recordings at 1 ℃ per temperature rise.
3. The results of analysis by SLAN software equipped with a PCR instrument revealed different genotypes based on the difference in Tm values exhibited by the formed hybridization peaks. The rs2285666 locus peak appears at 58 ℃ and is CC genotype, the peak appears at 50 ℃ and is TT genotype, and the peaks at the two positions are CT genotype. The success rate of typing reaches 100% in the melting curve chart of multiple samples.
4. The genomic DNA of 100 cases of oral epithelial cells is subjected to sanger sequencing at the same time, and the detection result completely accords with the detection result of the invention, so that the accuracy of the result of the method for detecting the ACE2 polymorphism is high.
Sequence listing
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<120> probe, primer and kit for detecting ACE2 gene polymorphism
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Claims (7)
1. A probe for detecting ACE2 gene polymorphism, the sequence of the probe is shown as SEQ ID No. 1; or modified by groups at both ends of the sequence of SEQ ID No. 1.
2. The probe of claim 1, wherein the modifying group is: the modification group of 5' is a fluorescent group CY 5; the modifying group at the 3' is a quenching group BHQ 2.
3. The probe of claim 1, wherein the probe comprises a loop sequence and a stem sequence, wherein the loop sequence is 6-21bp, and is flanked by two reverse complementary stem sequences.
4. A primer for detecting ACE2 gene polymorphism is characterized in that the sequence of the primer is shown as SEQ ID No. 2 and SEQ ID No. 3.
5. A kit for detecting polymorphism of ACE2 gene, characterized in that the kit comprises the probe of any one of claims 1 to 3 and/or the primer of claim 4.
6. The kit of claim 5, wherein the kit further comprises Taq enzyme, dNTP, and/or Mg2+, and instructions for use.
7. A method for detecting polymorphism of ACE2 gene, comprising the steps of:
(1) collecting a sample and extracting DNA;
(2) performing a fluorescent quantitative PCR reaction using the probe of any one of claims 1 to 3 and the primer of claim 4;
(3) analyzing the result by using the matched software of the PCR instrument, defining proper base lines and threshold values according to an amplification curve, and displaying different genotypes based on the difference of Tm values displayed by formed hybridization peaks; wherein the Tm value is 58 ℃ for the wild type, and the Tm value is 50 ℃ for the mutant type.
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| CN202011082369.3A CN114317694A (en) | 2020-10-12 | 2020-10-12 | Probe, primer and kit for detecting ACE2 gene polymorphism |
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| CN114317694A true CN114317694A (en) | 2022-04-12 |
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