CN114350790A - Method for detecting invasive periodontitis - Google Patents
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- CN114350790A CN114350790A CN202210074921.7A CN202210074921A CN114350790A CN 114350790 A CN114350790 A CN 114350790A CN 202210074921 A CN202210074921 A CN 202210074921A CN 114350790 A CN114350790 A CN 114350790A
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Abstract
The invention relates to the technical field of genes related to periodontitis in biotechnology, in particular to a detection method, primers, a specific probe, a kit and application of three genes related to susceptibility to periodontitis and single nucleotide polymorphic sites thereof. The invention provides genes ANKRD36C, SREK1IP1 and TMEM97 related to periodontitis, polymorphic sites thereof, a method for detecting the polymorphic sites, and application of the genes in prevention, auxiliary diagnosis, treatment and the like of periodontitis. The gene related to periodontitis and the nucleic acid sequence of the polymorphic site thereof provided by the invention are utilized to construct a kit for genetic diagnosis of periodontitis, can be applied to auxiliary diagnosis of periodontitis and judgment on whether an individual has susceptibility to periodontitis, and is beneficial to prevention, early diagnosis and treatment of periodontitis.
Description
Technical Field
The invention relates to application of single nucleotide polymorphisms rs147285461, rs750306056 and rs781830688 in screening patients with periodontitis in the field of biotechnology.
Background
Aggressive periodontitis (AgP) is a rapidly progressing inflammation occurring in the tissues surrounding the teeth, frequently occurring in clinically healthy individuals, manifested by a rapid loss of attachment and bone destruction occurring at an early stage. Most teeth are loosened and displaced due to a great loss of alveolar bone, thereby affecting masticatory function and hindering diet. Typical limiting AgP occurs after puberty, while widespread AgP occurs below 30 years of age. The current accepted prevalence of AgP is: the global population general movement trend is 1.6%, with the highest incidence rates in africa and south america, 4.2% and 4.0%, respectively; the overall prevalence in asia is 1.2%; european minimum, only 0.1%. The prevalence rate of the invasive periodontitis of adolescents in Shanghai is 0.81%. Invasive periodontitis is the large-area destruction of alveolar bone caused by the decrease of the resistance of the body to bacteria after human genes are mutated or genes show polymorphism. At present, the invasive periodontitis is caused by the mutation of the family cathepsin C (CTSC) gene, but a plurality of pathogenic genes are unknown due to the difference of race, living environment and the latitude on earth.
With the development of gene sequencing technology, people are continuously and deeply researching gene functions. It was found that one base among every 600 bases in the DNA sequence of the human genome has polymorphism, i.e., Single Nucleotide Polymorphism (SNP). A polymorphism represented by a SNP refers to a single base variation, which may be caused by a transition or transversion of a single base, or by an insertion or deletion of a base.
There were studies showing that the gene polymorphism at the IL-6-572 locus was significantly associated with chronic periodontitis, but there were also cases where, for example, Czech had no polymorphism at this locus and 32.3% of IL-6-572G allele carriers in the control group had not developed chronic periodontitis. (Guangzhen, Liu Jing jin, Ma Xin, Wu Dong hong, Yu Jie, Huang Guo Qiang the analysis of the interleukin 6 gene-572 site polymorphism and the susceptibility of severe chronic periodontitis, in the No. 7 of No. 43 at the end of 2008 of the Chinese journal of stomatology). Therefore, the development of a detection method with higher accuracy for judging the aggressive periodontitis is of great significance for clinical diagnosis.
In view of this, the invention is particularly proposed.
Disclosure of Invention
One of the objectives of the present invention is to provide the ANKRD36C, SREK1IP1 and TMEM97 genes associated with susceptibility to periodontitis, which addresses the deficiencies of the prior art.
The second purpose of the invention is to overcome the defects in the prior art and provide a primer for detecting the single nucleotide polymorphism related to the invasive periodontitis and a kit thereof.
The invention firstly provides a group of primers for detecting the relevant single nucleotide polymorphism of the invasive periodontitis, which comprise specific amplification primer pairs of single nucleotide polymorphism sites rs147285461, rs750306056 and rs 781830688.
Wherein, the rs147285461 specific amplification primer pair comprises:
an upstream primer: 5'-TGGCAGACTTGAGGTTTGAATCTAC-3' (SEQ ID NO:1)
A downstream primer: 5'-TGGCAAGTGAATAAAGCATGGGA-3' (SEQ ID NO: 2);
the specific amplification primer pair of rs750306056 comprises:
an upstream primer: 5'-ATACCCATCAAACCACTGGCCTT-3' (SEQ ID NO:3)
A downstream primer: 5'-TCCTAAACTCATAGGCCTGCTTTG-3' (SEQ ID NO:4)
The rs781830688 specific amplification primer pair comprises:
an upstream primer: 5'-CTGAACAAACAGATGCCATGGGGC-3' (SEQ ID NO:5)
A downstream primer: 5'-GCTCTTGTGCATTGCTGCCAGTG-3' (SEQ ID NO:6)
The primer can be used for preparing a kit for detecting the single nucleotide polymorphism related to the invasive periodontitis, in particular for preparing the kit for detecting the single nucleotide polymorphism related to the invasive periodontitis of Chinese Han nationality population.
Still another object of the present invention is to provide a kit for detecting an aggressive periodontitis-associated single nucleotide polymorphism, comprising: the primer pair for specifically amplifying single nucleotide polymorphism sites in the nucleotide sequences of rs147285461, rs750306056 and rs781830688 and PCR reaction liquid, wherein the PCR reaction liquid comprises dNTP mixed liquid, Taq DNA polymerase and PCR buffer liquid.
Furthermore, the kit can also selectively comprise a universal reagent for DNA extraction, a universal reagent for PCR amplification, proteinase K and the like.
The universal reagent for DNA extraction and the universal reagent for PCR amplification are not specific to the kit, and various universal reagents well known in the art can be used.
As the general reagent for DNA extraction, various reagents which are conventionally available in genomic DNA extraction kits can be used.
PCR amplification universal reagents, such as selected from: taq enzyme, PCR buffer, MgCl2, dNTPs or Taq PCR Master Mix.
Because the DNA extraction universal reagent and the PCR universal reagent can be purchased separately through market approaches or can be configured according to the prior art, the reagents which need to be assembled into the kit can be configured according to the actual needs of customers, and can also be assembled into the kit completely for the sake of convenience.
The kit is suitable for blood samples, genome DNA samples, tissue samples and the like.
The kit of the invention is suitable for the detection of samples for diagnostic or non-diagnostic purposes.
The invention further discloses a method for using the kit of the invention for diagnostic or non-diagnostic purposes, comprising the following steps:
step one, determining the nucleotides of single nucleotide polymorphic sites (rs147285461, rs750306056 and rs781830688) of ANKRD36C, SREK1IP1 and TMEM97 genes;
and secondly, amplifying the gene segments of ANKRD36C, SREK1IP1 and TMEM97 by adopting a molecular biology technology of Polymerase Chain Reaction (PCR).
And step three, detecting whether the ANKRD36C, SREK1IP1 and TMEM97 genes in the amplified fragments in the step two have single nucleotide polymorphism and the type of the single nucleotide polymorphism by using a sequencing technology.
In the second step, the polymerase chain reaction system is: carrying out amplification reaction on each sample in a system with the total volume of 25ul, wherein the system comprises 1ul of prepared genomic DNA diluent and 1ul of 10mM dNTP mixed solution, 1ul of each amplification primer, 2.5ul of 10 xTaq Buffer (with MgCl2) and 0.2ul of 5U/. mu.l Taq DNA polymerase, and finally adding ddH2O to prepare the total volume of 25 ul;
the polymerase chain reaction procedure was: starting at 94 ℃ for 5min, performing denaturation at 94 ℃ for 30s, performing annealing at 63 ℃ for 30s, performing extension at 72 ℃ for 30s, reducing the annealing temperature by 0.5 ℃ in each cycle, and performing 10 cycles; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 30 times; extension at 72 ℃ for 10 min.
In the third step, the sequencing technology can be Sanger sequencing or next generation sequencing technology.
Based on the research on the single nucleotide polymorphism of Han nationality aggressive periodontitis, the rs147285461, rs750306056 and rs781830688 sites are selected as the single nucleotide polymorphism detection sites for combined diagnosis of aggressive periodontitis, a typing method based on the combination of PCR detection reaction and sequencing technology is adopted, 2 specific primers are designed for each site aiming at three hot spot mutation sites of periodontitis patients, and a target gene segment PCR fragment is obtained through PCR amplification. The genotype of the template is analyzed by sequencing the fragment of interest. The kit has simple operation steps, high detection specificity and good stability, can correctly distinguish homozygote and heterozygote of each site, and has high repeatability in multiple tests; the time is short, and the sequencing result can be completed within one working day from the amplification of the fragments; the Sanger sequencing technology is adopted, the cost is low, and the method is suitable for detecting gene mutation of clinical patients, detecting heterozygote carriers of normal people and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 clinical intraoral photographs of a patient
FIG. 2 is a CBCT image examination result chart of clinical samples.
FIG. 3 exon sequencing data statistics, LGBb brother; LRCd father; LSLc mother; LYQa invasive periodontitis case samples.
FIG. 4 is an electrophoretogram for human genome extraction, wherein 1 represents a sample and M represents Marker SM 0331.
FIG. 5 is a graph of rs750306056 sequencing results.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Unless otherwise indicated, the experimental methods, detection methods, preparation methods, and reagents disclosed herein all employ conventional techniques or reagents of the art, which are conventional in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
The features and properties of the present invention are described in further detail below with reference to examples.
Primer sequences used in the examples:
the specific amplification primer pair of rs147285461 comprises:
an upstream primer: 5'-TGGCAGACTTGAGGTTTGAATCTAC-3' (SEQ ID NO:1)
A downstream primer: 5'-TGGCAAGTGAATAAAGCATGGGA-3' (SEQ ID NO: 2);
the specific amplification primer pair of rs750306056 comprises:
an upstream primer: 5'-ATACCCATCAAACCACTGGCCTT-3' (SEQ ID NO:3)
A downstream primer: 5'-TCCTAAACTCATAGGCCTGCTTTG-3' (SEQ ID NO:4)
The rs781830688 specific amplification primer pair comprises:
an upstream primer: 5'-CTGAACAAACAGATGCCATGGGGC-3' (SEQ ID NO:5)
A downstream primer: 5'-GCTCTTGTGCATTGCTGCCAGTG-3' (SEQ ID NO:6)
Example 1 obtaining a sample
One patient, infant patient, female, 15 years old, student, with good school score, was collected and kept on children. Oral examination revealed a total of 25 teeth in the mouth. Poor oral hygiene, supragingival tartar (+++), BOP (+), redness and swelling of the gingiva, probing depth of periodontal pocket of 3-7mm, and pus discharge around 35 and 36 teeth; bleeding index 3; 15, 22, 26, 32-36 loose III degrees, 37 and 46 loose I degrees, 21, 31 and 41 loose teeth are lacked (see figure 1). The deciduous teeth erupt normally without early shedding. CBCT shows that the alveolar bone is absorbed to the root length of 1/3-root apex, and the tooth germ of 18, 28, 38 and 48 teeth which is not completely developed is seen (see figure 2). The patients have moderate development, no abnormal development of skin, hair and nails, and normal sweat secretion. Normal jaw development and symmetrical facial form. Deny general systemic medical history, deny medical and food allergy history, deny family history (parents, grandparents, milk, heteroova, phoenix and brother are non-invasive periodontitis), deny recent marriage history in family. The diagnosis is as follows: aggressive periodontitis
The study was approved by the ethical committee of the affiliated oral hospital of Nanchang university (ethical approval No.: Kouzhen & Luen's No. 2020, 010), and all family members participating in the study signed informed consent to obtain peripheral blood of patients and related families.
Example 2 sample preparation (using Tiangen organism Medium blood genomic DNA extraction kit (0.5-3ml) (DP332))
1. Sampling: 5ml of blood of adolescent invasive periodontitis patients and family members thereof is extracted by an EDTA-K2 anticoagulation tube.
2. Mu.l of a solution of Proteinase K (20mg/ml) was added to a 15ml centrifuge tube.
3. Treating the materials: 3ml of blood sample was added and mixed in a 15ml centrifuge tube containing the above protease K solution.
3. 2.4ml of buffer GE was added to the centrifuge tube containing the blood sample, and mixed by shaking for 30 sec.
Standing at 4.65 deg.C for 10min, and shaking every 3min to assist cracking.
5. 2ml of absolute ethanol was added to the sample and mixed well, whereupon a flocculent precipitate may appear.
6. Half of the solution and flocculent precipitate obtained in the previous step were transferred to an adsorption column CB5 (the adsorption column was placed in a 15ml collection tube), centrifuged at 3,000rpm (. about.1,850 Xg) for 3min, the waste liquid was decanted, and adsorption column CB5 was returned to the collection tube.
7. And (4) transferring the residual solution in the step (6) into the same adsorption column, and repeating the operation in the step (6).
8. 2ml of buffer GD was added to adsorption column CB5, centrifuged at 5,000rpm (. about.4,500 Xg) for 1min, the waste liquid was decanted, and adsorption column CB5 was returned to the collection tube.
9. 2ml of buffer PW (previously checked for the presence of absolute ethanol) was added to adsorption column CB5, centrifuged at 5,000rpm (. about.4,500 Xg) for 1min, the waste solution was discarded, and adsorption column CB5 was returned to the collection tube.
10. 2ml of buffer PW was added to adsorption column CB5, centrifuged at 5,000rpm (. about.4,500 Xg) for 15min, the collection tube was discarded, and adsorption column CB5 was placed in a new 15ml centrifuge tube.
11. And (3) suspending and dropping 300 mu l of elution buffer TB into the middle part of the adsorption membrane, standing at room temperature for 5min, centrifuging at 5,000rpm (4,500 Xg) for 2min, and collecting the solution into a centrifuge tube to obtain the genome DNA.
Example 3 clinical validation Using human peripheral blood Whole exon secondary sequencing technology
ENST00000456556:p.Glu491fs/c.1470dupT、
ENST00000513458:p.Arg91fs/c.271dupA、
ENST00000226230:p.Ter177fs/c.528dupA。
ANKRD36C, in which a T is inserted at position 1470 of CDS, and the amino acids at positions 490 and later in the amino acid sequence encoded by the gene are changed; the mutation occurred at 95944647bp on chromosome two (chr 2); the direction of transcription of the gene is opposite to that of the chromosome, so that an insertion A is formed on the base sequence of the chromosome, and a T is formed on the sense strand of the gene; the frame shift mutation causes a T to be inserted into the 5' UTR region-159 of the gene, and the translation of the protein cannot be initiated.
SREK1IP1, the 271 th position of CDS of the gene is inserted with an A, which causes the 90 th position and the later position of the amino acid sequence coded by the gene to be changed; the mutation occurs at the 64728113bp position of chromosome five (chr 5); frame shift mutations result in the insertion of an A at position 373 of the non-coding transcript of the gene.
TMEM97, deletion of stop codon, insertion of A at position 528 of CDS of the gene, resulting in the change of the stop codon of the gene into a codon capable of encoding amino acid, resulting in the failure of timely termination of the amino acid sequence encoded by the gene, and lengthening of the protein encoded by the gene; this mutation occurred at 28326780bp on chromosome 17 (chr 17).
Example 4DNA quality testing
1) Detecting by electrophoresis (voltage of 120-180V) of 5 mu l of DNA solution 1% agarose and 1X TAE buffer solution, wherein a single band indicates that the DNA is complete and has no degradation, an obvious band indicates that the concentration can meet the PCR requirement, and the DNA electrophoresis chart extracted in the example 2 is as shown in figure 4, and the DNA is complete and has no degradation;
2) and detecting the concentration and the purity by a spectrophotometer, taking 1 mul to detect the OD value, wherein the OD 260/280 is 1.7-2.0, which shows that the DNA quality is better, and the DNA is polluted by protein less than 1.7 and RNA more than 2.0. There is generally a small amount of protein and RNA contamination that does not affect normal PCR.
Example 5PCR amplification
And respectively carrying out PCR amplification by using the genome DNA of each sample as a template and using specific amplification primer pairs of rs147285461, rs750306056 and rs 781830688.
PCR reaction components (25. mu.l system): the system comprises 1ul of prepared genome DNA, 1ul of 10mMdNTP mixed solution, 1ul of each amplification primer, 2.5ul of 10 xTaq Buffer (with MgCl2), 0.2ul of 5U/. mu.l Taq DNA polymerase and finally ddH2O, and the total volume is 25 ul.
The Touch-down PCR reaction conditions are as follows: starting at 94 ℃ for 5min, performing denaturation at 94 ℃ for 30s, performing annealing at 63 ℃ for 30s, performing extension at 72 ℃ for 30s, reducing the annealing temperature by 0.5 ℃ in each cycle, and performing 10 cycles; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 30 times; extension at 72 ℃ for 10 min.
Example 6Sanger sequencing
Taking 5 μ l of 1% agarose gel for electrophoresis of PCR products, and the electrophoresis parameters are as follows: electrophoresis observation at 150V, 100mA for 10-20 min
2. Recovery of target PCR strip cutting gel
3. Sequencing of the PCR products Using 3730XL sequencer available from ABI, USA
4. Data analysis, finding results in a result group, and analyzing with sequence analysis software is consistent with the above results.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. Application of a kit for detecting the polymorphism or genotype of ANKRD36C, SREK1IP1 and TMEM97 in human genome in screening patients with periodontitis.
2. The use of claim 1, wherein the kit comprises: and the primer pair specifically amplifies single nucleotide polymorphism sites in the nucleotide sequences of ANKRD36C, SREK1IP1 and TMEM 97.
3. The use of claim 2, wherein the primer pairs are three pairs, wherein the nucleotide sequence of the upstream primer of one primer pair is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; the nucleotide sequence of the upstream primer of the other primer pair is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4; the nucleotide sequence of the upstream primer of the other primer pair is shown as SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 6.
4. The use of claim 2, wherein the kit further comprises a PCR reaction solution comprising a dNTP mix, Taq DNA polymerase and PCR buffer.
5. A method for detecting single nucleotide polymorphic sites of ANKRD36C, SREK1IP1 and TMEM97 genes related to periodontitis susceptibility, which is characterized by comprising the following steps: it comprises the following steps:
step one, determining the nucleotides of single nucleotide polymorphic sites (rs147285461, rs750306056 and rs781830688) of ANKRD36C, SREK1IP1 and TMEM97 genes;
and secondly, amplifying the gene segments of ANKRD36C, SREK1IP1 and TMEM97 by adopting a molecular biology technology of Polymerase Chain Reaction (PCR).
And step three, detecting whether the ANKRD36C, SREK1IP1 and TMEM97 genes in the amplified fragments in the step two have single nucleotide polymorphism and the type of the single nucleotide polymorphism by using a sequencing technology.
6. The method for detecting the single nucleotide polymorphic sites of ANKRD36C, SREK1IP1 and TMEM97 genes related to periodontitis according to claim 5, wherein:
in the second step, the polymerase chain reaction system is: carrying out amplification reaction on each sample in a system with the total volume of 25ul, wherein the system comprises 1ul of prepared genomic DNA and 1ul of 10mMdNTP mixed solution, 1ul of each amplification primer, 2.5ul of 10 xTaq Buffer (with MgCl2) and 0.2ul of 5U/. mu.l Taq DNA polymerase, and finally adding ddH2O to prepare the total volume of 25 ul;
the polymerase chain reaction procedure was: starting at 94 ℃ for 5min, performing denaturation at 94 ℃ for 30s, performing annealing at 63 ℃ for 30s, performing extension at 72 ℃ for 30s, reducing the annealing temperature by 0.5 ℃ in each cycle, and performing 10 cycles; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, and circulating for 30 times; extension at 72 ℃ for 10 min.
7. The method for detecting the single nucleotide polymorphic sites of ANKRD36C, SREK1IP1 and TMEM97 genes related to periodontitis according to claim 5, wherein: in the third step, the sequencing technology can be Sanger sequencing or next generation sequencing technology.
8. The application of the mononucleotide polymorphic sites of ANKRD36C, SREK1IP1 and TMEM97 genes related to periodontitis susceptibility in auxiliary diagnosis of periodontitis and judgment on whether individuals have periodontitis susceptibility is characterized in that:
the mononucleotide polymorphic sites of the ANKRD36C, SREK1IP1 and TMEM97 genes are rs147285461, rs750306056 and rs 781830688.
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