CN112626199A

CN112626199A - Primer and method for detecting risk prediction site for converting chronic hepatitis into liver cirrhosis

Info

Publication number: CN112626199A
Application number: CN202110003237.5A
Authority: CN
Inventors: 陈雪青; 王淑一
Original assignee: Adicon Clinical Laboratories Center Inc
Current assignee: Adicon Clinical Laboratories Center Inc
Priority date: 2021-01-04
Filing date: 2021-01-04
Publication date: 2021-04-09

Abstract

The invention discloses a primer, a method and a kit for detecting a risk prediction site rs72613567: TA for converting chronic hepatitis into cirrhosis, wherein the primer, the method and the kit comprise a primer for amplifying the site rs 72613567; by adopting a Sanger sequencing technology, the method can be used for quickly detecting the rs72613567 site mutation condition in a chronic hepatitis patient. The detection result completed by the invention is accurate, and risk assessment and genetic guidance can be provided for preventing chronic hepatitis from being converted into liver cirrhosis.

Description

Primer and method for detecting risk prediction site for converting chronic hepatitis into liver cirrhosis

Technical Field

The invention belongs to the field of life science and biotechnology, and particularly relates to a primer for detecting the mutation of a risk prediction site rs72613567 for converting chronic hepatitis into cirrhosis, which can be used for quickly detecting the mutation of the rs72613567 site of HSD17B13 gene in a patient with chronic hepatitis by adopting the common PCR and Sanger sequencing technology.

Background

Chronic hepatitis is one of the major diseases worldwide, leading to a higher mortality rate. Common causes of chronic hepatitis include hepatitis virus (hepatitis B virus, hepatitis C virus) infection, long-term drinking, non-alcoholic fatty liver disease (NAFLD), autoimmune hepatitis, and the like. The course of the disease is fluctuating or progressive, and some patients may progress to cirrhosis if not treated properly. However, the course of the disease varies between individuals, and this variation is due, at least in part, to genetic risk factors. In recent years, the role of genetic factors in the progression of chronic liver disease has become one of the major research areas in the hepatology community.

Recent studies have found a correlation between single base mutation of 17 β -hydroxysteroid dehydrogenase 13(HSD17B13) and chronic liver injury. 17 β -hydroxysteroid dehydrogenases (17 β -HSDs) are a type of NAD (P) H/NAD (P) -dependent oxidoreductase named because they can oxidize the 17 th carbon of steroids. In the cofactor NAD (P) H/NAD (P)⁺With the aid of (1), 17 β -HSDs may activate or deactivate 18-carbon or 19-carbon steroids by oxidizing 17-hydroxy or reducing 17-ketone. Currently, in mammals, 14 members of this family have been reported, and are sequentially named 17 β -HSD1-14 according to the order of discovery. In 2007, Liu et al, Shanghai Dandan Genesis, cloned HSD17B13 from a cDNA library of human liver. The enzyme is highly expressed in liver, and among 14 members of the 17 beta-HSD family, only the chromosomes of HSD17B13 and HSD17B11 are located at 4q 22.1. This region is rich in a class of genes encoding retinol dehydrogenase, and thus the chromosomal location of HSD17B13 suggests that this enzyme may be involved in retinol metabolism.

One large-scale study identified associations between HSD17B13 variants and chronic liver injury in four independent cohorts using the exon sequence approach. The results show that HSD17B13 rs72613567 is associated with reducing the risk of alcoholic and non-alcoholic liver disease, including progression of chronic hepatitis to cirrhosis and steatosis to steatohepatitis. A recent population study in denmark also showed that carrying HSD17B13 rs72613567 variant was associated with a reduction in alanine Aminotransferase (ALT) levels and found that this ALT reduction effect was amplified by the genetic risk of fatty liver and alcohol consumption, suggesting that HSD17B13 rs72613567 has protective effects on alcoholic liver disease and NAFLD.

Functional studies have shown that rs72613567 consists of an adenine insertion (a-ins) located near the coding gene region (chr4:87310241, grch38.p7) which is located near the donor splice site of intron 6, resulting in frame shift and premature truncation of the HSD17B13 protein, resulting in reduced enzymatic activity. It has also been reported that the variation of rs72613567 in HSD17B13 gene, TA affects hepatic lipid homeostasis. Although the mechanism of action is not clear at present, the expression of HSD17B13 protein in the liver of a patient carrying rs72613567A-ins allele is obviously reduced, and the risk of the chronic liver disease progressing to cirrhosis is reduced. This beneficial effect may be a potential drug target in reducing liver damage and even in genetic susceptibility.

It is reported in the literature that the incidence of TA is low in Hispanic Americans and Africas (9% and 1-8% respectively), but high in east Asians and Europe (27-40% and 22-31% respectively). The development and detection method of rs72613567: TA can provide risk assessment and genetic guidance for preventing the chronic hepatitis from being converted into the liver cirrhosis, and can further promote the research of pathogenic mechanisms and the research and development of targeted drugs.

Sanger sequencing is one of the main means of gene detection and is also the gold standard of gene detection. The HSD17B13 rs72613567 TA mutation is detected by a sequencing method, so that risk assessment and genetic guidance can be provided for chronic hepatitis patients. The invention discovers that the rs72613567 site mutation of HSD17B13 gene is more common in Chinese population, and the incidence rate is 44.12%.

Disclosure of Invention

The invention aims to provide a primer for detecting the mutation of a risk prediction site rs72613567 for converting chronic hepatitis into cirrhosis, and the primer can be used for quickly detecting the mutation of the rs72613567 site of HSD17B13 gene in a patient with chronic hepatitis by adopting a PCR (polymerase chain reaction) technology. The primer for detecting the mutation condition of the risk prediction site rs72613567 for converting chronic hepatitis into liver cirrhosis comprises the following steps:

the primer for amplifying the rs72613567 site of the HSD17B13 gene has a base sequence as follows:

HSD17B13-F：TGTAAAACGACGGCCAGTATGGGATGTGGAGGAAGTG

HSD17B 13-R: AACAGCTATGACCATGCTATTGGTGTTTTAGTATTTGGGT are provided. Further, the kit also comprises a sequencing primer, wherein the base sequence of the sequencing primer is as follows:

M13 F：TGTAAAACGACGGCCAGT

M13 R：AACAGCTATGACCATG。

primer sequences HSD17B13-F and HSD17B13-R are primers for amplifying the risk prediction site rs72613567 base for converting chronic hepatitis into cirrhosis.

The invention also provides a method for detecting the mutation condition of the risk prediction site rs72613567 for converting chronic hepatitis into liver cirrhosis, which comprises the following steps:

1. extracting genome DNA in peripheral blood or muscle tissue;

2. amplifying the DNA extracted in the step 1 by using PCR;

3. sequencing the amplification product in the step 2;

4. judging a sequencing result, and determining whether the rs72613567 site of the HSD17B13 gene is mutated or not;

wherein the PCR amplification primers are:

HSD17B13-F：TGTAAAACGACGGCCAGTATGGGATGTGGAGGAAGTG

HSD17B13-R：AACAGCTATGACCATGCTATTGGTGTTTTAGTATTTGGGT。

further, the sequencing primer base sequence is:

M13 F：TGTAAAACGACGGCCAGT

M13 R：AACAGCTATGACCATG。

the invention also provides a kit for detecting the mutation of the risk prediction site rs72613567 for converting chronic hepatitis into cirrhosis, which is characterized by comprising the following components in parts by weight:

(i) blood/tissue DNA extraction reagent;

(ii) detection system PCR amplification reaction solution: comprises a primer for amplifying rs72613567 locus of HSD17B13 gene, and the base sequence is as follows:

HSD17B13-F：TGTAAAACGACGGCCAGTATGGGATGTGGAGGAAGTG

HSD17B13-R：AACAGCTATGACCATGCTATTGGTGTTTTAGTATTTGGGT；

(iii) sequencing system reagent: comprises a sequencing primer, and the base sequence of the sequencing primer is as follows:

M13 F：TGTAAAACGACGGCCAGT

M13 R：AACAGCTATGACCATG；

(iv) a positive control and a negative control.

Has the advantages that: the total number of the existing liver disease patients in China is about 4 hundred million, wherein about 9300 million chronic hepatitis B infected patients and about 1300 million hepatitis C patients exist, the incidence rate of chronic hepatitis in China is high, and a large number of chronic hepatitis patients develop diseases such as cirrhosis every year. rs72613567: TA, as a beneficial mutation, reduces the risk of chronic hepatitis developing into cirrhosis. The invention can provide risk assessment and genetic suggestion for chronic hepatitis patients by detecting the mutation condition of the risk prediction site rs72613567 for converting chronic hepatitis into cirrhosis, and has good economic and social benefits.

Secondly, the invention adopts PCR technology, and can optimize the amplification efficiency and construct a stable amplification system by adjusting the reaction conditions such as primer concentration, annealing temperature and the like. The method for detecting the mutation of the risk prediction site rs72613567 of the patient from the conversion of chronic hepatitis into cirrhosis by using the sequencing technology is mature, simple and convenient, high in detection sensitivity and good in specificity, and reduces the detection cost and difficulty compared with a fluorescent quantitative PCR method.

Drawings

FIG. 1 is an agarose gel electrophoresis chart of the amplification primers, wherein M is Marker DL 2000, 1-24 are blood samples 1-24, the primers are used for amplifying a target band, the target band is clear, and the size of a product is correct.

FIG. 2 is a sequence screenshot of HSD17B13 rs72613567 site without mutation in sample detection.

FIG. 3 is a sequence screenshot of sample detection HSD17B13 rs72613567 site with heterozygous mutation A/T.

FIG. 4 is a sequence screenshot of sample detection HSD17B13 rs72613567 site with homozygous mutation A/A.

Detailed Description

The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings. It should be noted that the conventional conditions and methods not described in the examples are generally employed by those skilled in the art according to the routine procedures: such as OsOb and Kingston, fourth edition, or following the manufacturer's suggested procedures and conditions.

Example 1

A primer for detecting the mutation of a risk prediction site rs72613567 for converting chronic hepatitis into cirrhosis is a specific amplification primer designed aiming at the rs72613567 mutation site of HSD17B13 gene, and the base sequence of the primer is as follows:

HSD17B13-F：TGTAAAACGACGGCCAGTATGGGATGTGGAGGAAGTG

HSD17B13-R：AACAGCTATGACCATGCTATTGGTGTTTTAGTATTTGGGT；

a kit for detecting the risk prediction site rs72613567 mutation of chronic hepatitis converted into cirrhosis comprises

(i) Blood/tissue DNA extraction reagent;

(ii) detecting a system PCR reaction solution;

(iii) sequencing system reagents;

(iv) a positive control and a negative control.

The blood/tissue DNA extraction reagent can be purchased from commercialized reagents such as Tiangen DNA extraction kit and the like.

The PCR amplification reaction solution of the detection system comprises: 2 times PCR Buffer; 2mM dNTPs; KOD FX DNA Polymerase (1U/. mu.l); HSD17B13 rs72613567: primers for upstream and downstream of TA site (5. mu.M).

The sequencing system reagent comprises: sequencing purification solution (ExoI:0.6U, CIP:1.2U), EDTA (125mmol), absolute ethanol, 75% ethanol, HIDI (highly deionized formamide), sequencing primers: upstream and downstream primers (3.2 μm) of rs72613567 of HSD17B13 gene were detected.

Example 2

The operation flow of the blood/cell/tissue genome DNA extraction kit (Tiangen organism):

(1) extracting tissue DNA from blood: 1) mu.l of blood was taken and added to 900. mu.l of erythrocyte lysate, mixed by inversion, left at room temperature for 5 minutes, and mixed by inversion several times in the meantime. Centrifuge at 12,000 rpm for 1min, aspirate the supernatant, leave the leukocyte pellet, add 200. mu.l of buffer GA, and shake until thoroughly mixed. 2) Add 20. mu.l proteinase K solution and mix well. 3) Add 200. mu.l buffer GB, mix well by inversion, stand at 70 ℃ for 10 minutes, clear the solution, centrifuge briefly to remove beads on the inner wall of the tube cap. 4) Add 200. mu.l of absolute ethanol, mix well with shaking for 15 seconds, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover. 5) Adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is put into a collecting pipe), centrifuging at 12,000 rpm for 30 s, pouring off waste liquid, and putting the adsorption column CB3 back into the collecting pipe. 6) Add 500. mu.l buffer GD (check whether absolute ethanol has been added before use) to adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, dump the waste and place adsorption column CB3 in the collection tube. 7) To the adsorption column CB3, 700. mu.l of a rinsing solution PW (previously used, whether or not absolute ethyl alcohol has been added) was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and the waste liquid was discarded, and the adsorption column CB3 was put into a collection tube. 8) To the adsorption column CB3, 500. mu.l of a rinsing solution PW was added, and the mixture was centrifuged at 12,000 rpm for 30 seconds, and then the waste liquid was discarded. 9) The adsorption column CB3 was returned to the collection tube, centrifuged at 12,000 rpm for 2 minutes, and the waste liquid was discarded. The adsorption column CB3 was left at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material. 10) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 100 mu l of elution buffer TE into the middle part of the adsorption membrane, standing for 2-5 minutes at room temperature, centrifuging for 2 minutes at 12,000 rpm, and collecting the solution into the centrifuge tube.

(2) Reagent preparation: preparing X mul of PCR reaction liquid of a detection system according to the parts of detected people, and subpackaging 18 mul of each part:

x18. mu.l reaction solution X (n specimen +1 part positive control +1 part negative control +1 part blank control)

And n is the number of detected samples.

(3) Sample adding: adding 2 mul DNA into the PCR reaction solution of the detection system; directly adding 2 mul of positive control substance and negative control substance into the positive control substance and the negative control substance; blank control was supplemented with 2. mu.l of physiological saline or nothing.

(4) Amplification: the detection is carried out on a conventional PCR instrument, and available instruments include ABI veriti (Applied Biosystems, USA) and the like. The reaction conditions were as follows:

the preparation method of the PCR amplification system reagent comprises the following steps:

wherein, the primer sequence is as follows:

note: f is an upstream primer, R is a downstream primer

(5) Electrophoresis: electrophoresis on 1.5% agarose gel at 120V for 30min, and observation on a gel imaging system.

As shown in FIG. 1, the electropherogram of the product obtained after amplification of 24 blood samples with the primers is obtained. The electrophorogram shows that the amplification of the invention is effective and the band is clear.

(6) Sanger sequencing:

take 9. mu.l of PCR product and 2. mu.l of purification system. Purification was performed according to the following procedure:

mu.l of the purified product was mixed with the upper and lower sequencing primers, respectively, according to the following system:

sequencing reaction program:

and (3) a precipitation link:

adding 2 mu l of 125mmol EDTA into the product after the sequencing reaction, and standing for 5 min; adding 15 mul of absolute ethyl alcohol, and mixing evenly by vortex; centrifuging at 3700rpm for 30 min; inverting, centrifuging for 15sec, adding 50ml 70% ethanol, and mixing by vortex; centrifuging at 3700rpm for 15 min; inverting and centrifuging for 15sec, and placing on a metal bath at 95 ℃; after addition of 10. mu.l CBL, denaturation was carried out for 5min and finally sequencing was carried out on a sequencer (ABI3730) at-20 ℃ for 2 min.

(7) And (5) judging a result: the sequencing results were aligned with the HSD17B13(NC _000004.12) reference sequence, respectively, and the results were reported as a function of the actual mutation.

Example 3

34 clinical peripheral blood samples were taken, and the genome was extracted, reagents were prepared, and tested as described in example 2. Each sample was added to 2. mu.l of the detection system PCR reaction solution. At the same time, positive and negative are made, and blank control is performed respectively. 15 samples with rs72613567 mutation were found after sequencing, and the mutation rate was 44.12%, which is consistent with literature reports. Among them, 14 samples were heterozygous for the mutation and 1 sample was homozygous for the mutation. The mutation conditions of the 34 samples are shown in the table, and the sequencing results of the normal samples and the positive samples are shown in FIGS. 2-4.

Sequence listing

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<120> primers and method for detecting risk prediction site for converting chronic hepatitis into liver cirrhosis

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tgtaaaacga cggccagtat gggatgtgga ggaagtg 37

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aacagctatg accatgctat tggtgtttta gtatttgggt 40

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tgtaaaacga cggccagt 18

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aacagctatg accatg 16

Claims

1. The primer for detecting the mutation condition of the risk prediction site rs72613567 for converting chronic hepatitis into liver cirrhosis is characterized by comprising the following steps of:

HSD17B13-F：TGTAAAACGACGGCCAGTATGGGATGTGGAGGAAGTG

HSD17B13-R：AACAGCTATGACCATGCTATTGGTGTTTTAGTATTTGGGT。

2. the primer according to claim 1, further comprising a sequencing primer having a base sequence of:

M13 F：TGTAAAACGACGGCCAGT

M13 R：AACAGCTATGACCATG。

3. the primer according to claim 1, wherein the primer sequences HSD17B13-F and HSD17B13-R are primers for amplifying the risk prediction site rs72613567 for converting chronic hepatitis into liver cirrhosis.

4. The method for detecting the mutation condition of the risk prediction site rs72613567 for converting chronic hepatitis into liver cirrhosis comprises the following steps:

(1) extracting genome DNA in peripheral blood or muscle tissue;

(2) amplifying the DNA extracted in the step 1 by using PCR;

(3) sequencing the amplification product in the step 2;

(4) judging a sequencing result, and determining whether the rs72613567 site of the HSD17B13 gene is mutated or not;

wherein the PCR amplification primers are:

HSD17B13-F：TGTAAAACGACGGCCAGTATGGGATGTGGAGGAAGTG

HSD17B13-R：AACAGCTATGACCATGCTATTGGTGTTTTAGTATTTGGGT。

5. the method of claim 4, wherein the sequencing primer base sequence is:

M13 F：TGTAAAACGACGGCCAGT

M13 R：AACAGCTATGACCATG。