CN112626203A

CN112626203A - Primer and method for detecting mutations of C.55C > G and C.238C > T sites of ATP8B1 gene

Info

Publication number: CN112626203A
Application number: CN202110066529.3A
Authority: CN
Inventors: 裘振亚; 王淑一
Original assignee: Hefei Aidikang Medical Laboratory Co ltd
Current assignee: Hefei Aidikang Medical Laboratory Co ltd
Priority date: 2021-01-19
Filing date: 2021-01-19
Publication date: 2021-04-09

Abstract

The invention discloses a primer for detecting c.55C > G and c.238C > T base mutation of an ATP8B1 gene, which comprises a primer for amplifying c.55C > G and c.238C > T sites of the ATP8B1 gene; the Sanger sequencing technology can be used for rapidly detecting the c.55C > G and c.238C > T site mutation conditions of the ATP8B1 gene in a patient with the progressive familial intrahepatic cholestasis syndrome 1. The detection result completed by the invention is accurate, and has important reference significance for clinical differential diagnosis of the progressive familial intrahepatic cholestasis type 1 patient.

Description

Primer and method for detecting c.55C & gtG and c.238C & gtT site mutation of ATP8B1 gene

Technical Field

The invention belongs to the field of life science and biotechnology, and particularly relates to a primer for detecting mutation of C.55C & gtG and C.238C & gtT sites of an ATP8B1 gene, which can be used for rapidly detecting mutation conditions of C.55C & gtG and C.238C & gtT sites of an ATP8B1 gene in a patient with progressive familial intrahepatic cholestasis syndrome 1 by adopting a common PCR (polymerase chain reaction) combined with a Sanger sequencing technology.

Background

Progressive familial intrahepatic cholestasis type 1 (PFIC 1), early named Byler's disease, is an autosomal recessive genetic disease caused by mutations in the ATP8B1 gene. The ATP8B1 gene is located on chromosome 18q21-22, has a total length of about 176.68kb, comprises 28 exons and codes FIC1 protein containing 1251 amino acid residues, the exons are dispersed into genome sequences of at least 70kb, and RNA transcripts with a length of about 6-7 kb can be expressed in most tissues and a large amount of pancreas, intestines and liver. ATP8B1mRNA contains 3753 coding nucleotides, flanked by a 5 'untranslated region of unknown length, a 2.1kb 3' translated region, and a poly-tail.

ATP8Bl belongs to the subfamily P-atpase 4, members of the subfamily P-atpase 4 are involved in protein trafficking and apoptosis, and ATP8Bl is the 1 st gene in the subfamily P-atpase 4 involved in human genetic or functional diseases. FIC1 protein is mainly distributed at brush border of intestinal epithelial cell, gastric small concave epithelial cell, apical membrane of pancreatic acinar cell, and apical membrane of hepatic cell, biliary duct and bile duct, and expressed in kidney and inner ear hair cell. FIC1, an aminophospholipid translocase, is responsible for the transfer of phosphatidylserine from the outer layer to the inner layer of a biological membrane, thus maintaining the asymmetry in lipid distribution on both sides of the membrane. PFIC1 patients usually develop clinical manifestations of progressive intrahepatic cholestasis within 1 year of age, which may progress to end-stage liver disease; the most prominent biochemical feature is that serum gamma-glutamyl transpeptidase (GGT) is not high all the time and is not parallel to the severe intrahepatic cholestasis. Because the FIC1 protein is expressed in a plurality of systems, patients with PFIC1 often have a plurality of extrahepatic manifestations, such as diarrhea, pancreatitis, nerve deafness, etc. The foreign literature estimates that the incidence of PFIC1 is approximately 1/100000 to 1/1000000, and no obvious difference exists between men and women. At present, China lacks large-sample multi-center epidemiological survey data about the incidence of the disease, and the diagnosed children are limited to individual large hospitals in central cities such as Beijing, Shanghai, Guangzhou and Wuhan, so that the number of patients is quite limited, and clinical diagnosis and treatment experiences also need to be further accumulated.

Disclosure of Invention

The invention aims to provide a primer for detecting c.55C > G and c.238C > T site mutation of an ATP8B1 gene, which can be used for quickly detecting the c.55C > G and c.238C > T site mutation conditions of an ATP8B1 gene in a patient with progressive familial intrahepatic cholestasis syndrome 1 by adopting a PCR (polymerase chain reaction) technology. The primer for detecting the mutation conditions of c.55C > G and c.238C > T sites of the ATP8B1 gene comprises the following components:

the primer for amplifying the c.55C > G and c.238C > T sites of the ATP8B1 gene has the base sequence as follows:

ATP8B1-EXON1-F:TGTAAAACGACGGCCAGTTAGGGAGGGAAAAAGGGAG

ATP8B1-EXON1-R:AACAGCTATGACCATGATCCCGGATCTCCAAAGT

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

further, the primer sequences ATP8B1-EXON1-F and ATP8B1-EXON1-R are primers for amplifying bases c.55 and c.238 of the ATP8B1 gene.

The invention also provides a method for detecting the mutation conditions of the c.55C & gtG and c.238C & gtT sites of the ATP8B1 gene, 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 the sequencing result, and determining whether the c.55 site and the c.238 site of the ATP8B1 gene are mutated;

wherein the PCR amplification primers are:

ATP8B1-EXON1-F:TGTAAAACGACGGCCAGTTAGGGAGGGAAAAAGGGAG

ATP8B1-EXON1-R:AACAGCTATGACCATGATCCCGGATCTCCAAAGT

further, the sequencing primer base sequence is:

M13 F:TGTAAAACGACGGCCAGT

M13 R:AACAGCTATGACCATG

the invention also provides a kit for detecting c.55C > G and c.238C > T site mutation of the ATP8B1 gene, wherein the kit comprises:

(i) blood/tissue DNA extraction reagent;

(ii) detection system PCR amplification reaction solution: the primer comprises a c.55C & gtG site and a c.238C & gtT site for amplifying an ATP8B1 gene, and the base sequence of the primer is as follows:

ATP8B1-EXON1-F:TGTAAAACGACGGCCAGTTAGGGAGGGAAAAAGGGAG

ATP8B1-EXON1-R:AACAGCTATGACCATGATCCCGGATCTCCAAAGT

(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 invention can simultaneously detect c.55C > G and c.238C > T2 mutations of the ATP8B1 gene by designing 1 pair of primers, and is efficient, simple and convenient. The method is not reported in the literature and is found for the first time. 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 c.55C > G and c.238C > T mutation hotspots of the ATP8B1 gene of a patient by using a sequencing technology can detect the c.55C > G and c.238C > T mutations of the ATP8B1 gene, and compared with a fluorescent quantitative PCR method, the method reduces the detection cost and difficulty. The fluorescent quantitative PCR method needs to design probes aiming at different mutation types, and has high cost and great detection difficulty.

Drawings

FIG. 1 is the agarose gel electrophoresis of the amplification primers, M is Marker DL 2000, 1-10 is the blood sample, the amplification of the primers is effective, the target band is clear, and the size of the product is correct.

FIG. 2 is a sequence diagram of sample 3 showing the detection of ATP8B1 c.55C > C/G heterozygous mutation.

FIG. 3 is a sequencing screenshot of sample 6 for detecting ATP8B1 c.238C > C/T hybrid and mutation.

Detailed Description

Example 1

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.

A primer for detecting c.55C > G and c.238C > T site mutation of an ATP8B1 gene, wherein the primer is a specific amplification primer designed aiming at the c.55C > G and c.238C > T mutation sites of the ATP8B1 gene, and the primer comprises the following components:

the primer for amplifying c.55C and c.238 base of ATP8B1 gene has the base sequence:

ATP8B1-EXON1-F:TGTAAAACGACGGCCAGTTAGGGAGGGAAAAAGGGAG

ATP8B1-EXON1-R:AACAGCTATGACCATGATCCCGGATCTCCAAAGT

a kit for detecting the mutation of C.55C > G and C.238C > T sites of ATP8B1 gene includes

(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); ATP8B1 upstream and downstream primers (10. 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: and upstream and downstream primers (3.2 mu M) for detecting c.55C > G and c.238C > T of the ATP8B1 gene.

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 was performed on a conventional PCR instrument, and available instruments include ABI veriti96 (Applied Biosystems, USA), etc. 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 20 cases of blood samples were amplified with the corresponding primers. Analysis by electropherograms showed that the amplification of the invention was efficient and the bands were 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: and respectively comparing the sequencing result with an ATP8B1 negative reference sequence, and reporting the result according to the actual mutation condition.

Example 3

10 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. After sequencing, the ATP8B1 c.55 site gene mutation condition is that the

samples

2, 4 and 10 have mutation, and the rest samples do not have mutation; the mutations of ATP8B1 c.238 sample No. 2 and No. 10, but not all the other samples. The mutation status of samples 1-10 is shown in the following table, the agarose gel electrophoresis results are shown in FIG. 1, and the positive sequencing graphs are shown in FIGS. 2-3.

	c.55C＞G	c.238C＞T
			1	-	-
2	+	+
			3	-	-
4	+	-
			5	-	-
6	-	-
			7	-	-
8	-	-
			9	-	-
10	+	+
			Detecting mutation rate this time	30％	20％
Literature reports mutation rates	Not reported	Not reported

Note: + a heterozygous or complete mutation, and-a negative mutation

Sequence listing

<110> Yidekang laboratory Co., Ltd for medical examination of Hefei Aidikang

Primer and method for detecting mutation of c.55C, G and c.238C and T sites of ATP8B1 gene

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgtaaaacga cggccagtta gggagggaaa aagggag 37

<210> 2

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aacagctatg accatgatcc cggatctcca aagt 34

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgtaaaacga cggccagt 18

<210> 4

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aacagctatg accatg 16

Claims

1. The primers for detecting the mutation conditions of c.55C > G and c.238C > T sites of the ATP8B1 gene are characterized in that the base sequence is as follows:

ATP8B1-EXON1-F:TGTAAAACGACGGCCAGTTAGGGAGGGAAAAAGGGAG

ATP8B1-EXON1-R:AACAGCTATGACCATGATCCCGGATCTCCAAAGT。

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

M13 F:TGTAAAACGACGGCCAGT

M13 R:AACAGCTATGACCATG。

3. the primer of claim 1, wherein the primer sequences ATP8B1-EXON1-F and ATP8B1-EXON1-R are primers for amplifying bases ATP8B1 gene c.55, c.238.

4. The method for detecting the mutation conditions of the c.55C > G and c.238C > T sites of the ATP8B1 gene 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 the sequencing result, and determining whether the c.55 and c.238 loci of the ATP8B1 gene are mutated; wherein the PCR amplification primers are:

ATP8B1-EXON1-F:TGTAAAACGACGGCCAGTTAGGGAGGGAAAAAGGGAG

ATP8B1-EXON1-R:AACAGCTATGACCATGATCCCGGATCTCCAAAGT。

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

M13 F:TGTAAAACGACGGCCAGT

M13 R:AACAGCTATGACCATG。