WO2010008071A1 - Method for screening of gene mutation by utilizing quantification technique - Google Patents

Abstract

Disclosed is a novel screening method for determining the presence or absence of a gene mutation in every exon of a gene associated with a congenital disease or a neoplastic disease rapidly and conveniently. First, a hetero-duplex product comprising an exon having a gene mutation therein is cleaved with an endonuclease specific to a heterozygostic site or a chemical substance capable of cleaving a mismatch. Next, nested primers for primers that are used for the formation of a PCR product of each exon are so designed that the nested primer are adjacent to introns and consensus regions, a real-time PCR is carried out, and the comparative quantification with a wild-type control is carried out.  In this manner, the presence or absence of an exon having any gene mutation therein can be detected. The method is a gene mutation screening method utilizing a quantitative PCR technique.  The method can be achieved rapidly, conveniently and at low cost without the need of using any expensive apparatus, because the method utilizes a quantitative technique.  The method can accurately detect even a mutation which occurs at the terminal of an exon and is hardly detected by conventional prior art techniques.  Therefore, the method is useful for the identification of a site having a gene mutation associated with a congenital disease or a neoplastic disease therein.

Description

Gene mutation screening method using quantitative method

The present invention is a novel method for screening gene mutations quickly and easily using a quantitative method using real-time PCR.

Because mutations and deletions in specific regions of genes cause onset of various congenital diseases and neoplastic diseases such as cancer, identification of mutation sites is not only useful for definitive diagnosis and decision of treatment policy, but also the disease mechanism It is also important for elucidation and development of therapeutic methods.
In order to carry out genetic tests for congenital diseases and neoplastic diseases, it is necessary to detect gene mutation sites for all responsible genes related to the diseases.
However, the number of responsible genes is not limited to one, and since one responsible gene may have many exons, it is not an easy task to detect gene mutation sites related to diseases.
Depending on the disease, testing requires enormous amounts of time, money, and labor, so it is not uncommon for genetic testing itself to be reduced.
Therefore, for the purpose of carrying out an efficient genetic test, a method is used in which exons are screened for each exon before sequencing.
Examples include SSCP (single-strand conformational polymorphism) method and DHPLC (Denaturing high performance liquid chromatography) method.
In addition, several methods are known as screening methods for detecting mutations by treating a heterozygous site-specific endonuclease after heteroduplex formation (for example, Patent Document 1).

JP-T 2002-512043 HUMAN MUTATION, 27 (12), 1163-1173 (2006)

The above SSCP and DHPLC have already been introduced in many facilities, but the disadvantage that the risk of false negative becomes high when the mutation site is located at the end of the exon has not been overcome.
On the other hand, as one screening method using heteroduplex heterozygous site-specific endonuclease treatment, there is a method in which endonuclease-treated PCR products (each exon) are labeled and then migrated to the capillary of an autosequencer.
Although this method can accurately detect mutations, it takes a lot of time and requires a long time for detection, and the cost of reagents is also expensive.
As another method, there is a method in which an endonuclease-treated PCR product (each exon) is directly run on an agarose gel.
In this method, if the mutation site is near both ends of the PCR product (each exon), it cannot be detected.
As a solution to this problem, a method has been proposed in which the primer is set to about 150 bases outside (region in the intron). However, since the intron is likely to be mutated, the probability of false positive is increased.
Thus, the present condition is that the screening method excellent in all of quickness, simplicity, accuracy, and economical efficiency does not exist yet (nonpatent literature 1).

In the method of the present invention, the heterozygous site of the heteroduplex is specifically cleaved, and then a nested primer is designed to be adjacent to the consensus region of the intron inside the duplex forming primer, and real-time PCR is performed. This is a method of screening for the presence or absence of mutations quickly, simply and inexpensively by carrying out comparative quantification with a wild type control (see FIG. 1).
According to the method of the present invention, even if the mutation site is located at the exon end, it can be easily detected, and various drawbacks of existing screening methods can be solved.
Hereinafter, the present invention will be described in detail.

The method of the present invention is a method comprising the following procedures.
Procedure 1: Primers are designed outside the consensus region of each exon and intron of the targeted disease gene and amplified by PCR.
Operation 2: For homozygous gene mutations (autosomal recessive genetic diseases, etc.), mix equal amounts of PCR products from healthy individuals (all wild type) and patient genes (including mutants). In the case of gene mutations (autosomal dominant genetic diseases, etc.), duplexes are formed using only the PCR products of patient genes.
Operation 3: A heterozygous site is cleaved.
Operation 4: Primers are designed so that the cleaved PCR product is adjacent to the outside of the consensus region of the intron, and each duplex is amplified and quantified by real-time PCR.
Operation 5: The presence or absence of a gene mutation is detected by observing a quantitative difference in comparison with a wild-type control.

<Operation 1>
The genetic diseases targeted by the method of the present invention include, for example, congenital factor VII deficiency, systemic fetus, congenital ichthyosis, Tay-Sachs disease, cystic fibrosis, phenylketonuria, galactosemia, sugar Autosomal dominant genetic diseases such as primary disease type I, Wilson's disease, Werner syndrome, cystic fibrosis, congenital muscular dystrophy, autosomal recessive diseases such as sickle cell anemia, congenital antithrombin deficiency, congenital protein S deficiency And sexual recessive genetic diseases such as hemophilia.
Screening is performed targeting each exon of these disease responsible genes.
The primer (duplex primer) in operation 1 is designed to be outside the consensus region of the targeted exon and intron.

Specifically, in the primer design, when the exon is large, the PCR is designed to be divided into a plurality of parts.
Further, in the boundary region between exons and introns, it may be set at least several bases (5 to 6 bases) or more outside the consensus sequence (sequence recognized by snRNPs during splicing), preferably several bases to 100 The base may be designed to be separated from the base, more preferably several tens to a hundred bases.
As a result, the quantification primer in operation 4 becomes a nested PCR (nested-PCR), and the specificity of the target is increased. In addition, the nonspecific exonuclease activity of the endonuclease used in operation 3 has an adverse effect on the quantification primer region. (The primer region is scraped off and the primer is not attached) can be avoided.
The heat resistant DNA polymerase used in PCR is preferably one having a proofreading function such as KOD DNA polymerase so as not to cause a replication error.
In addition, PCR conditions and the like may be in accordance with ordinary methods.

<Operation 2>
A patient specimen is used to form a duplex.
Specifically, in the case of homozygous gene mutations (autosomal recessive genetic diseases, etc.), a healthy person's gene (PCR product) and a patient's gene (PCR product) are mixed in a one-to-one relationship. In the case of gene mutations (autosomal dominant genetic diseases, etc.), only the patient's own gene (PCR product) is used to form duplexes.
As a result, only the homoduplex is formed in the exon having no mutation, and the ratio of homoduplex: heteroduplex = 1: 1 is formed in the exon having the mutation.

<Operation 3>
A heterozygous site-specific cleavage treatment is performed.
Specifically, when the formed duplex is treated with heterozygous site-specific endonuclease CEL1 or T7 nuclease or single-stranded DNA degrading enzyme S1 nuclease, only the heteroduplex is cleaved at the mismatch site. .
In addition, a method (Chemical Cleavage of Mismatch method) of cleaving the mismatch site using a chemical substance such as piperidine instead of an enzyme such as nuclease can be used.

<Operation 4>
Primers are designed so as to be adjacent to the outside of the intron consensus region of the cleaved PCR product (each exon), real-time PCR is performed, and each duplex is amplified and quantified.
As the template for real-time PCR, those that have been treated with nuclease in duplex and those that have not been treated are diluted in equal amounts (about 400 to 500 times).

The primer for quantification in this operation may be the same primer as the first duplex primer (operation 1).
Here, for the purpose of enhancing the specificity of real-time PCR and avoiding false positives when exonuclease activity occurs, the method of nested PCR is applied, and the quantification primer is further inside (several bases) than the duplex primer. It is preferable to design them up to about 100 bases apart.
In addition, by designing the quantitation primer so as to be adjacent to the outside of the intron consensus sequence, mutations in all exon regions including the intron consensus sequence can be detected.

<Operation 5>
When amplification and quantification are performed from both ends of each exon including the intron / consensus region by real-time PCR using a primer for quantification (Step 4) (using an intercalator fluorescent dye such as SYBR Green I), an exon with only a homoduplex (mutation) Exon without) does not decrease compared to the case without cleavage treatment, but exon containing heteroduplex (exon with mutation) is theoretically reduced to 50%. It can be detected.
When the amplification efficiency of real-time PCR is maximum, a difference of 50% corresponds to one cycle.
However, since a measurement error is included in the instrument although it is small, it is possible to perform screening more reliably by expressing a difference of 50% as a difference of one cycle or more.
Therefore, by utilizing the fact that intercalator fluorescent dyes have a PCR inhibitory action and increasing the concentration of intercalator fluorescent dyes, real-time PCR can be used to increase the number of cycles and facilitate screening. I can do it.
The same effect can be obtained by adding another PCR inhibitor.

Non-specific nuclease activity may occur in heterozygous site-specific endonucleases and single-stranded DNA degrading enzymes.
That is, even a homoduplex-only wild type control may decrease after the cleavage treatment.
In addition, since there may be a difference in amplification efficiency depending on the PCR conditions, the results will vary in the reduction rate without the wild type control.
Therefore, in order to eliminate non-specific activity such as nuclease and influence on amplification efficiency by PCR conditions, it is preferable to use the following two-step correction method.
(1) As with patient specimens, a wild-type control is prepared for each exon, and the duplex of each exon is quantified by dividing it into an equal amount and a non-cleaved one.
At this time, the duplex not cut is corrected as 100, and the cut duplex is quantified.
As a result, both the wild type control and the patient specimen obtain a reduction rate affected by nonspecific nuclease activity.
(2) Next, the reduction rate of each exon of the patient specimen is corrected with the reduction rate of each exon of the wild type control as 100.
By setting the result obtained by this correction to the true reduction rate of each exon, it is possible to eliminate the influence of nonspecific nuclease activity and the like from the result.

The method of the present invention is an autosomal recessive genetic disease (congenital factor VII deficiency, systemic fetus, congenital ichthyosis, Tay-Sachs disease, cystic fibrosis, phenylketonuria, galactosemia, glycogenosis type I , Wilson's disease, Werner syndrome, cystic fibrosis, congenital muscular dystrophy, sickle cell anemia, etc.) and congenital recessive genetic diseases (hemophilia, etc.).
The kit is a complete wild type (Wild Type) gene without heterogeneous mutation or single nucleotide polymorphism, disease-specific primer set, heterozygous site-specific nuclease enzyme or single-stranded DNA degrading enzyme, buffer A liquid or the like may be used as a component of the kit.
In the kit, the wild-type gene can also be used as a quantitative control.

When trying to identify genetic mutation sites in congenital diseases or cancer, all exons can be sequenced by screening for the presence or absence of mutations in each exon using a screening method such as the method of the present invention. Compared to the case of sequencing, time, labor, and cost can be greatly reduced.
Here, the techniques in the screening method of the present invention, which are not in the existing screening methods, and the effects of the invention are listed.

(1) The duplex PCR primer is designed outside, and after the endonuclease treatment, the quantitative primer is designed to be adjacent to the intron consensus region.
This way
i) Targeted gene specificity increases as nested PCR.
ii) can invalidate non-specific exonuclease activity of endonuclease,
iii) Can easily detect exon end mutations (resolves the main cause of false negatives),
iv) Since only regions necessary for screening (all exon regions including intron consensus sequences) can be checked, false positives are unlikely to occur.
(2) The non-specific nuclease activity of the endonuclease can be nullified by always calculating the ratio to the wild type (with the wild type as 100) using the wild type as a control and calculating the value with the correction value.
Corrections can be made to minimize other procedural or measurement effects.
(3) The method of the present invention is the first gene mutation screening method using a real-time PCR quantification method. By using the quantification method, even if there is a mutation at the exon end, it is the same as the mutation located at the center of the exon. Easy detection is possible.
Furthermore, it is possible to realize a rapid, simple, and inexpensive screening method utilizing the characteristics of real-time PCR.
(4) A kit can be made for all congenital diseases including autosomal dominant genetic diseases such as congenital antithrombin deficiency.

The outline of the new screening method using the quantitative method is shown. The different bases (difference between G and C, enclosed characters) in the base sequences of two types of plasmid DNAs (reference G and reference C) are shown. A heteroduplex product, which is a product similar to an exon containing a gene mutation, is quantified by real-time PCR after treatment with a heterozygous site-specific endonuclease (corrected as 100 for a duplex product treated with ultrapure water) . In the figure, “Homo” means a duplex product of only the wild type, and “Hetero” means a duplex product in which the wild type and the mutant type are mixed at 1: 1. The result of having detected the gene mutation located in the exon end by the method of this invention using the patient sample of protein S deficiency is shown (it corrected by setting wild type control to 100). The result of having detected the gene mutation located in the consensus area | region of an intron by the method of this invention using the patient sample of an antithrombin deficiency is shown (it corrected by setting wild type control to 100). The result of having implemented the method of this invention with respect to the sample which mixed the patient sample of the antithrombin deficiency and the normal sample (wild type) by 1: 1 is shown (it corrected by setting wild type control to 100). The result of detecting a gene mutation located in the central region of an exon by a method of the present invention using a single-strand DNA-degrading enzyme using a patient sample with antithrombin deficiency is shown (corrected assuming that the wild-type control is 100).

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.

Two types of plasmid DNAs containing the base sequences of reference G (SEQ ID NO: 1) and reference C (SEQ ID NO: 2) were used.
The only difference is the difference between the enclosing characters G and C in FIG.
(1) Primer set
Forward Primer: ACACCTGATCAAGCCTGTTCATTTGATTAC (SEQ ID NO: 3)
Reverse Primer: CGCCAAAGAATGATCTGCGGAGCTT (SEQ ID NO: 4)
Primers are common to PCR and real-time PCR.
(2) Duplex formation After obtaining PCR products of reference G and reference C (632 bp: Polymerase uses KOD) using the above primers, homoduplex (only reference G), heteroduplex (reference G, and Reference C was mixed 1 to 1).
・ Duplex formation conditions (use of thermal cycler)
95 ° C 2 min
95 ℃ ～ 85 ℃ 5 sec. (2 ℃ / sec.)
85 ℃ ～ 25 ℃ 600 sec. (0.1 ℃ / sec.)
4 ° C. for 2 min or more (3) Treatment with enzyme-treated CEL1 nuclease at 42 ° C. for 20 minutes and then at 4 ° C. for 2 minutes.
(4) Quantification by real-time PCR Real-time PCR was performed using the primer set of (1) above.
The following four samples are quantitative.
・ Homo + water → Homoduplex only treated with ultrapure water.
・ Homo + CEL1 → Homoduplex only treated with heterozygous site-specific endonuclease (CEL1).
・ Hetero + water → treated with ultrapure water in the presence of 1: 1 homoduplex and heteroduplex.
• Hetero + CEL1 → Enzyme-treated with heterozygous site-specific endonuclease (CEL1) in the presence of 1: 1 homoduplex and heteroduplex.
(4) Results As shown in FIG. 3, when homoduplex and heteroduplex are present at a ratio of 1: 1, the enzyme treatment with a heterozygous site-specific endonuclease and quantification are compared with the control of ultrapure water treatment. Since it decreases to about 50%, the presence or absence of mutation can be detected easily.

The method of the present invention was performed on a patient sample having protein S deficiency and having a heterozygous mutation (x mark) at the end of exon 2.
This is a very difficult part to detect with the prior art.
(1) PCR primer for primer set duplex formation (PROS1 gene, exon 2 amplification)
Forward Primer: GGCAGAAAATGATTTTAACTCTTATTGT (SEQ ID NO: 5)
Reverse Primer: TGGAAATGTTCAGTCTGTAGTTGTAT (SEQ ID NO: 6)
Real-time PCR primers
Forward Primer: ATATAAACTGATTGTTTCCTTC (SEQ ID NO: 7)
Reverse Primer: AGTTTCCATAAATGCTTACCGTT (SEQ ID NO: 8)
(2) Duplex formation Using the above PCR primer for duplex formation, after obtaining the PROS1 gene and exon 2 region of the healthy person DNA and patient DNA (polymerase uses KOD), homoduplex (only for healthy subject samples), Heteroduplexes (patient specimens only) were formed respectively.
Duplex formation conditions (use of thermal cycler): Same as Example 1.
(3) Enzyme treatment: Same as Example 1.
(4) Quantification by real-time PCR Real-time PCR was performed using the primer set for real-time PCR described in (1) above.
The following four samples are quantitative.
-Only wild-type duplex treated with ultrapure water.
-Enzymatic treatment of only wild-type duplex with heterozygous site-specific endonuclease (CEL1).
-Treated with ultrapure water in the presence of 1: 1 homoduplex and heteroduplex by patient specimens.
-Enzyme-treated with heterozygous specific endonuclease (SEL1) in the presence of 1: 1 homoduplex and heteroduplex by patient specimen.
(5) Results As shown in FIG. 4, in the patient specimen, since the fluctuation amount of the wild-type control is reduced to 100, it is reduced to 46.25%, which is close to the theoretical value (50%) when corrected. It was shown that even difficult exon end mutations can be easily detected.

The method of the present invention was carried out on a patient specimen having antithrombin deficiency and having a heterozygous mutation (x mark) at the end of the intron region consensus sequence adjacent to exon 5. This is a very difficult part to detect with the prior art.
(1) Primer set Duplex formation PCR primer (Antithrombin gene, exon 5 amplification)
Forward Primer: CCAAAGGATCTCTTAATCCAAACTGA (SEQ ID NO: 9)
Reverse Primer: GCATGCCTTAACACTGGAAAC (SEQ ID NO: 10)
Real-time PCR primers
Forward Primer: TCTGTGGATTGAAGCCAACTTT (SEQ ID NO: 11)
Reverse Primer: CTGCTGTTCATGCATCTCCT (SEQ ID NO: 12)
(2) Duplex formation After obtaining the antithrombin gene and exon 5 region (polymerase uses KOD) of normal and patient DNA using the above PCR primer for duplex formation, homoduplex (only for healthy subjects) , Heteroduplexes (patient specimens only) were formed respectively.
Duplex formation conditions (use of thermal cycler): Same as Example 1.
(3) Enzyme treatment: Same as Example 1.
(4) Quantification by real-time PCR Real-time PCR was performed using the primer set for real-time PCR described in (1) above.
The following four samples are quantitative.
-Only wild-type duplex treated with ultrapure water.
-Enzymatic treatment of only wild-type duplex with heterozygous site-specific endonuclease (CEL1).
-Treated with ultrapure water in the presence of 1: 1 homoduplex and heteroduplex by patient specimens.
-Enzyme-treated with heterozygous specific endonuclease (SEL1) in the presence of 1: 1 homoduplex and heteroduplex by patient specimen.
(5) Results As shown in FIG. 5, in the patient specimen, the amount of fluctuation of the wild type control is reduced to 42.43% which is close to the theoretical value (50%) when corrected as 100. It was shown that even very difficult mutations at the end of the intron consensus region can be easily detected.

Antithrombin deficiency has patterns of type I and type II depending on the amount of antigen. In the case of type II, there may be either autosomal dominant inheritance or autosomal recessive inheritance.
Therefore, assuming the type II, the patient specimen and the wild type were mixed 1: 1, and the method of the present invention was carried out.
As the patient sample, a sample having a heterozygous mutation (x mark) near the center of exon 4 was used.
(1) Primer set Duplex formation PCR primer (antithrombin gene, exon 4 amplification)
Forward Primer: ACACTATAATATGGATATGCTTGTGTCAAT (SEQ ID NO: 13)
Reverse Primer: GCTCCTTTCTATTCTTTCTCCAAC (SEQ ID NO: 14)
Real-time PCR primers
Forward Primer: CCTCCTATGAATGTTTGTGT (SEQ ID NO: 15)
Reverse Primer: CTTTTGGTCAGACTACCTT (SEQ ID NO: 16)
(2) Duplex formation After obtaining the antithrombin gene and exon 4 region (polymerase uses KOD) of healthy person DNA and patient DNA using the above PCR primer for duplex formation, homoduplex (only for healthy person samples) , Heteroduplexes (normal and patient duplexes mixed 1: 1) were each formed.
Duplex formation conditions (use of thermal cycler): Same as Example 1.
(3) Enzyme treatment: Same as Example 1.
(4) Quantification by real-time PCR Real-time PCR was performed using the primer set for real-time PCR described in (1) above.
The following four samples are quantitative.
-Only wild-type duplex treated with ultrapure water.
-Enzymatic treatment of only wild-type duplex with heterozygous site-specific endonuclease (CEL1).
-Treated with ultrapure water in the presence of a 3: 1 homoduplex and heteroduplex from healthy (wild-type) specimens and patient specimens.
-Enzyme-treated with heterozygous specific endonuclease (SEL1) in the presence of 3: 1 homoduplex and heteroduplex from healthy (wild-type) specimen and patient specimen.
(5) Results As shown in FIG. 6, in the patient specimen, since it decreases to 76.41% close to the theoretical value (75%) when the fluctuation amount of the wild type control is corrected to 100, congenital antithrombin deficiency It was shown that mutations can be easily detected by mixing patient specimens and healthy subject specimens 1: 1 even for mutations that are unknown whether homozygous or heterozygous, such as type II.

The method of the present invention was performed using a single-strand DNA degrading enzyme (S1 nuclease) on a patient specimen with antithrombin deficiency and a heterozygous mutation (x mark) near the center of exon 1.
(1) Primer set Duplex formation PCR primer (Antithrombin gene, exon 1 amplification)
Forward Primer: TGGGCTCTACACTTTGCTT (SEQ ID NO: 17)
Reverse Primer: GAGGTCATTCCTGTGAGTC (SEQ ID NO: 18)
Real-time PCR primers
Forward Primer: TCATCAGCCTTTGACCTCA (SEQ ID NO: 19)
Reverse Primer: GAGGTCACAAAACCCAGTAG (SEQ ID NO: 20)
(2) Duplex formation After obtaining the antithrombin gene and exon 1 region (polymerase uses KOD) of healthy person DNA and patient DNA using the above PCR primer for duplex formation, homoduplex (only for healthy person samples) , Heteroduplexes (patient specimens only) were formed respectively.
Duplex formation conditions (use of thermal cycler): Same as Example 1.
(3) Enzyme treatment: S1 nuclease, 23 ° C., 15 minutes (4) Quantification by real-time PCR Real-time PCR was performed using the primer set for real-time PCR described in (1) above.
The following four samples are quantitative.
-Only wild-type duplex treated with ultrapure water.
-Enzymatic treatment of only wild-type duplex with single-stranded DNA-degrading enzyme (S1 nuclease).
-Treated with ultrapure water in the presence of 1: 1 homoduplex and heteroduplex by patient specimens.
-Enzyme-treated with a single-strand DNA degrading enzyme (S1 nuclease) in the presence of 1: 1 homoduplex and heteroduplex by patient specimen.
(5) Results As shown in FIG. 7, in the patient specimen, the variation amount of the wild-type control is reduced to 64.22% compared to the theoretical value (50%) when corrected as 100. It was shown that screening with a single-strand DNA-degrading enzyme can be performed sufficiently by delineating the reduction rate as a criterion.

If you want to identify genetic mutation sites in congenital diseases, if you screen for the presence or absence of mutations in each exon using the screening method of the present invention, you can sequence only the exons that have mutations, which greatly reduces the time, labor, and cost. Can be reduced.
In addition, the method of the present invention has the advantage that it is quicker and simpler than the preceding screening technique and can accurately detect exon ends that are difficult to detect mutations with the current method.
The kit using the method of the present invention can screen for gene mutations quickly and easily, helps to simplify the current genetic test, and can contribute widely to gene medicine and people.

Claims (3)

A screening method for gene mutation, which comprises the following procedure.
(1) Primers are designed outside the consensus region of each exon and intron of the target disease gene and amplified by PCR.
(2) In the case of homozygous gene mutations, all wild-type healthy individuals and patient gene PCR products containing mutant exons are mixed in equal amounts, or in the case of heterozygous gene mutations, the patient gene Each of the PCR products is used to form duplexes.
(3) The heterozygous site is cleaved.
(4) Primers are designed to be adjacent to the outside of the intron consensus region, and each duplex is amplified and quantified by real-time PCR.
(5) The presence or absence of a gene mutation is detected by observing a quantitative difference compared to a wild-type control. The method for screening a genetic mutation according to claim 1, wherein in step (1), a primer is designed 5 or more bases outside the consensus region of each exon and intron of the target disease gene. The gene mutation according to claim 1 or 2, wherein the cleavage of the heterozygous site is cleavage by treatment with a heterozygous site-specific endonuclease or a single-stranded DNA-degrading enzyme, or cleavage using a chemical substance Screening method.

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