# US 10,370,730 B2 · Detection method for mutation in 93rd amino acid of hepatitis C virus NS5A protein, and detection kit for mutation in 93rd amino acid of hepatitis C virus NS54 protein

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,370,730 B2
- Document ID: US10370730B2
- Status: Granted
- Kind: B2
- Application number: 15/103,184
- Filed: 2014-12-11
- Published: 2018-06-14
- Granted: 2019-08-06
- Earliest claimed: 2013-12-11
- Assignees: Saitama Medical University
- Applicants: Saitama Medical University
- Inventors: Satoshi Mochida; Yoshihito Uchida; Jun-ichi Kouyama; Kayoko Naiki
- Web page: https://patentgrep.com/patent/US10370730B2

## CPC

- C12Q 1/707: {non-A, non-B Hepatitis, excluding hepatitis D}
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein, the method including: synthesizing cDNA using, as a template, hepatitis C virus RNA in a sample; and performing a real-time PCR with a cycling probe method using, as a template, the cDNA; wherein a primer set used in the real-time PCR is a certain primer set; and wherein probes used in the real-time PCR include certain probes.

## Claims

### Claim 1

A method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein, the method comprising:
synthesizing cDNA using, as a template, hepatitis C virus RNA in a sample; and
performing a real-time PCR with a cycling probe method using, as a template, the cDNA;
wherein a primer set used in the real-time PCR is a primer set which is designed to be able to amplify a region comprising the base sequence coding for the amino acid at position 93 of the hepatitis C virus NS5A protein, and wherein probes used in the real-time PCR comprise:
a probe consisting of the base sequence of SEQ ID NO: 1 or the complement thereof; and
at least one probe selected from
a probe consisting of the base sequence of SEQ ID NO: 2 or the complement thereof, and
a probe consisting of the base sequence of SEQ ID NO: 18 or the complement thereof:
(SEQ ID NO: 1)
aacgcat[a]ca
(SEQ ID NO: 2)
aacgcac[a]ca
(SEQ ID NO: 18)
acgcac[a]ca
wherein each of the SEQ ID NOs: 1, 2, and 18, a base described in brackets denotes RNA.

### Claim 2 (depends on 1)

The method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 1, wherein the probes used in the real-time PCR further comprise:
a probe consisting of the base sequence of SEQ ID NO: 9 or the complement thereof;
and at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 10 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 22 or the complement thereof:
(SEQ ID NO: 9)
aacgcgt[a]ca
(SEQ ID NO: 10)
aacgcgc[a]ca
(SEQ ID NO: 22)
acgcgc[a]ca
wherein each of the SEQ ID NOs: 9, 10, and 22, a base described in brackets denotes RNA.

### Claim 3 (depends on 2)

The method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 2, wherein the probes used in the real-time PCR further comprise:
a probe consisting of the base sequence of SEQ ID NO: 3 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 4 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 19 or the complement thereof:
(SEQ ID NO: 3)
aacgcat[a]ta
(SEQ ID NO: 4)
aacgcac[a]ta
(SEQ ID NO: 19)
acgcac[a]ta
wherein each of the SEQ ID NOs: 3, 4, and 19, a base described in brackets denotes RNA.

### Claim 4 (depends on 2)

The method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 2, wherein the probes used in the real-time PCR further comprise at least one of the following (1) and (2):
(1) a combination consisting of: a probe consisting of the base sequence of SEQ ID NO: 5 or the complement thereof; and
at least one of a probe consisting of the base sequence of SEQ ID NO: 6 or the complement thereof, and
a probe consisting of the base sequence of SEQ ID NO: 20 or the complement thereof;
(2) a combination consisting of: a probe consisting of the base sequence of SEQ ID NO: 7 or the complement thereof; and
at least one of a probe consisting of the base sequence of SEQ ID NO: 8 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 21 or the complement thereof:
(SEQ ID NO: 5)
aatgcat[a]ca
(SEQ ID NO: 6)
aatgcac[a]ca
(SEQ ID NO: 20)
atgcac[a]ca
(SEQ ID NO: 7)
aatgcat[a]ta
(SEQ ID NO: 8)
aatgcac[a]ta
(SEQ ID NO: 21)
atgcac[a]ta
wherein each of the SEQ ID NOs: 5, 6, 20, 7, 8, and 21, a base described in brackets denotes RNA.

### Claim 5 (depends on 2)

The method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 2, wherein the primer set used in the real-time PCR is the following (1):
(1) a primer set consisting of a primer consisting of the base sequence of SEQ ID NO: 11 and a primer consisting of the base sequence of SEQ ID NO: 12:
(SEQ ID NO: 11)
ggttccatgaggatcgttg
(SEQ ID NO: 12)
ccgtcacgtagtggaaatc.

### Claim 6 (depends on 1)

The method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 1, wherein the probes used in the real-time PCR further comprise:
a probe consisting of the base sequence of SEQ ID NO: 3 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 4 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 19 or the complement thereof:
(SEQ ID NO: 3)
aacgcat[a]ta
(SEQ ID NO: 4)
aacgcac[a]ta
(SEQ ID NO: 19)
acgcac[a]ta
wherein each of the SEQ ID NOs: 3, 4, and 19, a base described in brackets denotes RNA.

### Claim 7 (depends on 6)

The method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 6, wherein the probes used in the real-time PCR further comprise at least one of the following (1) and (2):
(1) a combination consisting of: a probe consisting of the base sequence of SEQ ID NO: 5 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 6 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 20 or the complement thereof;
(2) a combination consisting of: a probe consisting of the base sequence of SEQ ID NO: 7 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 8 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 21 or the complement thereof:
(SEQ ID NO: 5)
aatgcat[a]ca
(SEQ ID NO: 6)
aatgcac[a]ca
(SEQ ID NO: 20)
atgcac[a]ca
(SEQ ID NO: 7)
aatgcat[a]ta
(SEQ ID NO: 8)
aatgcac[a]ta
(SEQ ID NO: 21)
atgcac[a]ta
wherein each of the SEQ ID NOs: 5, 6, 20, 7, 8, and 21, a base described in brackets denotes RNA.

### Claim 8 (depends on 6)

The method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 6, wherein the primer set used in the real-time PCR is the following (1):
(1) a primer set consisting of a primer consisting of the base sequence of SEQ ID NO: 11 and a primer consisting of the base sequence of SEQ ID NO: 12:
(SEQ ID NO: 11)
ggttccatgaggatcgttg
(SEQ ID NO: 12)
ccgtcacgtagtggaaatc.

### Claim 9 (depends on 1)

The method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 1, wherein the probes used in the real-time PCR further comprise at least one of the following (1) and (2):
(1) a combination consisting of: a probe consisting of the base sequence of SEQ ID NO: 5 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 6 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 20 or the complement thereof;
(2) a combination consisting of:
a probe consisting of the base sequence of SEQ ID NO: 7 or the complement thereof;
and at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 8 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 21 or the complement thereof:
(SEQ ID NO: 5)
aatgcat[a]ca
(SEQ ID NO: 6)
aatgcac[a]ca
(SEQ ID NO: 20)
atgcac[a]ca
(SEQ ID NO: 7)
aatgcat[a]ta
(SEQ ID NO: 8)
aatgcac[a]ta
(SEQ ID NO: 21)
atgcac[a]ta
wherein each of the SEQ ID NOs: 5, 6, 20, 7, 8, and 21, a base described in brackets denotes RNA.

### Claim 10 (depends on 1)

The method for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 1, wherein the primer set used in the real-time PCR is the following (1):
(1) a primer set consisting of a primer consisting of the base sequence of SEQ ID NO: 11 and a primer consisting of the base sequence of SEQ ID NO: 12:
(SEQ ID NO: 11)
ggttccatgaggatcgttg
(SEQ ID NO: 12)
ccgtcacgtagtggaaatc.

### Claim 11

A kit for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein, the kit comprising:
a probe consisting of the base sequence of SEQ ID NO: 1 or the complement thereof;
and at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 2 or the complement thereof, and
a probe consisting of the base sequence of SEQ ID NO: 18 or the complement thereof:
(SEQ ID NO: 1)
aacgcat[a]ca
(SEQ ID NO: 2)
aacgcac[a]ca
(SEQ ID NO: 18)
acgcac[a]ca
wherein each of the SEQ ID NOs: 1, 2, and 18, a base described in brackets denotes RNA and the other bases denote DNA.

### Claim 12 (depends on 11)

The kit for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 11, wherein the kit further comprises:
a probe consisting of the base sequence of SEQ ID NO: 9 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 10 or the complement thereof, and
a probe consisting of the base sequence of SEQ ID NO: 22 or the complement thereof:
(SEQ ID NO: 9)
aacgcgt[a]ca
(SEQ ID NO: 10)
aacgcgc[a]ca
(SEQ ID NO: 22)
acgcgc[a]ca
wherein each of the SEQ ID NOs: 9, 10, and 22, a base described in brackets denotes RNA and the other bases denote DNA.

### Claim 13 (depends on 12)

The kit for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 12, wherein the kit further comprises:
a probe consisting of the base sequence of SEQ ID NO: 3 or the complement thereof; and at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 4 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 19 or the complement thereof:
(SEQ ID NO: 3)
aacgcat[a]ta
(SEQ ID NO: 4)
aacgcac[a]ta
(SEQ ID NO: 19)
acgcac[a]ta
wherein each of the SEQ ID NOs: 3, 4, and 19, a base described in brackets denotes RNA and the other bases denote DNA.

### Claim 14 (depends on 12)

The kit for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 12, wherein the kit further comprises at least one of the following (1) and (2):
(1) a combination consisting of: a probe consisting of the base sequence of SEQ ID NO: 5 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 6 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 20 or the complement thereof;
(2) a combination consisting of: a probe consisting of the base sequence of SEQ ID NO: 7 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 8 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 21 or the complement thereof:
(SEQ ID NO: 5)
aatgcat[a]ca
(SEQ ID NO: 6)
aatgcac[a]ca
(SEQ ID NO: 20)
atgcac[a]ca
(SEQ ID NO: 7)
aatgcat[a]ta
(SEQ ID NO: 8)
aatgcac[a]ta
(SEQ ID NO: 21)
atgcac[a]ta
wherein each of the SEQ ID NOs: 5, 6, 20, 7, 8, and 21, a base described in brackets denotes RNA and the other bases denote DNA.

### Claim 15 (depends on 12)

The kit for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 12, wherein the kit further comprises the following (1):
(1) a primer set consisting of a primer consisting of the base sequence of SEQ ID NO: 11 and a primer consisting of the base sequence of SEQ ID NO: 12:
(SEQ ID NO: 11)
ggttccatgaggatcgttg
(SEQ ID NO: I2)
ccgtcacgtagtggaaatc.

### Claim 16 (depends on 11)

The kit for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 11, wherein the kit further comprises:
a probe consisting of the base sequence of SEQ ID NO: 3 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 4 or the complement thereof, and
a probe consisting of the base sequence of SEQ ID NO: 19 or the complement thereof:
(SEQ ID NO: 3)
aacgcat[a]ta
(SEQ ID NO: 4)
aacgcac[a]ta
(SEQ ID NO: 19)
acgcac[a]ta
wherein each of the SEQ ID NOs: 3, 4, and 19, a base described in brackets denotes RNA and the other bases denote DNA.

### Claim 17 (depends on 16)

The kit for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 16, wherein the kit further comprises at least one of the following (1) and (2):
(1) a combination consisting of:
a probe consisting of the base sequence of SEQ ID NO: 5 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 6 or the complement thereof, and
a probe consisting of the base sequence of SEQ ID NO: 20 or the complement thereof;
(2) a combination consisting of: a probe consisting of the base sequence of SEQ ID NO: 7 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 8 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 21 or the complement thereof:
(SEQ ID NO: 5)
aatgcat[a]ca
(SEQ ID NO: 6)
aatgcac[a]ca
(SEQ ID NO: 20)
atgcac[a]ca
(SEQ ID NO: 7)
aatgcat[a]ta
(SEQ ID NO: 8)
aatgcac[a]ta
(SEQ ID NO: 2I)
atgcac[a]ta
wherein each of the SEQ ID NOs: 5, 6, 20, 7, 8, and 21, a base described in brackets denotes RNA and the other bases denote DNA.

### Claim 18 (depends on 16)

The kit for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 16, wherein the kit further comprises the following (1):
(1) a primer set consisting of a primer consisting of the base sequence of SEQ ID NO: 11 and a primer consisting of the base sequence of SEQ ID NO: 12:
(SEQ ID NO: 11)
ggttccatgaggatcgttg
(SEQ ID NO: 12)
ccgtcacgtagtggaaatc.

### Claim 19 (depends on 11)

The kit for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 11, wherein the kit further comprises at least one of the following (1) and (2):
(1) a combination consisting of: a probe consisting of the base sequence of SEQ ID NO: 5 or the complement thereof; and at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 6 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 20 or the complement thereof;
(2) a combination consisting of:
a probe consisting of the base sequence of SEQ ID NO: 7 or the complement thereof; and
at least one probe selected from a probe consisting of the base sequence of SEQ ID NO: 8 or the complement thereof, and a probe consisting of the base sequence of SEQ ID NO: 21 or the complement thereof:
(SEQ ID NO: 5)
aatgcat[a]ca
(SEQ ID NO: 6)
aatgcac[a]ca
(SEQ ID NO: 20)
atgcac[a]ca
(SEQ ID NO: 7)
aatgcat[a]ta
(SEQ ID NO: 8)
aatgcac[a]ta
(SEQ ID NO: 21)
atgcac[a]ta
wherein each of the SEQ ID NOs: 5, 6, 20, 7, 8, and 21, a base described in brackets denotes RNA and the other bases denote DNA.

### Claim 20 (depends on 11)

The kit for detecting a mutation in an amino acid at position 93 of a hepatitis C virus NS5A protein according to claim 11, wherein the kit further comprises the following (1):
(1) a primer set consisting of a primer consisting of the base sequence of SEQ ID NO: 11 and a primer consisting of the base sequence of SEQ ID NO: 12:
(SEQ ID NO: 11)
ggttccatgaggatcgttg
(SEQ ID NO: 12)
ccgtcacgtagtggaaatc.