# US 2016/0258010 A1 · Allele specific pcr assay for detection of nucleotide variants

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- Number: US 2016/0258010 A1
- Document ID: US20160258010A1
- Status: Published application
- Kind: A1
- Application number: 15/030,019
- Filed: 2014-10-20
- Published: 2016-09-08
- Earliest claimed: 2013-10-18
- Assignees: University of Pittsburgh—Of the Commonwealth System of Higher Education
- Applicants: University of Pittsburgh - Of the Commonwealth System of Higher Education
- Inventors: Constantinos Panousis; Elias Halvas; John Mellors
- Web page: https://patentgrep.com/patent/US20160258010A1

## CPC

- C12Q 1/6858: Allele-specific amplification
- C12Q 1/6851: Quantitative amplification
- C12Q 2525/186: incorporating a non-extendable or blocking moiety
- 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

Described herein are improved methods for detecting one or more alleles of a target nucleic acid molecule in a biological sample. The methods can be used, for example, for detecting low-frequency drug resistance mutations of a target nucleic acid molecule in a biological sample from a subject receiving the drug. In several embodiments, the subject is a subject with an HIV-1 infection, and the method is a method of detecting one or more drug-resistance mutations in an HIV-1 reverse transcriptase gene. Oligonucleotide primers for use in the disclosed methods, and compositions comprising same, are also provided.

## Claims

### Claim 1

A method of detecting a mutant first allele of a target nucleic acid molecule in a biological sample, comprising:
(A) amplifying the target nucleic acid molecule from the biological sample by real-time polymerase chain reaction (qPCR) using a test primer pair in a test amplification, comprising
a first plus primer comprising a locked nucleic acid at the 3′ end that is complementary to the mutant first allele of the target nucleic acid molecule, a mismatch nucleotide at the −1 position from the 3′ end that is not complementary to the target nucleic acid molecule, and remaining nucleotides that are complementary to the target nucleic acid molecule; and
a reverse primer of the test primer pair;
(B) amplifying the target nucleic acid molecule from the biological sample by qPCR using a control set of primers in a control amplification, comprising
the first primer pair; and
a first control primer comprising a locked nucleic acid at the 3′ end that is complementary to a wildtype first allele corresponding to the mutant first allele, and remaining nucleotides that are the same as the first plus primer;
(C) determining a threshold cycle (Ct) value of the test amplification and a Ct value of the control amplification; and
(D) comparing a difference between the Ct values of the test and control amplifications with a standard control generated using a mixture of target nucleic acid molecules comprising a pre-selected proportion of the mutant and wildtype first alleles to detect the mutant first allele of the target nucleic acid molecule in the biological sample.

### Claim 2 (depends on 1)

The method of claim 1, further comprising detecting a mutant second allele of the target nucleic acid molecule in the biological sample, wherein:
the reverse primer of the test primer pair is a second plus primer comprising a locked nucleic acid at the 3′ end that is complementary to the mutant second allele, a mismatch nucleotide at the −1 position from the 3′ end that is not complementary to the target nucleic acid molecule, and remaining nucleotides that are complementary to the target nucleic acid molecule;
the control set of primers further comprises a second control primer comprising a locked nucleic acid at the 3′ end that is complementary to a wildtype second allele corresponding to the mutant second allele, and remaining nucleotides that are the same as the second plus primer; and
the difference between the Ct values of the test and control amplifications are compared with a standard control generated using a mixture of target nucleic acid molecules comprising a pre-selected proportion of the mutant and wildtype first alleles and the mutant and wildtype second alleles to detect the mutant first and second alleles of the target nucleic acid molecule in the biological sample.

### Claim 3 (depends on 1)

The method of claim 1, wherein the control set of primers further comprises:
a third control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype first allele, and remaining nucleotides that are the same as the first plus primer; and
a fourth control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype first allele and is not the same as the locked nucleic acid of the third control primer, and remaining nucleotides that are the same as the first plus primer.

### Claim 4 (depends on 2)

The method of claim 2, wherein the control set of primers further comprises:
(a) a third control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype first allele, and remaining nucleotides that are the same as the first plus primer;
(b) a fourth control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype first allele and is not the same as the locked nucleic acid of the third control primer, and remaining nucleotides that are the same as the first plus primer;
(c) a fifth control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype second allele, and remaining nucleotides that are the same as the second plus primer;
(d) a sixth control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype second allele and is not the same as the locked nucleic acid of the fifth control primer, and remaining nucleotides that are the same as the second plus primer;
a combination of (a) and (b);
a combination of (c) and (d); or
a combination of (a), (b), (c), or (d).

### Claim 5 (depends on 1)

The method of claim 1, wherein detecting the mutant first allele of the target nucleic acid molecule in the biological sample comprises detecting a proportion of the target nucleic acid molecules in the sample comprising the mutant first allele in the biological sample, and wherein no more than 20% of the target nucleic acid molecules in the biological sample comprise the mutant first allele.

### Claim 6 (depends on 5)

The method of claim 5, wherein no more than 1% of the target nucleic acid molecules in the biological sample comprise the mutant first allele.

### Claim 7 (depends on 1)

The method of claim 1, comprising a first round amplification of template DNA from the biological sample prior to the test or control amplifications, wherein the first round amplification comprises use of a proof-reading DNA polymerase.

### Claim 8 (depends on 1)

The method of claim 1, wherein the mutant first allele, the mutant second allele, or both, is a drug resistant mutant allele of Human Immunodeficiency Virus (HIV)-1 or HIV-2.

### Claim 9 (depends on 8)

The method of claim 8, wherein the mutant first allele, the mutant second allele, or both, is a drug resistant mutant allele of HIV-1 reverse transcriptase.

### Claim 10 (depends on 9)

The method of claim 9, wherein the mutant first allele encodes one of the following mutations of HIV-1 reverse transcriptase: K65R, M184V, M184I, M41L, A62V, K65N, K65E, D67N, D67G, D67E, T69I, T69D, K70R, K70E, K70G, K70T, K70N, K70Q, L74V, L74I, V75I, V75M, V75T, F77L, L100I, K101P, K103N, K103S, V106M, Y115F, F116Y, Q151M, Y181C, Y181I, Y188L, G190S, G190A, L210W, T215Y, T215F, T215E, T215I, T215C, T215D, K219Q, K219E, K219N, K219R, P225H, or M230L.

### Claim 11 (depends on 9)

The method of claim 9, wherein the first plus primer, the first control primer, and the reverse primer, comprise or consist of the nucleic acid sequences set forth as one of:
(a) SEQ ID NOs: 1, 2, and 3, respectively, for detecting a K65R allele;
(b) SEQ ID NOs: 29, 41, and 3, respectively, for detecting a K65R allele;
(c) SEQ ID NOs: 40, 42, and 3, respectively, for detecting a K65R allele;
(d) SEQ ID NOs: 1, 2, and 37, respectively, for detecting a K65R allele;
(e) SEQ ID NOs: 29, 41, and 37, respectively, for detecting a K65R allele;
(f) SEQ ID NOs: 40, 42, and 37, respectively, for detecting a K65R allele;
(g) SEQ ID NOs: 1, 2, and 38, respectively, for detecting a K65R allele;
(h) SEQ ID NOs: 29, 41, and 38, respectively, for detecting a K65R allele;
(i) SEQ ID NOs: 40, 42, and 38, respectively, for detecting a K65R allele;
(j) SEQ ID NOs: 4, 5 and 3, respectively, for detecting a K70E allele;
(k) SEQ ID NOs: 4, 5, and 37, respectively, for detecting a K70E allele;
(l) SEQ ID NOs: 4, 5, and 38, respectively, for detecting a K70E allele;
(m) SEQ ID NOs: 6, 7, and 8, respectively, for detecting a M184V allele;
(n) SEQ ID NOs: 9, 10, and 10, respectively, for detecting a M184I allele;
(o) SEQ ID NOs: 11, 12, and 14, respectively, for detecting a K103N allele;
(p) SEQ ID NOs: 15, 12, and 14, respectively, for detecting a K103N allele;
(q) SEQ ID NOs: 16, 17, and 17, respectively, for detecting a Y181C allele; or
(r) SEQ ID NOs: 19, 20, and 20, respectively, for detecting a G190A allele.

### Claim 12 (depends on 9)

The method of claim 9, wherein the first and second alleles are selected from one of:
K65R and M184V, respectively;
K65R and M184I, respectively;
K65R and K103N, respectively;
K70E and M184V, respectively;
K70E and M184I, respectively;
K70E and K103N, respectively;
K103N and M184V, respectively; or
K103N and M184I, respectively.

### Claim 13 (depends on 2)

The method of claim 2, wherein
(a) the first and second mutant alleles are K65R and M184V mutant alleles of HIV-1 reverse transcriptase, respectively, and wherein the first plus primer, the first control primer, the second plus primer, and the second control primer, comprise or consist of the nucleic acid sequences set forth as SEQ ID NOs: 1, 2, 6, and 7, respectively; or
(b) the first and second mutant alleles are K65R and M184I mutant alleles of HIV-1 reverse transcriptase, respectively, and wherein the first plus primer, the first control primer, the second plus primer, and the second control primer, comprise or consist of the nucleic acid sequences set forth as SEQ ID NOs: 1, 2, 9, and 10, respectively.

### Claim 14 (depends on 1)

The method of claim 1, wherein the subject has an HIV-1 infection.

### Claim 15 (depends on 14)

The method of claim 14, wherein the subject is being treated with a combination of tenofovir and emtricitabine.

### Claim 16 (depends on 8)

The method of claim 8, further comprising identifying the subject as having a drug-resistant mutant allele of HIV-1 or HIV-2.

### Claim 17

An isolated nucleic acid molecule, comprising a plus primer comprising or consisting of the nucleic acid sequence set forth as any one of SEQ ID NOs: 1-2, 4-5, 6-7, 9-12, 15-17, 19-20, or 39-42.

### Claim 18

A composition, comprising:
(A) a primer pair comprising a forward and a reverse primer for amplifying a target nucleic acid molecule comprising a mutant first allele of a target nucleic acid; wherein
the forward primer is a first plus primer comprising a locked nucleic acid at the 3′ end that is complementary to the mutant first allele of the target nucleic acid molecule, a mismatch nucleotide at the −1 position from the 3′ end that is not complementary to the target nucleic acid molecule, and remaining nucleotides that are complementary to the target nucleic acid molecule; and
(B) a first control primer comprising a locked nucleic acid at the 3′ end that is complementary to a wildtype first allele corresponding to the mutant first allele, and remaining nucleotides that are the same as the first plus primer.

### Claim 19 (depends on 18)

The composition of claim 18, wherein the first plus primer, the first control primer, and the reverse primer, comprise or consist of the nucleic acid sequences set forth as:
(a) SEQ ID NOs: 1, 2, and 3, respectively;
(b) SEQ ID NOs: 29, 41, and 3, respectively;
(c) SEQ ID NOs: 40, 42, and 3, respectively;
(d) SEQ ID NOs: 1, 2, and 37, respectively;
(e) SEQ ID NOs: 29, 41, and 37, respectively;
(f) SEQ ID NOs: 40, 42, and 37, respectively;
(g) SEQ ID NOs: 1, 2, and 38, respectively;
(h) SEQ ID NOs: 29, 41, and 38, respectively;
(i) SEQ ID NOs: 40, 42, and 38, respectively;
(j) SEQ ID NOs: 4, 5 and 3, respectively;
(k) SEQ ID NOs: 4, 5, and 37, respectively;
(l) SEQ ID NOs: 4, 5, and 38, respectively;
(m) SEQ ID NOs: 6, 7, and 8, respectively;
(n) SEQ ID NOs: 9, 10, and 10, respectively;
(o) SEQ ID NOs: 11, 12, and 14, respectively;
(p) SEQ ID NOs: 15, 12, and 14, respectively;
(q) SEQ ID NOs: 16, 17, and 17, respectively; or
(r) SEQ ID NOs: 19, 20, and 20, respectively.

### Claim 20 (depends on 18)

The composition of claim 18, wherein the target nucleic acid molecule further comprises a second allele, and wherein
the reverse primer of the test primer pair is a second plus primer comprising a locked nucleic acid at the 3′ end that is complementary to the mutant second allele, a mismatch nucleotide at the −1 position from the 3′ end that is not complementary to the target nucleic acid molecule, and remaining nucleotides that are complementary to the target nucleic acid molecule; and
the composition further comprises a second control primer comprising a locked nucleic acid at the 3′ end that is complementary to a wildtype second allele corresponding to the mutant second allele, and remaining nucleotides that are the same as the second plus primer.

### Claim 21 (depends on 20)

The composition of claim 20, wherein the first plus primer, the first control primer, the second plus primer, and the second control primer, comprise or consist of the nucleic acid sequences set forth as
SEQ ID NOs: 1, 2, 6, and 7, respectively; or
SEQ ID NOs: 1, 2, 9, and 10, respectively.

### Claim 22 (depends on 18)

The composition of claim 18, further comprising:
a third control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype first allele, and remaining nucleotides that are the same as the first plus primer; and
the fourth control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype first allele and is not the same as the locked nucleic acid of the third control primer, and remaining nucleotides that are the same as the first plus primer.

### Claim 23 (depends on 22)

The composition of claim 22, further comprising
(a) a third control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype first allele, and remaining nucleotides that are the same as the first plus primer;
(b) a fourth control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype first allele and is not the same as the locked nucleic acid of the third control primer, and remaining nucleotides that are the same as the first plus primer;
(c) a fifth control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype second allele, and remaining nucleotides that are the same as the second plus primer;
(d) a sixth control primer, comprising a locked nucleic acid at the 3′ end that is not complementary to the mutant or wildtype second allele and is not the same as the locked nucleic acid of the fifth control primer, and remaining nucleotides that are the same as the second plus primer;
a combination of (a) and (b);
a combination of (c) and (d); or
a combination of (a), (b), (c), or (d).