Helper oligonucleotide for improved efficiency of amplification and detection/quantitation of nucleic acids

• US 10,443,095 B2
• Filed: 07/26/2017
• Issued: 10/15/2019
• Est. Priority Date: 08/02/2016
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Mehta, Rochak
• Primary Examiner(s): Woolwine, Samuel C
• Application Number:
  US15/660,686
• Publication Number:
  US 20180037945A1
• Time in Patent Office: 811 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/707 non-A, non-B Hepatitis, excluding hepatitis D
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/162 Helper probe

Abstract

Improved methods for the detection and quantitation of a target nucleic acid in a sample using a non-extending helper oligonucleotide are described. The methods include contacting nucleic acids in a sample with amplification reagents including one or more primers, one or more non-extending helper oligonucleotides, and one or more probes. The non-extending helper oligonucleotide facilitates and increases the target nucleic acid accessibility of one or more of the primers, result in greater accumulation of amplicon production, thereby increasing the efficiency and sensitivity of the amplification assay, including amplification assays for Hepatitis C Virus (HCV), for example, HCV Genotype 5. Kits, articles of manufacture, and reaction mixtures are also provided.

Claims

1. A method for detecting and/or quantitating a target nucleic acid of Hepatitis C Virus (HCV) Genotype 5 in a sample comprising:

(a) contacting nucleic acids in the sample with amplification reagents, the amplification reagents comprising;

at least an enzyme comprising DNA polymerase activity;

at least nucleoside triphosphate monomers or other nucleoside monomers;

at least one forward primer specific for the target nucleic acid, wherein the at least one forward primer comprises the sequence of SEQ ID NO;

1, and at least one reverse primer specific for the target nucleic acid, wherein the at least one reverse primer comprises the sequence selected from a group consisting of SEQ ID NOs;

2 and 3, for generating at least one amplicon;

at least one non-extending helper oligonucleotide, wherein;

(i) the non-extending helper oligonucleotide does not extend, (ii) the non-extending helper oligonucleotide anneals to part of the same portion of the target nucleic acid as at least one of the primers, and (iii) the non-extending helper oligonucleotide enhances the activity of at least one of the primers; and

at least one detectable probe specific for the amplicon or at least one DNA binding dye, wherein the at least one detectable probe comprises the sequence of SEQ ID NO;

4;

(b) incubating the nucleic acids with the amplification reagents for a period of time and under conditions sufficient for an amplification reaction to occur; and

(c) detecting the amplicon with the at least one detectable probe or the at least one DNA binding dye.

2. The method of claim 1, wherein the at least one non-extending helper oligonucleotide comprises a poly(A) sequence at the 3′

-end of the at least one non-extending helper oligonucleotide.

3. The method of claim 2, wherein the poly(A) sequence is between 4-12 nucleotides in length.

4. The method of claim 3, wherein the at least one non-extending helper oligonucleotide comprises the sequence of SEQ ID NO:

5.

5. A kit for amplifying and detecting and/or quantitating a target nucleic acid of HCV Genotype 5 in a sample comprising amplification reagents, the amplification reagents comprising:

(a) an enzyme comprising DNA polymerase activity;

(b) nucleoside triphosphate monomers or other nucleoside monomers;

(c) at least one forward primer and at least one reverse primer specific for the target nucleic acid;

(d) at least one non-extending helper oligonucleotide comprising the sequence of SEQ ID NO;

5, wherein;

(i) the non-extending helper oligonucleotide does not extend, (ii) the non-extending helper oligonucleotide anneals to part of the same portion of the target nucleic acid as at least one of the primers, and (iii) the non-extending helper oligonucleotide enhances the activity of at least one of the primers; and

(e) at least one detectable probe for the target nucleic acid or a DNA binding dye wherein the at least one forward primer comprises the sequence of SEQ ID NO;

1;

the at least one reverse primer comprises the sequence selected from a group consisting of SEQ ID NOs;

2 and 3; and

the at least one probe comprises the sequence of SEQ ID NO;

4.

6. A reaction mixture effective to amplify and detect and/or quantitate a target nucleic acid in a sample comprising amplification reagents comprising:

(a) a sample;

(b) an enzyme comprising DNA polymerase activity;

(c) nucleoside triphosphate monomers or other nucleoside monomers;

(d) at least one forward primer and at least one reverse primer specific for the target nucleic acid;

(e) at least one non-extending helper oligonucleotide comprising the sequence of SEQ ID NO;

5, wherein;

(i) the non-extending helper oligonucleotide does not extend, (ii) the non-extending helper oligonucleotide anneals to part of the same portion of the target nucleic acid as at least one of the primers, and (iii) the non-extending helper oligonucleotide enhances the activity of at least one of the primers; and

(f) at least one detectable probe for the target nucleic acid or a DNA binding dye wherein the at least one forward primer comprises the sequence of SEQ ID NO;

1;

the at least one reverse primer comprises the sequence selected from a group consisting of SEQ ID NOs;

2 and 3; and

the at least one probe comprises the sequence of SEQ ID NO;

4.