HEV Assay

• US 20130280697A1
• Filed: 04/17/2013
• Published: 10/24/2013
• Est. Priority Date: 04/18/2012
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Leying, Hermann, Myers, Thomas W., Newton, Nicolas, Russmann, Eberhard, San Filippo, Joseph, Schoenbrunner, Nancy, Wilts, Heike, Young, Karen K. Y., Zimmermann, Dirk, Wu, Xiaoning
• Granted Patent: US 9,702,017 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/707 non-A, non-B Hepatitis, excluding hepatitis D
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

A method of simultaneously amplifying genotypes 1, 2, 3 and/or 4 of HEV is disclosed comprising amplifying the genotypes 1, 2, 3 and/or 4 of HEV with one single none-degenerate forward primer partially overlapping the 5′UTR region of HEV and at least one reverse primer. Also disclosed are related methods comprising a probe, and kits for the detection of genotypes 1, 2, 3 and/or 4 of HEV.

Claims

1. A method for simultaneously amplifying genotypes 1, 2, 3 and/or 4 of HEV if present in a biological sample, comprising the steps of:

(a) isolating nucleic acids present in the biological sample;

(b) amplifying the nucleic acids isolated in step (a) using one non-degenerate forward primer and at least one non-degenerate reverse primer, wherein the forward and reverse primers are capable of simultaneously amplifying genotypes 1, 2, 3, and/or 4 of HEV.

2. The method of claim 1, wherein the forward primer comprises a sequence selected from SEQ ID NOS:

1-6 and the at least one reverse primer comprises a sequence selected from SEQ ID NOS;

7-14.

3. The method of claim 1, wherein the forward primer comprises SEQ ID NO:

6 and the at least one reverse primer comprises a sequence selected from SEQ ID NOS;

7-14.

4. The method of claim 1, further comprising two non-generate reverse primers, wherein the forward primer is SEQ ID NO:

6, and the reverse primers are SEQ ID NO;

13 and SEQ ID NO;

14.

5. The method of claim 1, wherein the nucleic acids are isolated by binding to a solid phase.

6. The method of claim 1, further comprising the step of:

(c) detecting amplified nucleic acid obtained in step (b) as an indication of the presence of at least one of genotypes 1, 2, 3 and/or 4 of HEV in the biological sample.

7. The method of claim 6, further comprising a probe under conditions sufficient for binding the probe to the amplified nucleic acids.

8. The method of claim 7, wherein the probe comprises at least 20 contiguous nucleotides of SEQ ID NOS:

15-19 or 25, or a complementary sequence thereof.

9. The method of claim 7, wherein the probe comprises at least 22 to 35 contiguous nucleotides of SEQ ID NOS:

15-19 or 25, or a complementary sequence thereof.

10. The method of claim 7, wherein the probe consists of a sequence selected from SEQ ID NOS:

15-19 or 25, or a complementary sequence thereof.

11. The method of claim 7, wherein the probe consists of a sequence selected from SEQ ID NO:

15-18 or 25.

12. The method of claim 7, wherein the probe comprises a fluorophore and a quencher.

13. The method of claim 12, wherein the fluorophore is coupled to the 5′

end of the probe, and the spacing between the fluorophore and the quencher comprises at least 9 nucleotides.

14. A kit for amplifying genotypes 1, 2, 3 and/or 4 of HEV if present in a biological sample, comprising:

(a) a template depended DNA polymerase, nucleotides, and

(b) one non-degenerate forward primer and at least one non-degenerate reverse primer, wherein the forward and reverse primers are capable of simultaneously amplifying genotypes 1, 2, 3, and/or 4 of HEV.

15. The kit of claim 14, wherein the forward primer comprises a sequence selected from SEQ ID NOS:

1-6 and the at least one reverse primer comprises a sequence selected from SEQ ID NOS;

7-14.

16. The kit of claim 14, wherein the forward primer is SEQ ID NO:

6 and the at least one reverse primer comprises a sequence selected from SEQ ID NOS;

7-14.

17. The kit of claim 15, further comprising a probe, wherein the probe comprises at least 22 to 35 contiguous nucleotides of SEQ ID NOS:

15-19 or 25, or a complementary sequence thereof.