Compositions and methods for detection of herpes simplex virus 1 and 2

Patent metadata

Field	Value
Publication number	US-9982313-B2
Application number	US-201213588943-A
Country	US
Kind code	B2
Filing date	Aug 17, 2012
Priority date	Aug 17, 2012
Publication date	May 29, 2018
Grant date	May 29, 2018

Abstract

Methods for the rapid detection of the presence or absence of Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers, probes targeting the genes for HSV-1 viral DNA polymerase B (HSV-1 Pol) and HSV-1 thymidine kinase C (HSV-1 TK), and also target the genes for HSV-2 thymidine kinase C (HSV-2 TK) and HSV-2 glycoprotein B (HSV-2 gB), along with kits are provided that are designed for the detection of HSV-1 and/or HSV-2.

First claim

What is claimed:

1. A method of detecting and discriminating HSV-1 and HSV-2 in a sample in a single PCR reaction, the method comprising:
performing an amplifying step comprising contacting the sample with two sets of HSV-1 specific primers to produce amplification products from two HSV-1 gene targets, if any HSV-1 nucleic acid is present in the sample; and two sets of HSV-2 specific primers to produce amplification products from two HSV-2 gene targets, if any HSV-2 nucleic acid is present in the sample;
performing a hybridizing step comprising contacting said amplification products from said two HSV-1 gene targets and from said two HSV-2 gene targets with two detectable HSV-1 probes, one for each HSV-1 gene target, and two detectable HSV-2 probes, one for each HSV-2 gene target; and
detecting the presence or absence of said amplification products from said two HSV-1 gene targets and from said two HSV-2 gene targets, wherein the presence of said amplification products from said two HSV-1 gene targets and from said two HSV-2 gene targets is indicative of the presence of HSV-1 and/or HSV-2 in the sample and wherein the absence of said amplification products from said two HSV-1 gene targets and from said two HSV-2 gene targets is indicative of the absence of HSV-1 and/or HSV-2 in the sample;
wherein said amplifying, hybridizing and detecting steps are performed in a single PCR reaction wherein a DNA polymerase having 5′ to 3′ exonuclease activity cleaves a fluorogenic probe hybridized to the amplification products during the PCR reaction, resulting in increased fluorescence, and
wherein said two sets of HSV-1 specific primers and two sets of HSV-2 specific primers comprise:
HSV-1 specific primer sets that comprise nucleic acid sequences of SEQ ID NOs: 1 and 2 or a complement thereof, that specifically amplify an HSV-1 DNA polymerase B (HSV-1 Pol) gene and that does not amplify an HSV-2 DNA polymerase (HSV-2 Pol) gene and HSV-1 specific primer sets that comprise nucleic acid sequences of SEQ ID NOs: 4 and 5 or a complement thereof, that specifically amplify an HSV-1 thymidine kinase C (HSV-1 TK) gene and that does not amplify an HSV-2 thymidine kinase C (HSV-2 TK) gene;
HSV-2 specific primer sets that comprise nucleic acid sequences of SEQ ID NOs: 7 and 8 or a complement thereof, that specifically amplify the HSV-2 TK gene and that does not amplify the HSV-1 TK gene and HSV-2 specific primer sets that comprise nucleic acid sequences of SEQ ID NOs: 10 and 11 or a complement thereof, that specifically amplify an HSV-2 glycoprotein B (HSV-2 gB) gene and that does not amplify an HSV-1 glycoprotein B (HSV-1 gB) gene,
wherein no target competition is observed at up to 10,000-folds target concentration differences between HSV-1 and HSV-2; and

wherein said two detectable HSV-1 and two HSV-2 probes comprise:
a detectable HSV-1 probe comprising the nucleic acid sequence of SEQ ID NO:3 or a complement thereof that is specific for an HSV-1 Pol amplification product, and a detectable HSV-1 probe comprising the nucleic acid sequence of SEQ ID NO: 6 or a complement thereof that is specific for an HSV-1 TK amplification product; and
a detectable HSV-2 probes comprising the nucleic acid sequence of SEQ ID NO: 9 or a complement thereof that is specific for an HSV-2 TK amplification product, and a detectable HSV-2 probe comprising the nucleic acid sequence of SEQ ID NO: 12 or a complement thereof that is specific for an HSV-2 gB amplification product.

2. The method of claim 1 , wherein each of the two detectable HSV-1 probes and two detectable HSV-2 probes is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety; and
wherein the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety of the two HSV-1 probes and/or two HSV-2 probes, wherein the presence or absence of fluorescence is indicative of the presence or absence of HSV-1 and/or HSV-2 in the sample.

3. The method of claim 2 , wherein said donor and acceptor fluorescent moieties are within no more than 5 nucleotides of each other on said probe.

4. The method of claim 3 , wherein said acceptor fluorescent moiety is a quencher.

Assignees

• Wu Xiaoning
• Young Karen
• Roche Molecular Systems Inc

Inventors

• Wu Xiaoning
• Young Karen

Classifications

• C12Q2561/101

  Taqman · CPC title

• C12Q2565/1015

  labels being on the same oligonucleotide · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/705 Primary

  for herpetoviridae, e.g. herpes simplex, varicella zoster · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title