Detection of human herpesviruses
The present invention provides methods, compositions, and kits related to nucleic acid detection assays for detecting human herpes virus. For example, the present invention provides detection assays for detecting human herpes virus subtypes HHV1- through HHV-8.
1 . A method of detecting the presence or absence of at least one human herpes virus subtype in a sample comprising;
a) contacting said sample with first and second oligonucleotides, wherein said first and second oligonucleotides are configured to form an invasive cleavage structure with a target sequence, wherein said target sequence comprises human herpes virus sequence, and
b) detecting the presence or absence of said at least one human herpes virus subtype in said sample.
2 . The method of claim 1 , wherein said detecting comprises observing a signal generated by cleavage of said first oligonucleotide, thereby identifying the presence of said at least one human herpes virus subtype in said sample.
3 . The method of claim 1 , wherein said at least one human herpes virus subtype is selected from the group consisting of: HHV-1, HHV-2, HHV-3, HHV-4, HHV-5, HHV-6, HHV-7, and HHV-8.
4 . The method of claim 1 , wherein said first oligonucleotide comprises a 5′ portion and a 3′ portion, wherein said 3′ portion is configured to hybridize to said target sequence, and wherein said 5′ portion is configured to not hybridize to said target sequence.
5 . The method of claim 1 , wherein said second oligonucleotide comprises a 5′ portion and a 3′ portion, wherein said 5′ portion is configured to hybridize to said target sequence, and wherein said 3′ portion is configured to not hybridize to said target sequence.
6 . The method of claim 1 , wherein said second oligonucleotide also serves as a first PCR primer configured to amplify said target sequence with a second PCR primer.
7 . The method of claim 6 , wherein said second oligonucleotide, which also serves as said first PCR primer, is selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:85.
8 . The method of claim 6 , further comprising contacting said sample with said second PCR primer.
9 . The method of claim 6 , wherein said second PCR primer is selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:65, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:84.
10 . The method of claim 1 , wherein said first oligonucleotide is selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:87.
11 . The method of claim 1 , further comprising contacting said sample with a FRET cassette.
12 . The method of claim 1 , wherein said target sequence comprises a PCR amplified product.
13 . The method of claim 1 , wherein said target sequence comprises genomic herpes virus nucleic acid.
14 . The method of claim 1 , wherein said first or second oligonucleotide comprises one or more mismatches with said target sequence.
15 . The method of claim 1 , wherein said first or second oligonucleotide does not hybridize to human genomic DNA under stringent conditions.
16 . The method of claim 1 , wherein said first and second oligonucleotides are configured such that a stable duplex between said first and second oligonucleotides and said target sequence is not formed.
17 . A kit for detecting the presence or absence of at least one human herpes virus subtype in a sample comprising;
a) first and second oligonucleotides configured to form an invasive cleavage structure with a target sequence, wherein said target sequence comprises human herpes virus sequences, and
b) a cleavage agent, wherein said cleavage agent is capable of cleaving said first oligonucleotide when said cleavage structure is formed.
18 . The kit of claim 17 , wherein said first oligonucleotide comprises a 5′ portion and a 3′ portion, wherein said 3′ portion is configured to hybridize to said target sequence, and wherein said 5′ portion is configured to not hybridize to said target sequence.
19 . The kit of claim 17 , wherein said second oligonucleotide comprises a 5′ portion and a 3′ portion, wherein said 5′ portion is configured to hybridize to said target sequence, and wherein said 3′ portion is configured to not hybridize to said target sequence.
20 . The kit of claim 17 , wherein said second oligonucleotide also serves as a first PCR primer configured to PCR amplify said target sequence with a second PCR primer.