Methods for detecting retroviruses
First Claim
1. A method for detecting in a single assay whether a sample contains an HIV retrovirus and/or an HTLV retrovirus, the method comprising:
- a) preparing cDNA by reverse transcribing nucleic acids in the sample;
b) ligating a first oligonucleotide linker to the cDNA to form an anchored-end cDNA;
c) amplifying the anchored-end cDNA to form amplified cDNA using primers that specifically anneal to the first oligonucleotide linker;
d) treating the amplified cDNA with a restriction enzyme to generate cDNA fragments;
e) ligating a second oligonucleotide linker to the cDNA fragments to form anchored-end cDNA fragments;
f) sequencing the anchored-end cDNA fragments using sequencing primers that specifically anneal to the second oligonucleotide linker; and
g) comparing the sequences with the known nucleotide sequences of HIV and/or HTLV retroviruses to determine if the sample contained an HIV retrovirus and/or an HTLV retrovirus.
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Accused Products
Abstract
Disclosed is a method for detecting human retroviral nucleic acids such as human immunodeficiency virus type I (HIV-1) nucleic acid, human T-cell leukemia virus type I (HTLV-I) nucleic acid, and human T-cell leukemia virus type II (HTLV-II) nucleic acid in a sample. In the method, the sample is treated with reverse transcriptase to generate cDNA, and the cDNA is subsequently analyzed to detect HIV-1, HTLV-I, and HTLV-II. The method may include performing PCR and the method may utilize specific primers. In addition, the method may utilize HTLV linkers that facilitate PCR amplification and sequencing. The cDNA may be treated with restriction enzymes before or after PCR amplification to facilitate sequencing and detection of HIV-1, HTLV-I, or HTLV-II nucleic acid.
14 Citations
15 Claims
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1. A method for detecting in a single assay whether a sample contains an HIV retrovirus and/or an HTLV retrovirus, the method comprising:
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a) preparing cDNA by reverse transcribing nucleic acids in the sample; b) ligating a first oligonucleotide linker to the cDNA to form an anchored-end cDNA; c) amplifying the anchored-end cDNA to form amplified cDNA using primers that specifically anneal to the first oligonucleotide linker; d) treating the amplified cDNA with a restriction enzyme to generate cDNA fragments; e) ligating a second oligonucleotide linker to the cDNA fragments to form anchored-end cDNA fragments; f) sequencing the anchored-end cDNA fragments using sequencing primers that specifically anneal to the second oligonucleotide linker; and g) comparing the sequences with the known nucleotide sequences of HIV and/or HTLV retroviruses to determine if the sample contained an HIV retrovirus and/or an HTLV retrovirus. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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Specification