Methods for detection and typing of nucleic acids

摘要

Disclosed are methods and kits for identifying and characterizing polynucleotide sequences in a sample which may include a heterogeneous sample. Some of the methods and kits are directed to the identification and characterization of a virus in a sample, which may include HIV capable of cause AIDS or AIDS-like symptoms. The virus may be HIV-1, and may also include drug resistant mutations. The methods may include reacting a mixture that includes, in addition to nucleic acid isolated from the sample, at least one oligonucleotide capable of specifically hybridizing to HIV nucleic acid where the oligonucleotide includes at least one non-natural base. In addition, the methods may include detection of one or more mutations in HIV nucleic acid that are associated with drug resistance.

主权项

1. A method for detecting the presence or absence of a mutation at a specific nucleotide position in a polynucleotide from a sample, comprising amplifying the polynucleotide with primers to detect the presence or absence of the mutation in the polynucleotide, the primers comprising:
(a) a first primer that is complementary to the polynucleotide at the specific nucleotide position of the mutation if the mutation is present in the polynucleotide; (b) a second primer that is complementary to the polynucleotide at the specific nucleotide position of the mutation if the mutation is absent in the polynucleotide; wherein one of the first primer and the second primer includes a non-natural base selected from K, X, H, J, M, N, iso-G and iso-C at one or more positions other than the specific nucleotide position of the mutation to create one or more non-adjacent mismatches across from a target base in a target specific sequence of the first primer and the second primer, and the other of the first primer and the second primer includes a non-natural base selected from K, X, H, J, M, N, iso-G and iso-C at two or more positions other than the specific nucleotide position of the mutation to create two or more non-adjacent mismatches across from a target base in a target specific sequence of the first primer and the second primer, and at least one of the first primer and the second primer includes a label.