CCR5 RNA transcription based amplification assay
AssigneeAkzo Nobel N.V.
InventorsJoseph W. Romano, Eun Mi Lee
Life of this patent
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Abstract
A method and a test kit for genotyping the CCR5 chemokine receptor. CCR5 transcript DNA is amplified using transcription based amplification. The amplification product is detected through the hybridization of appropriately labeled oligonucleotide wild type and mutant probes. In a liquid hybridization based ECL analysis, the mutant probe is a 16mer having the sequence 5′TCCATACATTAAAGAT3′ [SEQ ID NO:9]. The method and kit can be used to predict.
Claims18
A method of genotyping the CCR5 chemokine receptor to determine whether an individual is homozygous mutant, heterozygous or homozygous wild type, comprising:
- (a) providing CCR5 transcript RNA of an individual to be genotyped, wherein said CCR5 transcript RNA is obtained from whole blood or peripheral blood mononuclear cells of the individual;
- (b) amplifying said CCR5 transcript RNA using an isothermal transcription based amplification method;
- and
- (c) determining whether an amplification product of step (b) contains a wild type CCR5 allele, a mutant CCR5 allele or both wild type and mutant CCR5 alleles by independently hybridizing amplification product with labeled wild type and mutant specific probes, and detecting the resulting hybridization products.
An oligonucleotide useful as a mutant probe in a liquid hybridization based ECL (electrochemiluminescence) assay of the nucleic acid sequence based amplification product of CCR5 transcript RNA, said oligonucleotide having the sequence:
- 5′TCCATACATTAAAGAT3′[SEQ ID NO:9].
A method for predicting the susceptibility of an individual to infection by NSI (nonsyncytia-inducing) forms of HIV-1 (human immunodeficiency virus-1) and for predicting disease progression of an individual infected with NSI forms of HIV-1, comprising:
- (a) obtaining CCR5 transcript RNA of said individual, wherein said CCR5 transcript RNA is obtained from whole blood or peripheral blood mononuclear cells of the individual;
- (b) amplifying said CCR5 transcript RNA by using a transcription based amplification using a pair of oligonucleotides that target sequences of CCR5 gene spanning positions 450 through 650;
- (c) determining whether an amplification product of step (a) contains a wild type CCR5 allele, a mutant CCR5 allele or both wild type and mutant CCR5 alleles by independently hybridizing amplification product with labelled wild type and mutant specific probes and detecting the resulting hybridization products, and
- (d) predicting susceptibility of said individual to infection by NSI forms of HIV-1 and predicting disease progression depending on whether said individual is homozygous wild type, heterozygous or homozygous mutant for the CCR5 alleles.
A test kit for the diagnosis of infectability of individuals to NSI forms of HIV-1 comprising:
- (a) a pair of oligonucleotides for amplifying CCR5 RNA transcript by a transcription based amplification method,
- (b) an oligonucleotide DNA probe for detecting a wild type CCR5 allele, and
- (c) an oligonucleotide DNA probe for detecting a mutant CR5 allele.
Dependencies are inferred from the claim text.