| 主权项 |
1. A method for predicting quantitative tropism, quantitative tropism being the quantitative shift in HIV-1 co-receptor usage from CCR5 to CXCR4, from CXCR4 to CCR5, or from dual trophic virus to either CCR5 or CXCR4, in a patient infected by HIV comprising:
a) using a sample comprising viral genetic material from the patient; b) extraction of viral genetic material from said sample followed by single genome sequencing comprising the following steps:
1. amplification of the viral genetic material of a specific HIV region;2. analysis of amplicon integrity and pooling of samples;3. purification of the pooled amplicons;4. ligation of the pool of amplicons into a vector and transformation of the ligated product into competent cells;5. analysis of individual transformants obtained; and6. sequencing the resulting single clones to obtain a single clone genotype sequence; c) prediction of the specific tropism using said genotype sequence with a predictive algorithm comprising the following steps:
1. identifying the genetic pattern in said genotype sequence wherein at least one natural variability, acquired variability, drug selected mutation or mutation pattern is associated with the quantitative tropism,2. searching a genotype/phenotype correlative database for at least one genotype entry with a similar genetic pattern to at least one of the natural variability, acquired variability, drug selected mutation or mutation pattern identified in the genetic sequence in step c)1,3. obtaining the said at least one genotype entry with a similar genetic pattern with a matched phenotype in the correlative genotype/phenotype database, and4. predicting the HIV tropism from the database of the at least one genotype entry with a similar genetic pattern; d) analysis of clonal sequences without predictable phenotype in a single clone biological phenotyping assay, wherein the information obtained after said analysis is loaded into the correlative genotype-phenotype database used in step c), wherein the single clone biological phenotyping assay comprises the following steps:
1. generation of clonal partial or full-length HIV genome by using vector pHXB2D-≢NH2-V4-eGFP having SEQ ID NO: 6;2. transfection of mammalian cells with said genome either together with a suitable backbone to obtain recombinant HIV or directly as a full length HIV-1 genome; and3. infection of cell lines by said recombinant HIV to determine their biological phenotype wherein the infection process is occurring; e) prediction of the quantitative tropism based on the information obtained in steps c)1 to c)4 for every single sequence clone present in a sample of a HIV infected patient. |
