DESCRIPTION OF THE INVENTIONThis invention concerns a laboratory method for genotyping human immunodeficiency virus type 1 (HIV-1) through gene sequencing of pol. It allows to detect the nucleotide mutations conferring resistance to antiretroviral drugs active to pol.The present method allows to analyze also low viraemia samples at around 101 -102 copies HIV-RNA/ml.This method offers the advantage of being easily adaptable to both plasma and cellular samples and is therefore able to value "in advance" the genetic mutations potentially involved in the resistance phenomena to anti-retroviral drugs, even in patients with optimal/sub-optimal virologic control.The kit use the one-step RT-PCR technology, that offers very short processing times (within 5 hours).Finally, the use of one-step method for reverse transcription and the subsequent amplification of cDNA in dsDNA, limits the intervention and reduces the risk of sample’s contamination.The international guidelines show that a proper use of the resistance genotypic tests could improve the clinical outcomes of HIV-infected patients; more recently, panel of experts in the field have re-evaluated the recommendations on the use of such tests, suggesting a wider application in all clinical phases of the infection.Moreover, it is evident the necessity to have the use of analytic methods that are applicable to all viral variants actually known, with high sensitivity.The object of this invention is a method for genotyping HIV-1 which includes the following phases:a)HIV-1 RNA reverse transcription from biological samples using a specific anti-sense primer and subsequent PCR amplification of a dsDNA fragment, using a pair of external target-specific primers. The reverse transcription and amplification occur in a single step;b)sequencing of the product obtained in the phase a) (using "Big Dye terminator Cycle Sequencing" technology) in independent reactions using a set of primers, whose nucleotide sequences encompass a region located between HIV-1 env gene and 3'-LTR, which includes the whole gp41-env domain. In particular, the proposed method amplifies a genetic fragment included between the nucleotide positions 7300 and 9500 (referring to the HIV-1 subtype B sequence, HXB2 - GenBank Accession number K03455).c)assembling of each segment obtained during phase b) in a consensus sequence.Furthermore, according to a form of implementation of the method of the invention, when there is no evident PCR amplification in phase a) by RT-PCR, it is possible to improve the sensitivity with further step of amplification through a nested-PCR, using a pair of internal target-specific primers.Finally, the method can be applied indiscriminately:to plasma samples, for the detection of viral RNA circulating variants (expression of viral replication);to cellular samples, for the detection of viral cDNA variants integrated at lymphocyte level, even in subjects with effective virologic control (viral load <47 copies HIV-RNA/mL).
|Publication status||Published - 2007|