IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Molecular tools for the early detection and the epidemiological characterization of HIV-1 infections based on the strains circulating in Argentina
Autor/es:
PEREZ, G. R.; GARDIOL, DANIELA; TABORDA, MIGUEL A.; GIRI ADRIANA A
Lugar:
Viena
Reunión:
Conferencia; XVIII International AIDS Conference; 2010
Institución organizadora:
International AIDS Society
Resumen:
In the Americas,
different patterns of HIV-1 subtypes and recombinants distribution have been
found. In Argentina,
approximately 80% isolates are BF recombinants, almost 20% are B subtype and
scattered cases of A, C and F subtypes. This scenario represents a risk factor
for spreading unidentified infections due to differences in the
specificity/sensitivity of commercially available tests, and highlights the
need for developing diagnostic tools tailored on the strains circulating in
each region with affordable technology.
We developed
the CICHIV-1/HIV-1 RT-PCR assay, a method that includes a
competitive internal control (CICHIV-1) and combines RT-PCR, liquid
hybridization and EIA. First, we performed a genomic analysis using 1,745 HIV-1
subtypes and inter-subtypes listed at NCBI. A gag-gene fragment
presenting the lesser mismatches among strains circulating in Argentina and South America
was selected to design generic primers and probes. Primers specificity and
sensitivity were demonstrated with plasmids containing B or F subtype genomes.
Once primers functionality was achieved, the CICHIV-1 was
constructed. Briefly, sequences of the A1 region from Qβ
coliphage were replaced by the selected HIV-1 primers, using a pBR322
derivative in which the Qβ phage genome cDNA was cloned
downstream the T7 RNA polymerase promoter as template. The plasmid bearing the
modified Qβ genome was used to transform E. coli.
After induction, recombinant Qβ particles (CICHIV-1)
were obtained. CICHIV-1 is RNase resistant, stable at 4°C, economical to produce and
useful to verify the efficiency of the whole procedure, including RNA
extraction.
The CICHIV-1/HIV-1
RT-PCR assay fulfills the requirements for diagnostic tests for HIV-1 early
detection and/or molecular blood donor screening, that includes a CICHIV-1
consisting in a recombinant RNA packaged into capsids which hybridizes with the
same primers. This approach can be adapted for qualitative/quantitative detection
of other subtypes and inter-subtypes circulating in many developing regions,
contributing to a more effective HIV-1 epidemiological control.
Funding:
FIC-NIH(D43TW001037).