Co-amplification of multiple regions of the HIV-1 genome by the polymerase chain reaction: potential use in multiple diagnosis.
We have used the polymerase chain reaction (PCR) to detect by co-amplification, multiple regions of the HIV-1 genome in infected cells. Genomic RNA and DNA from productively infected H9 cells were independently extracted and amplified in reactions with and without reverse transcriptase respectively using primer pairs to the gag, env, tat and nef regions of the viral genome in the same reaction mixture. PCR-products were analysed by liquid hybridization with end labelled oligonucleotide probes followed by gel-electrophoresis (oligomer hybridization). The primer pairs were capable of detecting as few as 10 copies of RNA and 10-20 copies of integrated proviral DNA. The ability to co-amplify multiple target regions in the same incubation mixture provides a method for detecting and confirming the presence of HIV-1 in samples for which limited nucleic acid is available. In addition, in reconstitution experiments, the same method was used to detect HIV-1 and HTLV-I simultaneously with comparable sensitivity (20-40 gene copies each). This offers the possibility of simultaneous diagnosis of multiple viral infections, such as those that occur in AIDS, on the same sample preparation.
Hewlett
I K
IK
Division of Blood and Blood Products, Food and Drug Administration, Bethesda, Maryland 20892.
Ruta
M
M
Cristiano
K
K
Hawthorne
C A
CA
Epstein
J S
JS
D004279:DNA, Viral / Q000032:analysis*; D006800:Deltaretrovirus Infections / Q000175:diagnosis; D005784:Gene Amplification; D006678:HIV / Q000235:genetics*; D015368:Human T-lymphotropic virus 1 / Q000235:genetics; D006801:Humans; D012367:RNA, Viral / Q000032:analysis*
D004279:DNA, Viral; D012367:RNA, Viral