translation agency

Development and optimisation of in situ polymerase chain reaction detection of human immunodeficiency virus (HIV) RNA.
Ng SM; Vasak E; James A; Cooper DA; Delaney SF; McQueen PW; Dept. of
December 30, 1996
Annu Conf Australas Soc HIV Med. 1995 Nov 16-19;7:108 (abstract no.

The in situ hybridisation (ISH) method used in microscopy employs the reaction of specific probes with complementary sequences of nucleic acid at the individual cell level. In order to intensify the reaction product, amplification of low copy number nucleic acids by polymerase chain reaction (PCR) has been attempted recently by a small number of laboratories world-wide, combining these two techniques into in situ PCR. To apply this methodology for Human Immunodeficiency Virus-1 (HIV-1) viral-specific nucleic sequences in HIV-1 positive cells and tissue sections, we have developed and optimised the technique using oligonucleotide primers V3A and V3D and a digoxigenin-labelled V3B probe, sequences from the conserved region of the gp120 protein. The HIV-1 RNA was reverse transcribed to produce a cDNA copy and the in situ PCR reactions were performed using a slide thermal cycler. Peripheral blood mononuclear cells (PBMC) infected with HIV-1 (strain SF33) were used. We have optimised the technique for reagents concentrations of Taq polymerase, dNTPS, Mg++, and primers. We have also refined the substrate development conditions to increase the intensity of the reaction product by including a multilayered hapten-antibody protocol. The in situ PCR technique is suitable for tissue and cells attached to microscope slides. The reaction conditions have to be optimised for each nucleic acid sequence examined.

DNA Polymerases DNA Primers Evaluation Studies Human HIV Infections/VIROLOGY HIV-1/*GENETICS/ISOLATION & PURIF In Situ Hybridization Indicators and Reagents Leukocytes, Mononuclear/VIROLOGY Polymerase Chain Reaction/*METHODS RNA, Viral/BLOOD/*GENETICS ABSTRACT