J Korean Soc Virol.  1997 Mar;27(1):19-27.

One-Stage Polymerase Chain Reaction for the Comprehensive Detection of Type D Retrovirus Provial DNA

• Jeong YS

Abstract

To develop the polymerase chain reaction (PCR) for the detection of type D simian retrovirus (SRV) infection, an oligonucleotide primer pair was designed to hybridize to the sequences within euv gene of SRV subtype 1 (SRV-1). The 3'proximal env sequences annealing to the primers had been rather conserved among three different subtypes of SRV, SRV-1, SRV-2, and SRV-3 (Mason-Pfizer Monkey Virus: MPMV). The PCR using the primer pair targetingan an env region the successfully detected and amplified all three subtypes of SRV with excellent specificity after single round of reaction. The tests with peripheral blood mononuclear cells infected either with simian immunodeficiency virus or simian T-lymphotropic virus type 1, maior immunosuppressive viral agents together with SRV in simian, verified the specificity of the PCR by excluding any cross reactivity. Semiquantitative titration PCR, amplifying serially diluted plasmid DNA of each subtype, was performed to evaluate sensitivity limits of the reaction. Based on molecular weight of each cloned SRV genome, the PCR should be able to detect one SRV-infected cell per more than 5-7x104 uninfected cells after simple ethidium bromide staining of resulting products. The PCR must be very efficient screening sisters with its quickness, certainty, and sensitivity for SRV-infected animals used in human AIDS research model. Second round amplification of the reaction products from the first PCR, or Southern hybridization by radiolabeled probes shall render to compete its efficacy to ELISA which has been the most sensitive technique to screen SRV infection but with frequent ambiguity problem.

MeSH Terms

Animals
Betaretrovirus*
Clone Cells
DNA*
Enzyme-Linked Immunosorbent Assay
Ethidium
Genome
Haplorhini
Humans
Mason-Pfizer monkey virus
Mass Screening
Molecular Weight
Plasmids
Polymerase Chain Reaction*
Retroviruses, Simian
Sensitivity and Specificity
Siblings
Simian Immunodeficiency Virus
DNA
Ethidium