All issues > Volume 35(10); 1992

Original Article

J Korean Pediatr Soc. 1992;35(10):1402-1410. Published online October 15, 1992.

A Basic Study for Respiratory Syncytial Virus Detection Using Polymerase Chain Reaction

Yong Kweon YK Kim¹, Jong Duck JD Kim¹

¹Department of Pediatrics, Won Kwang University, College of Medicine, IRi, Korea

Abstract

Basic experimental approach for rapid and acurate detection of respiratory syncitial virus (RSV) infection at gene level has been performed using polymerase chain reaction (PCR). RSV RNA was extracted by RNAzolB methol from RSV which was cultured in Hep-2 cell line. Of RSV genome having about 8,000 base pairs, base pairs between 3237th and 3522nd were selected to make four kinds of primer: 5'-TCACGAAGGCTCCACATACA-3', 5'-TCCAAGGACACATTAGCGCA-3', 5'-TGCCCATGTTCCAATCATC-3' AND 5'-TAGTGAAGGTCCCTTGCGGT-3' Because RSV RNA is unstable, RSVcDNA for RSV RNA were made by reverse transcriptase and designed down stream primer in the presence of RNA inhibitor. By combination of two kinds of different primers, four kinds of different primer pairs were prepared and selected portion of RSV cDNA was amplified by PCR. Amplification carried out 50cycle for about 40minutes using Thermal Air Cycler. The RSV cDNA was detected by 1.5% agarose gel electrophoresis containing ethidium bromide to confirm DNA. The bands on position of 226, 186, 184, and 104 base pairs were obtained as expected. The nested amplification and negative control were used to rule out the false positive occured by contamination of other RNA and/or DNA.

Keywords :Respiratory syncytial virus, Polymerase chain reaction