Detection of Rubella Virus by Tri-Primer RT-PCR Assay and Genotyping by Fragment RT-PCR.

Polymerase chain reaction (PCR) is a widely used technique in the diagnosis of viral infections due to its low cost, high sensitivity, and specificity. Although the more advanced variations of PCR, such as real-time PCR and digital PCR are now available to researchers, conventional PCR is still used in many research studies. Here we describe the protocol for tri-primer diagnostic reverse transcription polymerase chain reaction for detection of rubella in throat swabs and further detailed protocol for a two fragment genotyping using two different sets of primers. In tri-primer diagnostic PCR, one forward and two reverse primers are used to detect clade I and clade II of the rubella virus. In the two fragments genotyping, each fragment of the genome is amplified, sequenced separately, and then the overlapping regions are aligned and full length sequence window is obtained.