Nested-PCR to Detect a Specific Viral Genomic Sequence

Retrieve the necessary reagents and keep them on ice in a clean room until ready to use. When ready, thaw and vortex the reagents. Next, prepare 48 microliters of first-round PCR mix for each sample in a 1.5-milliliter centrifuge tube, as outlined in Table 2 of the text protocol, and divide the reaction mix into 0.2-milliliter PCR tubes.

In a PCR workstation in a template room, add a 2-microliter sample of the cDNA into the first-round PCR mix. Include two microliters of CVS-11 cDNA as a positive control and two microliters of double-distilled water as a negative control. Then, transfer the sealed tubes into a PCR thermal cycler and cycle using the parameters listed in Table 3 of the text protocol.

To begin, prepare 48 microliters of second-round PCR mix for each sample in a 1.5-milliliter centrifuge tube, as outlined in Table 4 of the text protocol, and divide the reaction mix into 0.2-milliliter PCR tubes. Add two microliters of the first-round PCR product into the second-round PCR mix. Include double-distilled water as a negative control for this round of PCR. Then, perform PCR thermal cycling using the parameters listed in Table 3 of the text protocol.