Real-Time Reverse Transcription-Polymerase Chain Reaction (rRT-PCR)

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The page below is a sample from the LabCE course Zika Virus: Overview and Laboratory Testing. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

Learn more about Zika Virus: Overview and Laboratory Testing (online CE course)
Real-Time Reverse Transcription-Polymerase Chain Reaction (rRT-PCR)

PCR is a method developed by Kary Mullis in the 1980s. There are four main components to the PCR method:
  • DNA template - the sample DNA that contains the target sequence.
  • DNA polymerase - an enzyme that synthesizes new strands of DNA complementary to the target sequence.
  • Primers - short pieces of single-stranded DNA that are complementary to the target sequence.
  • Nucleotides (dNTPs or deoxynucleotide triphosphates) - single units of the four bases, which are the building blocks for new DNA strands.
The initial reaction begins with a high temperature being applied to the original double-stranded DNA molecule to separate the strands from each other (a process called denaturation). Then, the enzyme DNA polymerase is used to synthesize new stands of DNA which are complementary to the target sequence. A short piece of single-stranded DNA (the primer) that is complimentary to the target sequence is then used. The DNA polymerase is then used to add nucleotides (based on the primer) to delineate a specific region of the template sequence for amplification. At the end of the PCR reaction, the specific sequence will be synthesized (amplified) into billions of copies called amplicons. The upper image on the right is an overview of a typical PCR reaction.
Reverse transcription PCR (RT-PCR) is a PCR method that converts sample RNA into complimentary-DNA (cDNA), using the enzyme reverse transcriptase (refer to the lower image on the right, showing an overview of the RT-PCR method).
PCR and RT-PCR methods do have limitations. Inhibitors of PCR may be present in the sample and reagent limitations may occur, which can make the end-point quantification of the product somewhat difficult. To monitor the amplification of a targeted DNA during the PCR reaction and not at its end, rRT-PCR is being used. rRT-PCR amplifies a specific target sequence in the sample, then monitors the amplification progress using fluorescent technology. As the number of DNA amplification copies increases during the PCR reaction, there is also an increase in the fluorescence. This offers the distinct advantage of obtaining real-time monitoring of the PCR reaction and allows for quantitative analysis of the DNA expression.
For Zika virus identification, the rRT-PCR test is used on serum collected during the first two weeks after symptom onset. Also, rRT-PCR should also be conducted on urine samples collected less than 14 days after symptom onset. Urine should always be collected with a patient-matched serum specimen. A positive rRT-PCR result on any sample typically confirms Zika virus infection and usually, no additional testing is indicated. A negative rRT-PCR result does not exclude Zika virus infection, and serum should be analyzed by IgM antibody (serological) testing.