Introduction of Real Time RT-PCR into the Global Polio Laboratory Network

A new state-of-the-art diagnostic method - real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) - has been introduced into the Global Polio Laboratory Network (GPLN). The introduction of rRT-PCR offers several key programmatic advantages to global poliovirus surveillance by:

1. permitting rapid and reliable identification of poliovirus isolates
2. reducing personnel hands-on time for poliovirus identification
3. providing data output in a computerized format
4. minimizing the risk of sample contamination that accompany conventional PCR
5. leading to development of new screening assays for vaccine-derived polioviruses (VDPVs)
6. enabling routine use of PCR reagents capable of detecting specific wild poliovirus genotypes
7. setting the stage for direct poliovirus detection by biochemical amplification methods
8. opening the way for widespread use of rRT-PCR in surveillance for other vaccine-preventable diseases.

The basic PCR setup consists of two short synthetic single-stranded DNA “primers” that bind to target sequences in an RNA (or DNA) “template”. PCR assays are run as a series of programmed thermal cycles (usually 2–3 minutes/cycle) that are repeated 25–40 times:

1. “denaturation” at high temperature (usually 95ºC) to disrupt the base-pairing of the double-stranded template and separate the strands
2. “annealing” at lower temperatures (in the range of 44ºC–50ºC) to permit the primers to base-pair with the templates
3. “primer extension” at elevated temperatures (in the range of 60ºC– 65ºC), whereby a complementary copy of the template is made by extension of the primer by a thermostable DNA polymerase.

The two primers target complementary strands of the double-stranded DNA template, and at each cycle the number of DNA PCR amplification products (“amplicons”) effectively doubles. The chain lengths of the amplicons are determined by the spacing of the primer binding sites along the templates. Specificities of primer binding are determined by the strength of the base pairs formed between the primers and the nucleic acid templates, and primers can be designed to target any genomic interval. Thus, by a rapid in vitro reaction mimicking natural DNA replication, large amounts of DNA copies can be amplified from minute quantities (as little as one molecule) of original template nucleic acid. When the template is RNA, an initial primer extension step is performed by reverse transcriptase (RT) which makes a DNA copy of the RNA sequence. The basic conventional RT-PCR assay described above has been widely used with high proficiency within the GPLN to identify poliovirus isolates. Primer pairs have been prepared with varying specificities: panEntero (identifies all enteroviruses); panPolio (identifies all polioviruses); SeroPV1, SeroPV2, and SeroPV3 (identifies polioviruses by serotype); and Sab1, Sab2, Sab3 (identifies Sabin strains). Specific amplicons from each primer pair set had different chain lengths, and could easily be separated by gel electrophoresis and visualized.

The original conventional RT-PCR assays to the “real-time” format, whereby the PCR reaction is continuously monitored by special thermal cycling instruments equipped with multichannel fluorescence detectors. In the RT-PCR format, a specific “probe” is included in the reaction, along with the primers. The probe is another short synthetic DNA chain that can form specific stable base pairs with the amplicon. The target sequences on the template for the probe are flanked by the target sequences for the “forward” and “reverse” primers. The probe contains a chemical “fluorophore” (fluoresces when excited by input light) at one (5’) end and a chemical “quencher” (absorbs [“quenches”] the fluorescent light from the fluorophore) at the opposite (3’) end. In rRT-PCR, extension from the primer of the DNA copy along the template causes displacement of the probe from the template and probe degradation. Probe degradation causes the fluorophore and quencher to separate (“uncouple”), so that the fluorescent light emitted by the fluorophore is no longer quenched. At each rRT-PCR cycle, the amount of fluorescent light emitted effectively doubles until the reaction reaches a plateau.

The rRT-PCR format has been field-tested in four GPLN laboratories (CDC-Atlanta, NIH-Islamabad, ERC-Mumbai, NICD Johannesburg) and is poised for wider application within the GPLN.