Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network

Abstract

Surveillance and detection of polioviruses (PV) remain crucial to monitoring eradication progress. Intratypic differentiation (ITD) using the real-time RT-PCR kit is key to the surveillance workflow, where viruses are screened after cell culture isolation before a subset are verified by sequencing. The ITD kit is a series of real-time RT-PCR assays that screens cytopathic effect (CPE)-positive cell cultures using the standard WHO method for virus isolation. Because ITD screening is a critical procedure in the poliovirus identification workflow, validation of performance of real-time PCR platforms is a core requirement for the detection of poliovirus using the ITD kit. In addition, the continual update and improvement of the ITD assays to simplify interpretation in all platforms is necessary to ensure that all real-time machines are capable of detecting positive real-time signals. Four platforms (ABI7500 real-time systems, Bio-Rad CFX96, Stratagene MX3000P, and the Qiagen Rotor-Gene Q) were validated with the ITD kit and a redesigned poliovirus probe. The poliovirus probe in the real-time RT-PCR pan-poliovirus (PanPV) assay was re-designed with a double-quencher (Zen™) to reduce background fluorescence and potential false negatives. The updated PanPV probe was evaluated with a panel consisting of 184 polioviruses and non-polio enteroviruses. To further validate the updated PanPV probe, the new assay was pilot tested in five Global Polio Laboratory Network (GPLN) laboratories (Madagascar, India, Philippines, Pakistan, and Democratic Republic of Congo). The updated PanPV probe performance was shown to reduce background fluorescence and decrease the number of false positives compared to the standard PanPV probe.

Fig 1. ITD 5.0 Assay results for stratagene (Mx3005P), ABI7500, bio-rad (CFX96) and Rotor-Gene Q (Qiagen).

Assay performance against a standard virus panel (N = 184). (A) PanEV assay, 171 of 184 isolates; (B) Pan PV assay, 158 of 184 isolates; (C) Sabin 1 assay 28 of 184 isolates; (D) Sabin 2 assay 44 of 184 isolates; (E) Sabin 3 assay 43 of 184 isolates; (F) WPV1 assay 44 of 184 isolates; (G) WPV3-I assay 30 of 184 isolates; (H) WPV3-II assay 36 of 184 isolates; and (I) the PV Type 2 assay 50 of 184 isolates. The PanEV assay targets the 5’NTR, all other assays target the VP1 capsid region. The WPV3-I and WPV3-II assays target the WPV3 WEAF-B and SOAS variants, respectively.

Fig 2. Comparison of the background fluorescence on the Rotor-Gene Q.

Three updated PanPV probes with Zen^™ quencher were tested along with the current PanPV probe to ascertain baseline background levels against synthetic control RNA. Fluorescence at cycle six was selected as representative of background levels for each assay.

Fig 3. Comparison of the background fluorescence on the Rotor-Gene Q between PanPV and Zen8PV.

Background fluorescence levels of the PanPV and updated Zen8PV assays were measured against a virus panel of 32 polioviruses wild type 1. Fluorescence at cycle six was selected as representative of background levels for each assay and sample.

Fig 4. Comparative raw fluorescence data between PanPV and updated PanPV from six poliovirus isolates.

Percentage of raw background fluorescence values from standard PanPV assay, purple broken lines (n = 4) and purple solid lines (n = 2) that overlap at the maximum detectable signal (where samples increased 100%). Green lines are the fluorescence measured from the updated PanPV probes tested with the same isolates in the Rotor-Gene Q.

Fig 5. Pilot testing of poliovirus isolates with PanPV and updated PanPV probes.

A total of 293 poliovirus isolates were run with both PanPV and Zen8PV probes. Ct values are shown for wild poliovirus type 1 (purple); Sabin 1 (red); Sabin 3 (green); poliovirus type 2 (blue). The cross depicts the mean Ct value with standard deviation of all positive isolates.