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Particle Agglutination Method for Poliovirus Identification
Published on: April 20, 2011
Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3
Deepa K Sharma1, Uma P Nalavade, Jagadish M Deshpande
1Enterovirus Research Centre (ICMR), Mumbai, India.
Background & Objectives:
The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use.
Methods:
Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre.
Results:
No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus.
Interpretation & Conclusions:
rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.
Insights
New real-time reverse transcription-polymerase chain reaction (rRT-PCR) assays accurately detect wild poliovirus 1 and 3 (WPV1 and WPV3) in various mixtures. These validated assays are crucial for confirming wild poliovirus presence, even with vaccine strains.
Area of Science:
- Virology
- Molecular Biology
- Public Health
Background:
- Current real-time reverse transcription-polymerase chain reaction (rRT-PCR) assays are effective for poliovirus serotype identification but struggle with intratypic differentiation, particularly distinguishing wild and vaccine strains.
- Accurate differentiation is critical for polio eradication efforts, necessitating improved diagnostic tools.
Purpose of the Study:
- To develop specific real-time reverse transcription-polymerase chain reaction (rRT-PCR) assays for wild poliovirus 1 (WPV1) and wild poliovirus 3 (WPV3).
- To validate these assays for detecting genetic variations of WPV1 and WPV3 circulating in India.
Main Methods:
- Design of specific primers and probes for rRT-PCR based on VP1 sequences of WPV1 and WPV3 strains isolated in India since 2000.
- Evaluation of assay specificity using diverse poliovirus strains (WPV1, WPV3, Sabin strains) and non-polio enteroviruses (NPEVs).
- Determination of assay sensitivity through serial dilutions of WPV1 and WPV3 stock suspensions.
Main Results:
- The developed rRT-PCR assays demonstrated high specificity, showing no cross-reactivity with Sabin strains, intertypic wild polioviruses, or 27 types of NPEVs.
- All WPV1 and WPV3 strains isolated in India since 2000 were successfully amplified by the specific assays.
- The assays detected WPV1 and WPV3 at low concentrations (10^4.40 and 10^4.00 CCID50/ml, respectively), even in mixtures with Sabin strains or other wild polioviruses.
Conclusions:
- Validated rRT-PCR assays for WPV1 and WPV3 can effectively detect all genetic variations of these wild polioviruses circulating in India over the past decade.
- These specific assays, when used with existing methods, provide complete confirmation of wild poliovirus presence in intratypic mixtures, aiding polio surveillance.
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