Abstract
BackgroundGlobal polio eradication efforts rely in part on molecular methods of detecting polioviruses, both wild and vaccine strains, from human and environmental samples. Previous assays used for detection of Sabin oral polio vaccine (OPV) in fecal samples have been labor and time intensive and vary in their sensitivity and specificity.MethodsWe developed a high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay able to detect all 3 OPV strains in fecal samples. The assay used a KingFisher Duo Prime system for viral RNA isolation and extraction. Positive samples were retested and Sanger sequenced for verification of Sabin serotype identity.ResultsThe 95% lower limit of detection was determined to be 3 copies per reaction for Sabin 1 and 3 and 4 copies per reaction for Sabin 2, with no cross-reactivity between the 3 serotypes and their primers. A total of 554 samples (3.6%) were positive, with 304 positive samples (54.9%) containing >1 serotype. Of the positive samples, 476 (85.9%) contained enough RNA to be sequenced, and of these all sequences were Sabin serotypes. The previous assay we used could process 48 samples in a 10-hour period, whereas the new assay processed >100 samples in 6 hours.ConclusionsThe new high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay allowed for sensitive and specific detection of OPV serotypes while greatly decreasing sample handling and processing time. We were able to sequence 72.4% of the 210 positive samples in the cycle threshold range of 35–37.
Highlights
MethodsWe developed a high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay able to detect all 3 oral polio vaccine (OPV) strains in fecal samples
Global polio eradication efforts rely in part on molecular methods of detecting polioviruses, both wild and vaccine strains, from human and environmental samples
The 95% lower limit of detection was determined to be 3 copies per reaction for Sabin 1 and 3 and 4 copies per reaction for Sabin 2, with no cross-reactivity between the 3 serotypes and their primers
Summary
We developed a high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay able to detect all 3 OPV strains in fecal samples. The assay used a KingFisher Duo Prime system for viral RNA isolation and extraction. Positive samples were retested and Sanger sequenced for verification of Sabin serotype identity. Each village received a different amount of OPV coverage as part of the study: 70% of households in Capoluca, 30% in Campo Grande, and 10% in Tuxpanguillo. In each village, recruited families were randomized to receive of OPV based on these percentages, resulting in 155 vaccinated households across the 3 localities. Health information, travel and visit details, and records for vaccinations received by any children
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
