Clinical infectious diseases : an official publication of the Infectious Diseases Society of America · 2018
Lab Protocol Paper: Use of a High-throughput, Multiplex Reverse-transcription Quantitative Polymerase Chain Reaction Assay for Detection of Sabin Oral Polio Vaccine in Fecal Samples.
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van Hoorebeke Christopher, Huang ChunHong, Leary Sean, Holubar Marisa, Altamirano Jonathan, Halpern Meira S, Sommer Marvin, Maldonado Yvonne
Аннотация
BACKGROUND: Global 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.
METHODS: 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.
RESULTS: 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. 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.
CONCLUSIONS: The 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.
METHODS: 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.
RESULTS: 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. 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.
CONCLUSIONS: The 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.