The poliovirus detection assay was developed specifically for this study and was a multiplex 2-step reverse transcription, quantitative polymerase chain reaction assay (RT-qPCR). Ribonucleic acid was extracted from frozen stool samples using the MagMAX Viral RNA Isolation Kit on the Kingfisher Duo Prime instrument (both from Thermo Scientific Inc.), and reverse transcription utilized the SuperScript III Reverse Transcriptase (Thermo Fisher Scientific Inc.). Real-time polymerase chain reaction was performed in a total volume of 20 microliters, consisting of 5 microliters of complementary deoxyribonucleic acid (cDNA) and 15uL of iQ Multiplex Powermix with Sabin 1, 2, and 3 specific primers and probes.
Amplification and detection was performed on the CFX384 Real-Time System (BioRad). Samples were run in triplicate, and a sample was considered positive if 2 out of 3 reactions crossed the cycle threshold (CT) in less than 37 cycles. To account from sample handling variability during real-time polymerase chain reaction testing, Sabin 1, 2 and 3 positive controls, a negative control, and No Template Control (NTC) were then run together on 384 well plates with the unknown samples above.
Finally, genetic sequencing using the Sanger technique was performed on these OPV-positive samples to rule out the possibility of false positives due to other enteroviruses [4 (link)].
Amplification and detection was performed on the CFX384 Real-Time System (BioRad). Samples were run in triplicate, and a sample was considered positive if 2 out of 3 reactions crossed the cycle threshold (CT) in less than 37 cycles. To account from sample handling variability during real-time polymerase chain reaction testing, Sabin 1, 2 and 3 positive controls, a negative control, and No Template Control (NTC) were then run together on 384 well plates with the unknown samples above.
Finally, genetic sequencing using the Sanger technique was performed on these OPV-positive samples to rule out the possibility of false positives due to other enteroviruses [4 (link)].
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