GENOTYPE-SPECIFIC RNA PROBES FOR DIRECT IDENTIFICATION OF WILD POLIOVIRUSES BY BLOT HYBRIDIZATION

We have developed RNA probes for the direct identification of wild pol iovirus isolates by blot hybridization. The probes are complementary t o sequences of the first 30 to 32 codons of VP1, which evolve more ext ensively (similar to 1.5-fold) than the rest of VP1. To illustrate our general approach, we describe the design of probes specific to each o f four major genotypes recently endemic (1981 to 1991) to the Americas : Andean type 1, Brazil type 1, Brazil type 3, and Central America-Mex ico type 3, A wild isolate of each genotype was selected according to molecular and epidemiologic criteria to be representative of the princ ipal lineages in circulation, Variable VPI sequences of the representa tive isolates were amplified by the reverse transcriptase PCR and were inserted into a plasmid vector containing a T7 promoter. The in vitro transcripts, labeled with digoxigenin, served as probes. These formed stable hybrids only with RNAs of isolates of the corresponding genoty pes, Hybrids were detected by a sensitive chemiluminescence assay, cap able under normal diagnostic conditions of detecting specific wild pol iovirus sequences in samples containing up to a 100-fold excess of Sab in vaccine strain-related sequences of the same serotype.