The 18S rRNA and COWP gene sequences of the 11 patient isolates listed in the table have been submitted to GenBank and assigned accession numbers AY030084 to AY030093 and AF411631 to AF411633.
Twenty-five sporadic isolates were also characterized by using a second locus on the COWP gene.
As well, the PCR/RFLP profile of the COWP gene from the cervine genotype isolate was identical to that obtained from human genotype 1 isolates.
To confirm that the observed variation in the ITS 1 RLFP patterns was not due to heterogeneous products amplified from different copies of rDNA, further characterization of a select number of sporadic isolates was performed with the 18S rRNA as well as the COWP genes.
PCR of two 18S rDNA fragments (reaction 1 [8,17] and reaction 2 ), COWP (5), TRAP-C1 (6), and TRAP-C2 (4) gene fragments, as well as restriction digestion with RsaI for the COWP and TRAP-C1 genes, was performed.
For the nested-COWP procedure (N-COWP), a 769-bp fragment of the COWP gene was amplified with primers BCOWPF (5'-ACCGCT TCTCAACAACCATCTTGTCCTC-3') and BCOWPR (5'-CGCACCTGTTCCCACTCAATGTA AACCC-3'), which encompasses the 553-bp fragment (5).
For the N-COWP, E-COWP, COWP, 18S rDNA 1, 18S rDNA 2, TRAP-C1, and TRAP-C2 gene fragments, 5-[micro]l aliquots of the PCR products were analyzed by electrophoresis in 1% agarose-ethidium bromide gels.
Analysis of the published genotype 2 COWP gene sequence (GenBank accession numbers Z22537) led to design of two primers (BCOWPF and BCOWPR) to amplify a predicted E-COWP 769-bp fragment, which includes the 553-bp fragment amplified by the previously described Cry15/Cry9 primers (5).