2.4. Internal Control

In the triplex assay, a single-copy calreticulin gene, conserved in I. scapularis, was used as an internal control. The probe spans an exon–exon junction of calreticulin gene to avoid tick genomic DNA detection because the gene coding region has only one intron with a conserved position [33]. The internal control was tested with tick DNA, RNA and total nucleic acid, confirming its specificity for I. scapularis tick RNA, not DNA.