TABLE 1.
Primers used to create control constructs and those used for MAPREC and real-time PCR assays
| Purpose and name of primer or probe | Sequence (5′ to 3′)a | Location in genome |
|---|---|---|
| Creation of control constructs for MAPREC | ||
| S3+432/472C | GAGCTACATGAGAGTCCTCCGGCCCCTGAATGCGGCGAATC | 432-472 |
| S3+432/472T | GAGCTACATGAGAGTCCTCCGGCCCCTGAATGCGGCGAATT | 432-472 |
| S3-721 | TCAAACAATTTCAATAG | 721-704 |
| Creation of control constructs for real-time assays | ||
| S3+454/471 | CCCCTGAATGCGGCGAAT | 454-472 |
| S3-509 | CTGGCTGCTGGGTTGCA | 509-493 |
| Reverse transcription-PCR step in MAPREC | ||
| S3+432 | GAGCTACATGAGAGTCCTCCGGCCCCTGAATGCGGCGAAT | 432-471 |
| S3-721 | TCAAACAATTTCAATAAG | 721-704 |
| Real-time assay for revertantsb | ||
| S3+454/472C | CCCCTGAATGCGGCGAAGC | 454-472 |
| S3-509 | CTGGCTGCTGGGTTGCA | 509-493 |
| Probe 473/490 | 6FAM TAA CCA TGG AGC AGG CA MGB | 473-490 |
| Real-time assay for nonrevertantsc | ||
| S3+454/472T | CCCCTGAATGCGGCGAACT | 454-472 |
| S3-509 | CTGGCTGCTGGGTTGCA | 509-493 |
| Probe 473/490 | 6FAM TAA CCA TGG AGC AGG CA MGB | 473-490 |
a
Underlining shows nucleotide 468, which was mismatched in all forward primers to allow an EcoRI digest when the nonrevertant was present. This mismatch was included in the real-time assays. Italics show mismatches of nucleotides 471 and 472 in the forward primers for the real-time assays for nonrevertants and revertants to increase specificity in distinguishing nonrevertant from revertant OPV3 strains. 6FAM, 6-carboxyfluorescein; MGB, minor groove binder.
b
Detection and quantification of revertants in clinical samples.
c
Detection and quantification of nonrevertants in clinical samples.