TABLE 1.
Nucleotide sequence polymorphisms in the primer and probe regions used for three FRET probe-based real-time PCR assays among common Cryptosporidium species and genotypesa
| Assay | Species and/or genotype | Primer region, sequence | Probe region, sequence |
|---|---|---|---|
| 18S-LC1 | − | F4, 5′-GGAAGGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG-[fluorescein] |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, [Red 640]-5′-GTCACATTAATTGTGATCCGTAAAG-phosphate | ||
| C. hominis/C. cuniculus | F4, 5′-GGAAGGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, 5′-GTCACATTAATTGTGATCCGTAAAG | ||
| C. meleagridis | F4, 5′-GGAAAGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, 5′-GTCACATAAATTGTGATCCGTAAAG | ||
| C. parvum | F4, 5′-GGAAGGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, 5′-GTCACATTTAATGTGATCCGTAAAG | ||
| C. canis | F4, 5′-GGAAAGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, 5′-GTCACATAAAATGTGATCCGTAAAG | ||
| C. felis | F4, 5′-GGAAAGGTCGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, 5′-GTCACAAAATAAATT-ATTGTGATCCGTAAAG | ||
| W1 | F4, 5′-GGAAGGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, 5′-GTCACATTTATTGTGATCCGTAAAG | ||
| W13 | NA | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, 5′-GTCACATTAAAATGTGATCCGTAAAG | ||
| W7 | F4, 5′-GGAAGGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, 5′-GTCACATCAAAAAATGTGATCCGTCAAAG | ||
| C. andersoni/C. muris | F4, 5′-GGAAGGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTGTC | P6, 5′-GTCGCATCAGAGATGCGATCCGTAAAG | ||
| C. ubiquitum | F4, 5′-GGAAGGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, 5′-GTCACATATAAA-TGTGATCCGTAAAG | ||
| C. bovis | F4, 5′-GGAAGGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTATT | P6, 5′-GTCACAT-AAT-GTGATCCGTAAAG | ||
| C. baileyi | F4, 5′-GGAAGGGTTGTATTTATTAGATAAAG | P7, 5′-AAGCTGATAGGTCAGAAACTTGAATG | |
| R3, 5′-CTTGTCACTACCTCCCTGTGTC | P6, 5′-GTCACATAAAT-GTGATCCGTAAAG | ||
| 18S-LC2 | − | F2, 5′-AAGTATAAACCCCTTTACAAGTA | P3, 5′-GAAATTTTACTTTGAGAAAATTAGAGTG-[fluorescein] |
| R2, 5′-TATTATTCCATGCTGGAGTATTC | P4 [Red 640]-5′-TTAAAGCAGGCATATGCCTTGAAT-phosphate | ||
| C. parvum/C. hominis/C. meleagridis/C. cuniculus/W1 | F2, 5′-AAGTATAAACCCCTTTACAAGTA | P3, 5′-GAAATTTTACTTTGAGAAAATTAGAGTG | |
| R2, 5′-TATTATTCCATGCTGGAGTATTC | P4, 5′-TTAAAGCAGGCATATGCCTTGAAT | ||
| C. andersoni | F2, 5′-AAGTATAAACCCCTTTACGAGTA | P3, 5′-GAAATTTTACTTTGAGAAAATTAGAGTG | |
| R2, 5′-TATTATTCCATGCTGGAGTATTC | P4, 5′-TTAAAGCAGGCAACTGCCTTGAAT | ||
| W7 | F2, 5′-AAGTATAAACCCCTTTACAAGTA | P3, 5′-GAAATTTTACTTTGAGAAAATTAGAGTG | |
| R2, 5′-TATTATTCCATGCTGTAGTATAC | P4, 5′-TTAAAGCAGGCATTAGCCTTGTAT | ||
| C. ubiquitum | F2, 5′-AAGTATAAACCCCTTTACAAGTA | P3, 5′-GAAATTTTACTTTGAGAAAATTAGAGTG | |
| R2, 5′-TATTATTCCATGCTGGAGTATTC | P4, 5′-TTAAAGCAGGCATTAGCCTTGAAT | ||
| W13 | F2, 5′-AAGTATAAACCCCTTTACAAGTA | P3, 5′-GAAATTTTACTTTGAGAAAATTAGAGTG | |
| R2, 5′-TATTATTCCATGCTGAAGTATTC | P4, 5′-TTAAAGCAGGCTATTGCCTTGAAT | ||
| C. muris | F2, 5′-AAGTATAAACCCCTTTACGAGTA | P3, 5′-GAAACTTTACTTTGAGAAAATTAGAGTG | |
| R2, 5′-TATTATTCCATGCTGGAGTATTC | P4, 5′-TTAAAGCAGGCAACTGCCTTGAAT | ||
| C. bovis | F2, 5′-AAGTATAAACCCCTTAACAAGTA | P3, 5′-GAAACTTTACTTTGAGAAAATTAGAGTG | |
| R2, 5′-TATTATTCCATGCTGGAGTATTC | P4, 5′-TTAAAGCAGGCTATTGCCTTGAAT | ||
| C. baileyi | F2, 5′-AAGTATAAACCCCTTTACAAGTA | P3, 5′-GAAACTTTACTTTGAGAAAATTAGAGTG | |
| R2, 5′-TATTATTCCATGCTGGAGTATTC | P4, 5′-TTAAAGCAGGCTATTGCCTTGAAT | ||
| C. canis | F2, 5′-GAGTATAAACCCCTTTACAAGTA | P3, 5′-GAAACTTTACTTTGAGAAAATTAGAGTG | |
| R2, 5′-TATTATTCCATGCTCTAGTATTC | P4, 5′-TTAAAGCAGGCTTTTGCCTTGAAT | ||
| C. felis | F2, 5′-AAGTATAAACCCCTTTACAAGTA | P3, 5′-GAAATTTTACTTTGAGAAAATTAGAGTG | |
| R2, 5′-TATTATTCCATGCTGGAGTATTC | P4, 5′-TTAAAGCAGGCTTTTGCCTTGAAT | ||
| hsp90 | − | F5, 5′-ATTAACTCTCTCTATTCTCAGAA | P5, 5′-CTTCTCCAATGCATCTGCAGAATT-[fluorescein] |
| R6, 5′-CATACCGATACCAGTATCAG | P6 [Red 640]-5′-AGTAACTCTCTAAGAAATACGTCT-phosphate | ||
| C. hominis | F5, 5′-ATTAACTCTCTCTATTCTCAGAA | P5, 5′-CTTCTCCAATGCATCTGCAGAATT | |
| R6, 5′-CATACCGATACCAGTATCAG | P6, 5′-AGTAACTCTCTAAGAAATACGTCT | ||
| C. meleagridis | F5, 5′-ATTAACTCTCTCTATTCTCAGAA | P5, 5′-CTTCTCCAATGCATCTGCAGAATT | |
| R6, 5′-CATACCGATACCAGTATCAG | P6, 5′-AGTAACTCTCTGAGAAATACGTCT | ||
| C. parvum | F5, 5′-ATTAACTCTCTCTATTCTCAGAA | P5, 5′-CTTCTCCAATGCATCTGCAGAATT | |
| R6, 5′-CATACCGATACCAGTATCAG | P6, 5′-AGTAACTCTCTGAGAAATACATCT | ||
| C. andersoni | F5, 5′-ATTAACTCTCTCTATTCTCAGAA | P5, 5′-CTTTTCTAGTGCATCTGCAGAGTT | |
| R6, 5′-CATACCAACACCAGTATCAG | P6, 5′-AGTAACTCTCTCAAGAAGACATCC | ||
| C. muris | F5, 5′-ATTAACTCTCTCTATTCTCAGAA | P5, 5′-CTTCTCTAGTGCATCTGCAGAGTT | |
| R6, 5′-CATACCAACACCAGTATCAG | P6, 5′-AGTAACTCTCTCAAGAAGACATCC | ||
| C. canis | F5, 5′-ATTAACTCTCTCTATTCTCAGAA | P5, 5′-CTTTTCCAATGCATCAGCAGAGTT | |
| R6, 5′-CATACCAATACCTGTATCAG | P6, 5′-AATAATTCTCTGAGAAATACATCT | ||
| C. ubiquitum | F5, 5′-ATTAACTCTCTCTATTCTCAGAA | P5, 5′-CTTCTCCAGTGCATCTGCAGAATT | |
| R6, 5′-CATACCGATACCAGTATCAG | P6, 5′-AGTAGTTCTCTAAGAAAAACATCT |
a
The nucleotide mismatches to the sequences of primers and probes are underlined (the underlined hyphens represent deletions). W1, deer mouse genotype III; W7, muskrat genotype I; W13, skunk genotype; −, deletions; NA, not available (the nucleotide sequence of the forward primer region was unavailable in GenBank).