TABLE 1.
Refined set of PHAT gene probes
| Gene probe name | Gene function | Template straina | Sequence (5′-3′)b
|
PCR annealing temp (°C) | Amplicon size (bp) | Source or reference | |
|---|---|---|---|---|---|---|---|
| Primer 1 | Primer 2 | ||||||
| chuA | Outer membrane heme/hemoglobin receptor | CFT073 | ATCGCGGCGTGCTGGTTCTTGT | AGCCAGTTGGTCGGGTTGATAGTC | 59 | 985 | 1 |
| yjaA | Hypothetical protein | CFT073 | TGAAAGCAAACGTGAAGTGTC | TGCTTAAACATTGCCAGGAC | 50 | 324 | 1 |
| tspE4.C2 | Anonymous DNA fragment | CFT073 | CTTCCCGCTCTCCAGGCAACAT | GTGAGACAGAAACGCGGGTAGATA | 57 | 781 | 1 |
| stx1 | Shiga toxin 1 | TW07814 | AGGGCGTGGAGGATGTCAAGAATA | TGCCGGACACATAGAAGGAAACTC | 57 | 915 | 15 |
| stx2 | Shiga toxin 2 | TW07814 | GTTCCGGAATGCAAATCAGTCGTC | ATTTTTCACCAGTCGCCCACAC | 57 | 946 | 15 |
| eae | Intimin, attaching and effacing adherence protein | TW07892 | TTCGCAATGGTAATACTCTGGTTC | CCGGTTTGCTTTTCCCTTCC | 54 | 857 | 15 |
| bfp | Bundle-forming pili | TW03875 | AGAGAAGAACCCTGGAT | TCGACCTGAAATGTTGC | 45 | 411 | 15 |
| lt | Heat-labile toxin | TW07814 | TCGATGATGCTACCCCTGAAAAAC | CGCTCCGACCTGACCAAATG | 56 | 975 | 15 |
| ipaH | Invasion plasmid antigen | TW06117 | TTACCCGAGACGTTAGAAGA | CAGAGCGCCGGTATCATTATCG | 50 | 830 | 15 |
| virF | Transcription-al activator of virulence loci | TW06117 | AAGGTGTTCAATGACGGTTAGC | GCATGCGAATATCAAGAAGGA | 50 | 560 | 15 |
| aafII | Aggregative adherence fimbria II | TW04393 | CCAGGTGCGGCAATCAGAGT | TGGCAGGGCAGTATCGTTCAT | 54 | 988 | 15 |
| c0286 | Hypothetical protein | CFT073 | TTGCTTCCCTGCGATTATTTAGTG | CTGCGGTTCTGGCTGTGATGGATT | 54 | 930 | This study |
| c0311 | Hypothetical protein | CFT073 | AGTGTTTCTGCCTTTCCTG | GACTTCGTACATACCGCTCTG | 50 | 286 | This study |
| c0340 | Hypothetical protein | CFT073 | AGGATGTTCTGGAGGAGTGC | CAGGGATATGACGAGTTGTTC | 54 | 528 | This study |
| c1164 | Hypothetical protein ycdT | CFT073 | TCGGTATTGTTTGTGGGTGGATTT | TTGGCCGATGCTGATTGAACTGTC | 52 | 1,024 | This study |
| c1600 | Putative periplasmic iron binding protein, SitA | CFT073 | TCGGAAATCGTACTCTCGCTAAA | GCTGCAGAAGTCTCATCCATAACC | 54 | 599 | This study |
| c3389 | Hypothetical protein | CFT073 | TGCCTGCGGTGGATAACAAC | AGAACGCGGAACAAGACTGATTT | 55 | 839 | This study |
| c3680 | Hypothetical protein | CFT073 | CGGAAGTGGTCAACGGGCATAC | TTCGTCTTCATAGCGCTCTTTACC | 54 | 698 | This study |
| S3187 | Hypothetical protein | 2457T | GTGGTCGACCCCGGTTACTTTTT | GGATTCATTATTCGGGGTCTATTC | 54 | 488 | This study |
| iroN | Iron-related siderophore receptor | CFT073 | AAGTCAAAGCAGGGGTTGCCC | GACGCCGACATTAAGACGCAG | 65 | 700 | 8 |
| ompT | Outer membrane protease | CFT073 | AGCAGTAGTGATGAAGTTAT | CGGACATTAGTCTTGGAACT | 54 | 800 | 5 |
| hly | Alpha-hemolysin, lyses red blood cells | CFT073 | AGATGATGAGCTTCAGGTTC | ATCCGACTTGTACTATGTGC | 54 | 1500 | 5 |
| kpsMT | Group II capsule | CFT073 | GCGCATTTGCTGATACTGTTG | CATCCAGACGATAAGCATGAG | 52 | 519 | 5 |
| aer | Aerobactin, aerotaxis receptor | CFT073 | CGTACAGTATCCCGTGAAGAGTTT | AAGGGCATCAGTGAGGGAAGTATT | 65 | 764 | 5 |
All strains are E. coli except forS. flexneri strain 2457T.
The primers for the following probes were designed from E. coli CFT073 locus numbers: chuA (c4308), yjaA (c4968), tspE4.C2 (c4836), c0286, c0311, c0340, c1164, c1600, c3386, c3389, and c3680. The primers for the following probes were designed from the S. flexneri 2457T locus number S3187. The primers from the following probes were designed from the indicated GenBank accession numbers: stx1 (J93SLT1), stx2 (X07865), bfp (AB024946, AF119170, EC13FPGNS, U21784), lt (ECLT87, ECOELTBH), eae (AJ298279, AJ308550, AJ308551, AJ308552, M58154, U38618, U66102, Z11541, AB040740, AF025311, AF065628, AF116899, AF200363, AF449418, AF530555, AF530556, AF530557, AJ271407), ipaH (AF386526, AL391753, AF391753, AF348706, M76445, M32063), virF (AY206433, AF386526, AF348706, AL391753, X16661, X58464, M29172), and aafII (AF114828). Template strains identified by the prefix TW are from T. Whittam's collection (National Food Safety and Toxicology Center, Michigan State University).