TABLE 1.
PCR primers used for the detection of pathogen-specific gene markers in DNA extracted from manure, first separator pit water, and lagoon water
| Organism | PCR targeta | Primer sequenceb |
|---|---|---|
| Campylobacter jejuni | ceuE | F: CCTGCTACGGTGAAAGTTTTGC |
| R: GATCTTTTTGTTTGTGCTGC | ||
| Campylobacter coli | 23S rDNA | F: TATTCCAATACCAACATTAGT |
| R: TACAGCGTGGACTACCAGGGT | ||
| Clostridium perfringens | α toxin | F: GTTGATAGCGCAGGACATGTTAAG |
| R: CATGTAGTCATCTGTTCCAGCATC | ||
| β toxin | F: ACTATACAGACAGATCATTCAACC | |
| R: TTAGGAGCAGTTAGAACTACAGAC | ||
| ɛ toxin | F: ACTGCAACTACTACTCATACTGTG | |
| R: CTGGTGCCTTAATAGAAAGACTCC | ||
| ι toxin | F: GCGATGAAAAGCCTACACCACTAC | |
| R: GGTATATCCTCCACGCATATAGTC | ||
| E. coli | O157 rfb | F: CGTGATGATGTTGAGTTG |
| R: AGATTGGTTGGCATTACTG | ||
| LT1 | F: TGGATTCATCATGCACCACAAGG | |
| R: CCATTTCTCTTTTGCCTGCCATC | ||
| VT1 (stx1) | F: ACGTTACAGCGTGTTGCTGGGATC | |
| R: TGTGGCTGGGTTCGTTAATACGGC | ||
| VT2 (stx2) | F: TGTTGGCTGGGTTCGTTAATACGG | |
| R: TCCGTTGTCATGGAAACCGTTGTC | ||
| STI | F: TTTCCCCTCTTTTAGTCAGTCAACTG | |
| R: GGCAGGATTACAACAAAGTTCACAG | ||
| STII | F: CCCCCTCTCTTTTGCACTTCTTTCC | |
| R: TGCTCCAGCAGTACCATCTCTAACCC | ||
| CNF1 | F: GGCGACAAATGCAGTATTGCTTGG | |
| R: GACGTTGGTTGCGGTAATTTTGGG | ||
| eaeA | F: TGAGCGGCTGGCATGAGTCATAC | |
| R: TCGATCCCCATCGTCACCAGAGG | ||
| K99 | F: TATTATCTTAGGTGGTATGG | |
| R: GGTATCCTTTAGCAGCAGTATTTC | ||
| F41 | F: GCATCAGCGGCAGTATCT | |
| R: GTCCCTAGCTCAGTATTATCACCT | ||
| Listeria spp. | iap | F: ATGAATATGAAAAAGCAAC |
| R: TTATACGCCACCGAAGCCAAC | ||
| M. avium subsp. paratuberculosis | IS900 | F: GAAGGGTGTTCGGGGCCGTCGCTTAGG |
| R: GGCGTTGAGGTCGATCGCCCACGTGAC | ||
| Salmonella sp. | inv | F: CTGTTGAACAACCCATTTGT |
| R: CGGATCTCATTAATCAACAAT | ||
| Staphylococcus aureus | sea | F: TTGGAAACGGTTAAAACGAA |
| R: GAACCTTCCCATCAAAAACA | ||
| seb | F: TCGCATCAAACTGACAAACG | |
| R: GCAGGTACTCTATAAGTGCC | ||
| sec | F: GACATAAAAGCTAGGAATTT | |
| R: AAATCGGATTAACATTATCC | ||
| sed | F: CTAGTTTGGTAATATCTCCT | |
| R: TAATGCTATATCTTATAGG | ||
| tsst1 | F: ATGGCAGCATCAGCTTGATA | |
| R: TTTCCAATAACCACCCGTT | ||
| Yersinia spp. | 16S rDNA | F: GCGGCAGCGGGAAGTAGTTA |
| R: TACAGCGTGGACTACCAGGGT |
a
Limits of detection were as follows: E. coli and C. perfringens, 10 pg; Yersina spp. and Salmonella sp., 10 fg (10 pg correlates to 104 cells, and 10 fg correlates to 10 cells).
b
F, forward; R, reverse.