Table 1.
Multiplex PCR assays, target pathogens, genomic regions and primers. All reverse primers were synthesized with 5′GCA GTA TAT CGC TTG ACA 3′, the reverse complementary sequence of the Red Universal Reporter Probe, at the 5′ end. In addition to the pathogen specific primers, each assay consisted of DENV specific primers, (F) AAACCGTGCTGCCTGTAG/(R) TCTCTCCCAGCGTCAA (Sudiro et al., 1997) as internal control primers. †includes two primers.
| Organism | Genomic region | Primer Sequence (5'->3') | Amplicon size (bp) | Reference | |
|---|---|---|---|---|---|
| Assay for BRDC associated bacteria | |||||
| H. somni | 16S ribosomal DNA (rDNA) | F | GTGATGAGGAAGGCGATTAGT | 416 | Angen et al., 2009 (modified) |
| R | TTCGGGCACCAAGTRTTCA | ||||
| P. multocida | Putative transcriptional regulator gene, Pm0762 | F | TTGTGCAGTTCCGCAAATAA | 567 | Liu et al., 2004 |
| R | TTCACCTGCAACAGCAAGAC | ||||
| M. bovis | 16S rDNA | F | CCTTTTAGATTGGGATAGCGGATG | 360 | Wiggins et al., 2007 |
| R | CCGTCAAGGTAGCATCATTTCCTAT | ||||
| M. haemolytica (all serotypes) | Leukotoxin A (lktA) | F | GTCCCTGTGTTTTCATTATAAG | 385 | Klima et al., 2014a, b |
| R | CACTCGATAATTATTCTAAATTAG | ||||
| M. haemoytica (pathogenic serotypes) | Transferring-binding protein B (tbpB) | F | CTACTTGCTGCTTGTTCCTC | 452 | Klima et al., 2014a, b |
| R | CCATGTGCACCTGTTCTCAAA | This study | |||
| UDP-N-acetyl-D-glucosamine- 2-epimerase (nmaA) | F | AAGCCGTTTCAACATTAGCGT | 396 | This study | |
| R | CATCGCCATAAGGGTTGTGA | Klima et al., 2014a, b | |||
| Assay for BRDC associated viruses | |||||
| BoHV-1 | Glycoprotein B | F | TGAGGCCTATGTATGGGCAGTT | 432 | Klima et al., 2014a, b |
| R | GGACACAACAAACAATGCGG | ||||
| BRSV | G attachment glycoprotein | F | ACACATCAATYCAAAGCACCACAC | 374 | This study |
| R | GCTRGTTCTGTGGTGGRTTGTTGTC | Vilcek et al., 1994 (modified) | |||
| BPI-3 | M gene | F | TGTCTTCCACTMGATAGAGGGATAAAATT | 203 | Klima et al., 2014a, b |
| R† | CCTTTTTCATCTAGAATCTGAACTACTCC/ CCTTTCTCATCTAAGATCTGGACMACC | This study | |||
| BVDV/Hobi | 5’ untranslated region (UTR) | F† | CATACCTTCAGTAGGACGAGC/ CATGCCCRYAGTAGGACTAGC | 280 | This study/ Deregt et al., 2006 |
| R | ATGTGCCATGTACAGCAGAG | ||||
| BCoV | N | F | GCCGATCAGTCCGACCAATC | 407 | Tsunemitsu et al., 1999 |
| R | AGAATGTCAGCCGGGGTAT | ||||
| Assay for BED associated bacteria/protozoa | |||||
| C. perfringens | Alpha toxin (plC) | F | CTATGGGCTGGGGCATCAACT | 480 | This study |
| R | AACATGTCCTGCGCTATCAAC | ||||
| E. bovis/ E. zuernii | Internal transcribed spacer-1 (ITS-1) | F | AAAGGATGCAAAAGTCGTAACAC | 535-514 | Kawahara et al., 2010 (modified) |
| R | TGCAATTCACAATGCGTATCG | ||||
| C. parvum | Cryptosporidium oocyst wall protein (COWP) | F | CAAATTGATACCGTTTGTCCTTCTG | 348 | Guy et al., 2003 |
| R | AGGGCAGACAGGTTGAGTTGG | This study | |||
| S. e. Dublin | Hypothetical protein coding sequence SeD_A1118 | F | CCGACCTTGCCGTGTATCTCA | 408 | This study |
| R | GCTGCCGACTGTTACCGAGAA | ||||
| S. e. Typhimurium | Putative cytoplasmic protein coding sequence STM4493 | F | GAGCGTGCGGCGATGTTAGT | 572 | This study |
| R | CGGGTACTGCTGGTTGAAGGT | ||||
| E coli (EHEC) | Intimin (eaeA) | F | GACCCGGCACAAGCATAAGC | 384 | Blais et al., 2012 |
| R | CCACCTGCAGCAACAAGAGG | ||||
| Shiga like toxin-1 (stx-1) | F | GTCATTCGCTCTGCAATAGGTA | 559 | Frank et al., 1998 (modified) | |
| R | TTCCCCAGTTCAATGTAAGATCA | Ok et al., 2009 (modified) | |||
| E. coli (ETEC) | K99 antigen coding gene (K99) | F | CGACTACCAATGCTTCTGCGAA | 439 | Cho et al., 2010 (modified) |
| R | TTAGACGGAGCGCGGTCATC | This study | |||
| E. coli (EPEC) | Heat-labile toxin (Lt) | F | CGGAGGTCTTATGCCCAGAGG | 481 | This study |
| R | CCAGGGTTCTTCTCTCCAAGCT | West et al., 2007 (Modified) | |||
| Assay for BED associated viruses | |||||
| BRoV A/C | Outer capsid protein VP6 | F | GCATGGATGAAATGGTTAGAGA | 467 | This study |
| R | TGGATTGAAGTACCATGTAGT | ||||
| BRoV B | VP6 | F | ACAGTGAATGCTTGCGTCAG | 584 | This study |
| R | GCTTCCATGCCTGAAACACA | ||||
| BToV | Nucleocapsid phosphoprotein gene (N) | F | CCAAATGCTATGCCATTTCAGC | 297 | This study |
| R | TGCAGTCTCATTTGCCATCAT | ||||
| BVDV/Hobi | 5’ untranslated region (UTR) | F† | CATACCTTCAGTAGGACGAGC/ CATGCCCRYAGTAGGACTAGC | 280 | This study/ Deregt et al., 2006 |
| R | ATGTGCCATGTACAGCAGAG | ||||
| BCoV | N | F | GCCGATCAGTCCGACCAATC | 407 | Tsunemitsu et al., 1999 |
| R | AGAATGTCAGCCGGGGTAT | ||||