TABLE 3

Primers used to differentiate P. larvae and B. laterosporus strains

Target genePrimer sequenceaDirectionPrimer concn (μM)Annealing temp (°C)PurposeExpected size (bp)Reference or source
16S rRNA5′-AGAGTTTGATCMTGGCTCAG-3′Forward49.745216S rRNA gene∼1,50043
5′-CCGTCAATTCMTTTRAGTTT-3′Reverse38.52Universal primer
P. larvae rpoB5′-ATAACGCGAGACATTCCTAA-3′Forward67.852Amplifies P. larvae rpoB∼40039
5′-GAACGGCATATCTTCTTCAG-3′Reverse82.8
B. laterosporus rpoB5′-GCAGGTAAACTGGTCCAGAGCG-3′Forward49.7669.1Amplifies B. laterosporus rpoB∼900This study
5′-CACCTGTTGATTTATCAATCAGCG-3′Reverse45.8
P. larvae ftsA5′-AAATCGGTGAGGAAGACATT-3′Forward52.852Amplifies P. larvae ftsA∼50039
5′-TGCCAATACGGTTTACTTTA-3′Reverse74.6
B. laterosporus ftsA5′-CAGTCAAAATGATCTTATTGTCAGCATCG-3′Forward46.5455Amplifies B. laterosporus ftsA∼1,200This study
5′-CATCTTTTCAATGAAATTCCCTTGAG-3′Reverse48.68
P. larvae thiG5′-GCCAAGGCTTCGGGGCGGC-3′Forward47.4265Amplifies P. larvae thiG∼800This study
5′-CTCATTTATTTACTTCCAGACTGATGC-3′Reverse45.72
B. laterosporus thiG5′-GCATTCCTTCTATCGGACTACTAGC-3′Forward4455+bAmplifies B. laterosporus thiG∼775This study
5′-CGGATACGACTTTACGAATTGGAGAG-3′Reverse49.2
P. larvae Vagabond 15′-CTGGATGTGGCCCAAACCTTTC-3′Forward36.365Amplifies P. larvae genomic region∼1,500This study
5′-CCTGCACGGCAAAGGAAGGC-3′Reverse34.76
P. larvae Vagabond 25′-AAAAAAAATAGTGAGGTTGGCACAG-3′Forward30.860Amplifies P. larvae genomic region∼750This study
5′-CTGTGTAGAGAATGAATACGGAAT-3′Reverse44.06
  • a M and R, degenerate bases in primer sequences.

  • b +, an occasional increase in the annealing temperatures (60°C) was needed in order to eliminate sporadic, spurious bands.