The barcoding protocol for gyrB for Erwinia amylovora
Conventional PCR gyrB for Erwinia amylovora.
The test is based on PCR amplification and sequencing of 632 bp (amplicon including primers) of the gyrB gene for identification of Erwinia amylovora. The test is a new development from Brady et al., 2008 with new primers targeting a conserved region of the gyrB gene of Erwinia species. The gyrB gene encodes a type II topoisomerase that catalyzes the ATP-dependent reduction of supercoiling in double-stranded DNA. It relaxes tension which builds up during unwinding for replication and prevents breaking of DNA. It is located on the chromosome.
The bacterial gyrB gene is a protein coding sequence (CDS) for which translation table 11 (The Bacterial, Archaeal and Plant Plastid Code) applies.
Primer sequences and their application are given in the table below.
Primer name | Primer sequence (5’- 3’ orientation)* | Primer used for | |
PCR | Sequencing | ||
gyrB-Ea-F | caggaaacagctatgaccATGTSASCGGYGAAACYGA | x | |
gyrB-Ea-R | tgtaaaacgacggccagtCGGRTKTTCCAGCAGRTAYTC | x | |
M13rev-29 | caggaaacagctatgacc | x | |
M13uni-21 | tgtaaaacgacggccagt | x | |
*Lower-case characters indicate the universal M13 tails. These tails play no role in amplification of the target but are used for generating cycle sequence products.
Master mixes are prepared according to the table below.
Reagent | Working concentration | Volume per reaction (µL) | Final concentration |
Molecular grade water | N.A. | 5.5 | N.A. |
Bioline MyFi Mix* | 10 x | 12.5 | 1 x |
gyrB-Ea-F | 10 µM | 1.0 | 0.2 µM |
recA-R | 10 µM | 1.0 | 0.2 µM |
Subtotal | 20.0 | ||
Genomic DNA extract | 10 ng/µL | 5.0 | |
Total | 25.0 |
* The MyFiTM Mix is supplied as a 2x formulation containing MyFi DNA Polymerase, dNTPs, MgCl2 and enhancers at optimal concentrations. Other verified PCR master mixes containing a polymerase with proof reading activity are also fit for purpose.
Thermocycler profile: 2 min at 95°C then 30x [20 s at 95°C, 20 s at 60°C, 1 min at 72°C], 5 min at 72°C, hold at 4°C.
Amplicon sequencing is done with the M13 tails.
The gyrB amplicon (632 bp including primers) extends from position 464 to position 1074 included on the positive strand of the gene (2409 bp).
The gyrB barcode (580 nt) extends from position 495 to position 1074 included on the positive strand of the gene. The recA barcode starts with GCGGGTGCG and ends with CTGCTGTCT.
See appendix 7 of PM7/129 for guidance on data-analysis.
Performance characteristics
Analytical sensitivity
Cell pellets of pure cultures are used for the DNA extraction (section 2.1).
DNA concentration of approximately 1.1 ng/µL is sufficient to generate an amplicon that can be sequenced, leading to a consensus sequence with a HQ (Phred > 40) of at least 84%.
Analytical specificity
The gyrB amplicon was produced for 97 strains of Erwinia amylovora from worldwide origin and from various plant hosts.
The gyrB PCR is however not exclusive for Erwinia amylovora. Other Erwinia species will also produce the recA amplicon but can be reliably differentiated by the DNA barcode sequence.
Selectivity
Selectivity does not apply as pure cultures are used.
Reproducibility data
The DNA samples were processed by different persons. Therefore in this situation the reproducibility is identical to diagnostic sensitivity.