Primers and PCR conditions for MLST of P. multocida
The primers and the PCR conditions used in the establishment of this MLST scheme are presented below. PCR conditions may need to be modified for use in other laboratories. Since the same primers are used for the initial amplification, and for sequencing, it is important that PCR conditions are used which result in the amplification of the desired single fragment. This may involve some optimisation of the annealing temperature.
House keeping genes utilized in the scheme and the fragment size of the amplified products:
| adk (Adenylate kinase) | 570 bp |
| est (Esterase) | 641 bp |
| pmi (Mannose-6-Phosphate Isomerase) | 739 bp |
| zwf (Glucose-6-Phosphate Dehydrogenase) | 808 bp & 614 bp |
| mdh (Malate Dehydrogenase) | 620 bp |
| gdh (Glutamate dehydrogenase) | 702 bp |
| pgi (Phospho Glucose Isomerase) | 784 bp |
Primer sets designed for the amplification and sequencing of seven P. multocida housekeeping genes.
| Gene | Primer Sequence (5' to 3') | Amplicon Size (bp) | |
|---|---|---|---|
| Forward | Reverse | ||
| adk | TTTTTCGTCCCGTCTAAGC | GGGGAAAGGGACACAAGC | 570 |
| est | TCTGGCAAAAGATGTTGTCG | CCAAATTCTTGGTTGGTTGG | 641 |
| pmi | TGCCTTGAGACAGGGTAAGC | GCCTTAACAAGTCCCATTCG | 739 |
| zwf-1 | AATCGGTCGTTTGACTGAGC | TGCTTCACCTTCAACTGTGC | 808 |
| mdh | ATTTCGGGATCAGGGTTAGC | GGAAAACCGGTAATGGAAGG | 620 |
| gdh | ATCGACTTCTTCCGCAGACC | GCGGGTGATATTGGTGTAGG | 702 |
| pgi | ACCACGCTATTTTTGGTTGC | ATGGCACAACCTCTTTCACC | 784 |
| zwf-2 * | TGTTAGGTGTGGCAAGAACG | TTGCAACAAATGGTTTTGGA | 614 |
*The primer set zwf-2 is only used if the first primer set (zwf-1) fails to amplify the desired product.
Initially, six isolates of P. multocida were chosen randomly to determine that each primer set could produce a single amplicon. PCR reactions over a range of annealing temperatures were performed in order to determine the optimal Tm for each primer set.
The 50 µl PCR reaction mixture contained 1.5 mM MgCl2, 50 mM KCl and 10 mM Tris HCl (pH 8.3) in the buffer, 200 µM of each dNTP (dATP, dCTP, dGTP, dTTP), 0.4 µM of each primer and 1.25 units Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany). Two µl of genomic DNA was used as template.
Master mix for 50 µl reaction
| Reagents | Volume (µl) | Notes |
|---|---|---|
| H2O | 30.0 | |
| 10x reaction buffer | 5.0 | 1.5 mM MgCl2 |
| PCR primer 1 | 2.0 | 10 µM Primer stock = 0.4 µM Final concentration |
| PCR primer 2 | 2.0 | 10 µM Primer stock = 0.4 µM Final concentration |
| dNTP mix | 8.0 | 1.25 mM stock = 200 µM final concentration of each nucleotide |
| Taq polymerase | 1.0 | 1.25 units per reaction |
| Total | 48.0 |
Mix well by votexing and then spot spin and aliquot 48 µl into each PCR tube. Then add 2 µl of respective DNA to each tube.
dNTPs
dATP, dCTP, dGTP, dTTP
We used Boehringer Mannheim (Roche) (Cat # 1277049) dNTPs which are 100 mM.
A stock solution containing 1.25 mM of each dNTP by adding 5 µl of each dNTP to 380 µl DNA-grade H2O was prepared.
Using 16 µl of this stock solution in a 100 µl reaction volume or 8 µl in a 50 µl reaction volume gives 200 µM final concentration of each nucleotide.
A 4 µl aliquot of the PCR product was mixed with 1 µl loading buffer and then run in 2% DNA-grade agarose gel containing ethidium bromide (1 µl/µg) in 1%TAE buffer at 80 V for about 40 minutes and visualised under UV light. Ready-load 100 bp DNA ladder (Invitrogen 10380-012) was used in all gels to determine fragment size.
PCR cycle parameters
| STEP 1: | 94 °C | 5 minutes (Initial Denaturation) |
| STEP 2: | 94 °C | 30 seconds (Denaturation) |
| 48 °C | 30 seconds (Annealing) | |
| 72 °C | 1 minute (Extension) | |
| STEP 3: | Repeat STEP 2 into 30 cycles | |
| STEP 4: | 72 °C | 5 minutes |
| STEP 5: | Hold at 4 °C | |
PCR product clean-up
Each PCR reaction is done in duplicates.
- 2 × 50 µl PCR reaction products will be combined to give 100 µl product.
- Method of clean up - QIAquick PCR Purification Kit Protocol-using a Microcentrifuge (page-18, 2001 hand book).
- To determine the concentration of the sample the cleaned up product will be run on a 1% gel along with Low DNA mass ladder. (2 µl PCR sample and 2 µl Ladder)
Sequencing reaction
| Purified product (100 ng/µl) | = 4 µl |
| Primer (25 ng/µl) | = 1 µl |
| BDT 3.1 | = 4 µl |
| BDT Buffer/ H2O | = 1 µl |
| Total volume | = 10 µl |
NOTE: The amount of purified product often varies depending on the product concentration. So when less than 4 µl template is used buffer or water will be adjusted to give final volume 10 µl.
Sequencing cycle parameters
| Step-1 | 94 °C for 5minutes (Denaturation) |
| Step-2 | 96 °C for 10 seconds |
| 50 °C for 5 seconds (Annealing) | |
| 60 °C for 4 minutes (Extension) | |
| Step-3 | Repeat Step-2 into 30 cycles |
| Step-4 | Hold at 4 °C |