Multiplex PCR mixa
Locus
Primer sequences (5′-3′)b
Allele size range (bp)c
M1
Bruce30
Fw: PET – TGACCGCAAAACCATATCCTTC
Rw: TATGTGCAGAGCTTCATGTTCG
119–199
Bruce08
Fw: PET – ATTATTCGCAGGCTCGTGATTC
Rw: ACAGAAGGTTTTCCAGCTCGTC
312–384
Bruce11
Fw: 6FAM – CTGTTGATCTGACCTTGCAACC
Rw: CCAGACAACAACCTACGTCCTG
257–1076
Bruce45
Fw: 6FAM – ATCCTTGCCTCTCCCTACCAG
Rw: CGGGTAAATATCAATGGCTTGG
133–187
Bruce19
Fw: NED – GACGACCCGGACCATGTCT
Rw: ACTTCACCGTAACGTCGTGGAT
79–205
M2
Bruce06
Fw: NED – GATTGCGGAACGTCTGAACT
Rw: TAACCGCCTTCCACATAATCG
312–714
Bruce42
Fw: VIC – CATCGCCTCAACTATACCGTCA
Rw: ACCGCAAAATTTACGCATCG
164–914
M3
Bruce12
Fw: NED – CGGTAAATCAATTGTCCCATGA
Rw: GCCCAAGTTCAACAGGAGTTTC
302–452
Bruce18
Fw: PET – TATGTTAGGGCAATAGGGCAGT
Rw: GATGGTTGAGAGCATTGTGAAG
130–186
Bruce55
Fw: PET – TCAGGCTGTTTCGTCATGTCTT
Rw: AATCTGGCGTTCGAGTTGTTCT
193–553
Bruce21
Fw: 6FAM – CTCATGCGCAACCAAAACA
Rw: GTGGATACGCTCATTCTCGTTG
431–463
Bruce04
Fw: VIC – CTGACGAAGGGAAGGCAATAAG
Rw: TGGTTTTCGCCAATATCAACAA
313–473
M4
Bruce07
Fw: NED – GCTGACGGGGAAGAACATCTAT
Rw: ACCCTTTTTCAGTCAAGGCAAA
134–246
Bruce09
Fw: VIC – GCGGATTCGTTCTTCAGTTATC
Rw: GGGAGTATGTTTTGGTTGTACATAG
124–292
Bruce43
Fw: 6FAM – TCTCAAGCCCGATATGGAGAAT
Rw: TATTTTCCGCCTGCCCATAAAC
170–194
Bruce16
Fw: 6FAM – ACGGGAGTTTTTGTTGCTCAAT
Rw: GGCCATATCCTTCCGCAATA
227–353
6.
10× forward primer mixes (M1fw, M2fw, M3fw, and M4fw mixes) (see Note 1 ):
(a)
M1fw: Mix and dilute 4.4 μl of each 50 μM primer stock solutions [“Bruce08 Fw (PET labelled),” “Bruce11 Fw (6FAM labelled),” “Bruce45 Fw (6FAM labelled),” “Bruce30 Fw (PET labelled),” and “Bruce19 Fw (NED labelled)”] in a final volume of 110 μl of TE buffer or Mol Bio grade water to obtain a 2 μM (10×) primer mix working solution (see Note 2 ).
(b)
M2fw: Mix and dilute 50 μM primer stock solutions [8.8 μl of “Bruce06 Fw (NED labelled),” 1.1 μl of “Bruce42 Fw (VIC labelled),” and 1.1 μl of “Bruce42 Fw (unlabelled)”] in a final volume of 110 μl of TE buffer or Mol Bio grade water to obtain a 4.0, 0.5, and 0.5 μM (10×) primer mix working solution, respectively (see Note 2 ).
(c)
M3fw: Mix and dilute 50 μM primer stock solutions [3.3 μl of “Bruce12 Fw (NED labelled),” 1.1 μl of “Bruce55 Fw (PET labelled),” 1.1 μl of “Bruce55 Fw (unlabelled),” 1.1 μl of “Bruce18 Fw (PET labelled),” 1.1 μl of “Bruce18 Fw (unlabelled),” 4.4 μl of “Bruce21 Fw (6FAM labelled),” and 4.4 μl of “Bruce04 Fw (VIC labelled)”] in a final volume of 110 μl of TE buffer or Mol Bio grade water to obtain a 1.5, 0.5, 0.5, 0.5, 0.5, 2.0, and 2.0 μM (10×) primer mix working solution, respectively (see Note 2 ).
(d)
M4fw: Mix and dilute 50 μM primer stock solutions [3.3 μl of “Bruce07 Fw (NED labelled),” 4.4 μl of “Bruce09 Fw (VIC labelled),” 4.4 μl of “Bruce16 Fw (6FAM labelled),” and 3.3 μl of “Bruce43 Fw (6FAM labelled)”] in a final volume of 110 μl of TE buffer or Mol Bio grade water to obtain a 1.5, 2.0, 2.0, and 1.5 μM (10×) primer mix working solution, respectively (see Note 2 ).
7.
10× reverse primer mixes (M1rw, M2rw, M3rw, and M4rw mixes) (see Note 1 ).
(a)
M1rw: Mix and dilute 4.4 μl of each 50 μM primer stock solutions [“Bruce08 Rw (unlabelled),” “Bruce11 Rw (unlabelled),” “Bruce45 Rw (unlabelled),” “Bruce30 Rw (unlabelled),” and “Bruce19 Rw (unlabelled)”] in a final volume of 110 μl of TE buffer or Mol Bio grade water to obtain a 2 μM (10×) primer mix working solution (see Note 2 ).
(b)
M2rw: Mix and dilute 50 μM primer stock solutions [8.8 μl of “Bruce06 Rw (unlabelled)” and 2.2 μl of “Bruce42 Rw (unlabelled)”] in a final volume of 110 μl of TE buffer or Mol Bio grade water to obtain a 4.0 and 1.0 μM (10×) primer mix working solution, respectively (see Note 2 ).
(c)
M3rw: Mix and dilute 50 μM primer stock solutions [3.3 μl of “Bruce12 Rw (unlabelled),” 2.2 μl of “Bruce55 Rw (unlabelled),” 2.2 μl of “Bruce18 Rw (unlabelled),” 4.4 μl of “Bruce21 Rw (unlabelled),” and 4.4 μl of “Bruce04 Rw (unlabelled)”] in a final volume of 110 μl of TE buffer or Mol Bio grade water to obtain a 1.5, 1.0, 1.0, 2.0, and 2.0 μM (10×) primer mix working solution, respectively (see Note 2 ).
(d)
M4rw: Mix and dilute 50 μM primer stock solutions [3.3 μl of “Bruce07 Rw (unlabelled),” 4.4 μl of “Bruce09 Rw (unlabelled),” 4.4 μl of “Bruce16 Rw (unlabelled),” and 3.3 μl of “Bruce43 Rw (unlabelled)”] in a final volume of 110 μl of TE buffer or Mol Bio grade water to obtain a 1.5, 2.0, 2.0, and 1.5 μM (10×) primer mix working solution, respectively (see Note 2 ).
8.
Hi-Di formamide.
9.
POP-7 polymer, a separation matrix for performing DNA sequencing and fragment analysis on the Applied Biosystems genetic analyzer instruments (Life Technologies, USA).
10.
Anode and cathode buffers, reagents to support electrophoresis on Applied Biosystems genetic analyzer instruments (Life Technologies, USA).
11.
GeneScan 1200 LIZ size standard (Life Technologies, USA).
12.
DNA samples from one or more reference strains having known MLVA profiles, for comparison (e.g., B. melitensis biovar 1 strain 16 M). Be sure to use purified DNAs (A260/A280 ratio ≥1.8).
13.
DNA samples to be tested. Be sure to use purified DNAs (A260/A280 ratio ≥1.8).
2.3 Equipment and Instruments
1.
PCR plate cooling block or ice.
2.
Complete “clean set” (1,000 μl, 200 μl, 100 μl, 20 μl, and 10 μl) of single-channel pipettes for PCR master mix setup.
3.
10 μl single-channel pipettes for DNA solutions.
4.
8-channel pipettes (200 μl and 10 μl) for DNA solutions.
5.
Thermocycler with heated lid.
6.
Heat block (or thermocycler), capable of operating at 95 °C.
7.
Centrifuge and rotors adapted to 0.2 ml PCR tubes, 1.5 ml microcentrifuge tubes, and 96-well v-bottom PCR plates.
8.
Mixer.
9.
Capillary electrophoresis genetic analyzer (e.g., we use ABI PRISM genetic analyzer, either 3130 or 3500 series) (Life Technologies, USA) (see Note 3 ).
10.
Capillary arrays, 50 cm.
2.4 Software
1.
Data collection software, the operative software to drive the Applied Biosystems genetic analyzer instruments.
2.
GeneMapper 4.1 (Life Technologies, USA).
3 Methods
3.1 PCR Setup
1.
Defrost all PCR components and keep them on ice during preparation.
2.
Briefly vortex and spin down the primer mixes in their amber tubes.
3.
Place a 96-well v-bottom PCR plate or the required number of 0.2 ml PCR tubes in a PCR block or on ice.
4.
Mark four 1.5 ml microcentrifuge tubes as M1, M2, M3, and M4, respectively, for each of the four multiplex master mixes to be prepared.
5.
Prepare each master mix in the correspondent 1.5 ml microcentrifuge tube using the appropriate volumes (see Table 2), preferably introducing them in the following order: Mol Bio water, Q solution, 2× Type-it microsatellite PCR Master Mix, and primers.
Table 2
PCR master mixes composition
Component | Volume per reaction (μl)a | Final concentration |
|---|---|---|
2× Type-it microsatellite PCR Master Mix | 5 | 1× |
Q solution | 1 | 0.5× |
10× forward primer mixb | 1 | 1× |
10× reverse primer mixb | 1 | 1× |
Mol Bio grade water | 1 | – |
DNA template solution | 1 | 10–50 ng |
Final volume (μl) | 10 | – |
6.
Vortex the master mixes for 1–2 s.
7.
Leave the DNA clean room.
8.
To optimize the workflow and facilitate the dispensation of DNA samples, it is useful to include 32 samples in each PCR run: 28 DNA samples to be tested, two positive reference DNA samples for positive controls, and two no-template negative controls.
9.
Due to differences in the PCR cycling conditions, the M2 PCR should be run separately from M1, M3, and M4 PCRs.
10.
In a single 96-well plate or tube rack, dispense M1, M3, and M4 master mixes: 9 μl of each master mix in each well/tube, for each sample to be tested, dedicating distinct columns to each kind of master mix. Using a multichannel pipette, add 1 μl of each DNA sample (1–50 ng) to each master mix, keeping row positions constant across columns.
11.
In a separate 96-well plate or tube rack, dispense 9 μl of M2 master mix in each well/tube for each sample to be tested. Using a multichannel pipette, add 1 μl of each DNA sample (1–50 ng) to the master mix, keeping row positions constant across columns.
12.
Keep the plates/racks on ice.
13.
Run the PCRs under the following thermocycling conditions: initial activation step at 95 °C for 5 min followed by 30 cycles (for M1, M3, and M4 PCRs) or 26 cycles (for M2 PCR) of denaturation (at 95 °C for 30 s), annealing (at 60 °C for 90 s) and extension (at 72 °C for 30 s) steps, and a final extension step at 60 °C for 45 min and 20 °C for 120 min. Keep the products at 4 °C after the amplification reactions.
3.2 Dilution of PCR Products
1.
Prepare as many U-bottom bulk tubes as the wells used in PCR reactions.
2.
Insert the tubes in their storage racks.
4.
For each sample, add 2 μl of the PCR product from each reaction (M1, M2, M3, and M4).
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