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This method has been developed by the pertussis reference laboratory of National Public Health Institute (KTL), Turku, Finland and the protocol described below was adapted from Mäkinen J, Mertsola J, Viljanen MK, Arvilommi H and He Q. Rapid typing of Bordetella pertussis pertussis toxin gene variants by LightCycler Real-time PCR and fluorescence resonance energy transfer hybridisation probe melting curve analysis. J Clin Microbiol 2002;40(6):2213-6.
Figure 1. Workflow for typing ptxS1 alleles. According to new nomenclature, ptxA stands for ptxS1, and ptxA1, 2, 3 and 4 stand for ptxS1A, B, D and E, respectively.
 Table 1. Primers and probes used
Melting curve analysis of FRET probes:
Differentiation of ptxA1 from ptxA2, 3 and 4 |
Position |
Length (bp) |
GC (%) |
Tm (°C) |
|
Primers |
BP S |
CCC gAA AAC ATC CgC Agg gTA A |
514-535 |
22 |
54.5 |
72.3 |
|
|
BP Rmt1 |
CCg ATA TCg ACC TTg CC |
703-686 |
17 |
58.8 |
74.0 |
|
Product |
|
190 |
|
66.4 b |
|
Probes |
QJ3BDE |
Cgg TgC CAT gCg CAC CAA TgT-F a |
690-670 |
21 |
61.9 |
|
|
|
QJ4BDE |
LC Red640-CCg ACg ATC gAC gCT ACg gAC CTT Cg-P a |
668-643 |
26 |
65.4 |
|
|
|
Melting curve analysis of FRET probes:
Differentiation of ptxA4 from ptxA2 and 3 |
Position |
Length (bp) |
GC (%) |
Tm (°C) |
|
Primers |
BP S |
CCC gAA AAC ATC CgC Agg gTA A |
514-535 |
22 |
54.5 |
72.3 |
|
|
BP Rmt2 |
gCg CCC ACC ACC ggT gC |
701-685 |
17 |
82.4 |
76.2 |
|
Product |
|
188 |
|
66.4 c |
|
Probes |
QJ5E |
CAC ggA gTA TCC CAA CgC TCg C-F a |
575-597 |
22 |
63.6 |
|
|
|
QJ6E |
LC Red705-CgT CAg CCA gCA gAC TCg CgC CA-P a |
600-622 |
23 |
69.6 |
|
|
|
|
|
|
|
|
|
a F = Fluorescein labelled, P = Phosphorylated; b Mean of Tm of PCR products from ptxA2, 3 and 4 (SD=0.45°C); c Mean of Tm of PCR product from ptxA4 (SD=0.17°C)
Table 2. PCR master mix
Melting curve analysis of FRET probes:
Differentiation of ptxA1 from ptxA2, 3 and 4 |
Volume (µl) |
Final conc. |
|
LightCycler-FastStart DNA Master Hybridization Probes a |
2 |
1× |
|
MgCl2 stock solution (25 mM) a |
2,4 |
3 mM |
|
Primer BP S (20 µM) |
0,4 |
0,4 µM |
|
Primer BP Rmt1 (20 µM) |
0,4 |
0,4 µM |
|
Probe QJ3BDE (20 µM) |
0,2 |
0,2 µM |
|
Probe QJ4BDE (20 µM) |
0,2 |
0,2 µM |
|
DMSO |
1 |
5 % |
|
H2O a |
11,4 |
- |
|
Total volume |
18 b |
|
|
|
Melting curve analysis of FRET probes:
Differentiation of ptxA4 from ptxA2 and 3 |
Volume (µl) |
Final conc. |
|
LightCycler-FastStart DNA Master Hybridization Probes a |
2 |
1× |
|
MgCl2 stock solution (25 mM) a |
2,4 |
3 mM |
|
Primer BP S (20 µM) |
0,4 |
0,4 µM |
|
Primer BP Rmt2 (20 µM) |
0,4 |
0,4 µM |
|
Probe QJ5E (20 µM) |
0,2 |
0,2 µM |
|
Probe QJ6E (20 µM) |
0,2 |
0,2 µM |
|
DMSO |
1 |
5 % |
|
H2O a |
11,4 |
- |
|
Total volume |
18 b |
|
a Included in LightCycler-FastStart DNA Master Hybridization Probes –kit, Roche Diagnostics GmbH, Mannheim, Germany, cat.no. 2239272
b A 2-µl volume of sample DNA (3 ng/µl) was added to 18 µl of the LightCycler master mix in the LightCycler glass capillaries. Heated bacteria can also be used as a sample.
Table 3. PCR reaction condition for the FRET probe assay to differentiate ptxA1 from ptxA2, 3 and 4
|
Denaturation |
|
Cycles |
1 |
|
|
|
Type |
None |
|
|
|
Target temperature (°C) |
95 |
|
|
|
Incubation time (s) |
600 |
|
|
|
Temp. transition rate (°C/s) |
20 |
|
|
|
Amplification |
|
Cycles |
40 |
|
|
|
Type |
Quantification |
|
|
|
|
Segment 1 |
Segment 2 |
Segment 3 |
|
Target temperature (°C) |
95 |
55 |
72 |
|
Incubation time (s) |
10 |
10 |
9 |
|
Temp. transition rate (°C/s) |
20 |
20 |
20 |
|
Acquisition mode |
None |
Single |
None |
|
Gains |
None |
F2/1 |
None |
|
Melting curve |
|
Cycles |
1 |
|
|
|
Type |
Melting curves |
|
|
|
|
Segment 1 |
Segment 2 |
Segment 3 |
|
Target temperature (°C) |
95 |
50 |
94 |
|
Incubation time (s) |
0 |
30 |
0 |
|
Temp. transition rate (°C/s) |
20 |
20 |
0,1 |
|
Acquisition mode |
None |
None |
Continuous |
|
Gains |
None |
None |
F2/1 |
|
Cooling |
|
Cycles |
1 |
|
|
|
Type |
None |
|
|
|
|
Segment 1 |
|
|
|
Target temperature (°C) |
40 |
|
|
|
Incubation time (s) |
30 |
|
|
|
Temp. transition rate (°C/s) |
20 |
|
|
Table 4. PCR reaction condition for the FRET probe assay to differentiate ptxA4 from ptxA2 and 3
|
Denaturation |
|
Cycles |
1 |
|
|
|
Type |
None |
|
|
|
Target temperature (°C) |
95 |
|
|
|
Incubation time (s) |
600 |
|
|
|
Temp. transition rate (°C/s) |
20 |
|
|
|
Amplification |
|
Cycles |
42 |
|
|
|
Type |
Quantification |
|
|
|
|
Segment 1 |
Segment 2 |
Segment 3 |
|
Target temperature (°C) |
95 |
57 |
72 |
|
Incubation time (s) |
10 |
10 |
9 |
|
Temp. transition rate (°C/s) |
20 |
20 |
20 |
|
Acquisition mode |
None |
Single |
None |
|
Gains |
None |
F3/1 |
None |
|
Melting curve |
|
Cycles |
1 |
|
|
|
Type |
Melting curves |
|
|
|
|
Segment 1 |
Segment 2 |
Segment 3 |
|
Target temperature (°C) |
95 |
50 |
94 |
|
Incubation time (s) |
0 |
35 |
0 |
|
Temp. transition rate (°C/s) |
20 |
20 |
0,1 |
|
Acquisition mode |
None |
None |
Continuous |
|
Gains |
None |
None |
F3/1 |
|
Cooling |
|
Cycles |
1 |
|
|
|
Type |
None |
|
|
|
|
Segment 1 |
|
|
|
Target temperature (°C) |
40 |
|
|
|
Incubation time (s) |
30 |
|
|
|
Temp. transition rate (°C/s) |
20 |
|
|
Figure 2. Melting curves of different ptxA alleles and negative control from the FRET probe assays to differentiate ptxA1 from ptxA2, 3 and 4 (A) and to differentiate ptxA4 from ptxA2 and 3 (B). According to new nomenclature, ptxA stands for ptxS1, and ptxA1, 2, 3 and 4 stand for ptxS1A, B, D and E, respectively.
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