Detailed laboratory protocol for rapid identification of Bordetella pertussis pertactin gene variants using LightCycler Real-time PCR combined with melting curve analysis and gel electrophoresis

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, Viljanen MK, Mertsola J, Arvilommi H and He Q. Rapid identification of Bordetella pertussis pertactin gene variants using LightCycler Real-time polymerase chain reaction combined with melting curve analysis and gel electrophoresis. Emerg Infect Dis 2001;7(6):952-8. Figure 1. Workflow for typing Prn alleles Table 1. Primers and probes used a Position numbers indicate the position of bases relative to the first start codon of prn1. b Mean of Tm of PCR products from prn1-5 type of strains (SD=0.54°C). No specific amplification of strains with prn6-8. c Length of PCR product 260, 275, 260, 245, and 245 bp for prn1, prn2, prn3, prn4, and prn5. d Mean of Tm 58.78°C (SD=0.26°C), 56.31°C (SD=0.44°C), 56.59°C (SD=0.09°C), and 56.57°C (SD=0.29°C) for prn1, prn2, prn3, and prn4, respectively. No specific binding with prn5. e F = Fluorescein labelled, P = Phosphorylated Table 2. PCR master mix a Included in LightCycler-FastStart DNA Master SYBR Green I -kit, Roche Diagnostics GmbH, Mannheim, Germany, cat.no. 2239264 b Included in LightCycler-FastStart DNA Master Hybridization Probes -kit, Roche Diagnostics GmbH, Mannheim, Germany, cat.no. 2239272 c 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 allele-specific amplification Figure 2. Melting curves from the allele-specific amplification, showing the presence of amplified products from prn1-5 and the absence of amplification from prn6-8 (prn6 represents prn6-8) and negative control without template DNA. Table 4. PCR reaction condition for melting curve analysis of FRET probes Figure 3. Melting curves of prn1-5 alleles and negative control without template DNA from FRET probe assay. Figure 4. Gel electrophoresis. Products from FRET probe assay run in 2 % agarose gel, 120 V for 2 hours, and stained with EtBr. Lane 1, 50 bp DNA ladder; lane 2, prn2 strain (275 bp); lane 3, prn3 strain (260 bp); lane 4, prn4 strain (245 bp) and lane 5, negative control without template DNA.