Computational protocol: Evidence of a Louse Borne Outbreak Involving Typhus in Douai, 1710 1712 during the War of Spanish Succession

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Protocol publication

[…] The Douai specimens were further examined with a suicide nested PCR protocol after conventional phenol chloroform DNA extract . The program PerlPrimer version 1.1.6 was used to design PCR primers. The first suicide nested-PCR targeted a 206-base pair fragment of the Rickettsia prowazekii DNA invertase Pin-like protein (ORF0698; gi|3861237|emb|AJ235273.1|) by combining external primers RpDet-F1 5′-GTTGGATATATAAGGGTTTC-3′ and RpDet-R2 5′-CCGAGTCTATCTAATTTCCA-3′ and internal primers RpDet-F3 5′-ATGATCGTCAAGTGTTCGAT-3′ and RpDet-R4 5′-TAGACAGTCGCCATCTTGTA-3′; the final expected PCR product was 152 bp (). This region had never been amplified in our laboratory. The first round PCR mix contained 1.6 µL MgCl2, 0.5 µL bovine serum albumin (BSA), 2 µL Master Mix (Light Cycle® FastStart DNA Master Sybr Green, Roche Applied Science, France), 1 µL of a 10 µM solution of RpDet-F1, 1 µL of a 10 µM solution of RpDet-R2, 8.9 µL sterile water and 5 µL DNA (experimental tube) or 5 µL sterile water (negative control tube). The first round of amplification was done using a LightCycler™ apparatus (Roche Diagnostics) and the following conditions: 10-min activation at 95°C followed by 45 cycles of 15-sec denaturation at 95°C, 20-sec hybridisation at 55°C and 25-sec elongation at 72°C. PCR products were recovered by centrifugation of capillaries at 380 g for 2 minutes. The nested PCR was performed in the capillary containing 1.6 µL MgCl2, 0.5 µL BSA, 2 µL Master Mix, 1 µL of 10 µM solution of RpDet-F3; 1 µL of 10 µM solution of RpDet-R4; 8.9 µL sterile water and 5 µL first round PCR product. The second amplification in the LightCycler™ apparatus used the following conditions: activation at 95°C for 10 minutes and 45 cycles of denaturation at 95°C for 15 seconds, hybridisation at 57°C for 20 seconds, elongation at 72°C for 25 seconds. Nested PCR products were detected on a 2% agarose gel (Invitrogen™, Paisley, Scotland) in the presence of molecular weight marker VI (Boehringer Mannheim, Germany).A second suicide nested-PCR targeted a 187-base pair fragment of the R. prowazekii glutamine amidotransferase-like protein (rpr_ORF0700; gi|3861237|emb|AJ235273.1|) by combining external primers Rpro-F1 5′-ACTGTTATTACCGATCTTGCCA-3′ and Rpro-R1 5′-TGGTTGATGCTAGGTTATTTGG-3′ and internal primers Rpro-F11 5′-GTATTAAGAATTTGATGCCACCA-3′and Rpro-R11 5′-GTTATTAGTCCAAATGACGTGAA-3′; the final expected PCR product was 130 bp (). This region had never been amplified in our laboratory. The first round of PCR was performed using a HotSartTaq DNA Polymerase Kit (Qiagen) with 0.8 µL MgCl2, 0.2 µL HotStart Taq, 2.5 µL 10X PCR buffer, 2.5 µL dNTP, 0.5 µL BSA, 0.5 µL of a 10 µM solution of each Rpro-F1 and Rpro-R1, 12.5 µL sterile water and 5 µL DNA (experimental tube) or 5 µL sterile water (negative control tube). The first round of amplification was done in an ABI GeneAmp™ 2700 thermocycler (Applied Biosystems, CA, USA) under the following conditions: 10-min activation at 95°C and 45 cycles of 30-sec denaturation at 95°C, 45-sec hybridisation at 58°C and 90-sec elongation at 72°C. The PCR products were purified using the Millipore plate protocol and suspended into 40 µL water. The second round of PCR was performed using 0.8 µL MgCl2, 0.2 µL HotStart Taq, 2.5 µL 10X PCR buffer, 2.5 µL dNTP, 0.5 µL BSA, 0.5 µL Rpro-F11, 0.5 µL Rpro-R11, 12.5 µL sterile water and 5 µL first round PCR product. The second amplification was performed using the following conditions: 10-min activation at 95°C and 45 cycles of 30-sec denaturation at 95°C, 45-sec hybridisation at 62°C and 90-sec elongation at 72°C. Nested PCR products were detected on a 2% agarose gel (Invitrogen™, Paisley, Scotland) in the presence molecular weight marker VI (Boehringer Mannheim, Germany).Nested PCR products were purified using the Millipore plate protocol and suspended in 50 µL water. The sequencing reaction was carried out in a tube containing 3 µL Big Dye Terminator, 0.5 µL forward or reverse primer, 3.5 µL purified PCR product and 3 µL sterile water using the following conditions: activation at 95°C for 5 minutes followed by 25 cycles consisting of 30-sec denaturation at 96°C, 20-sec hybridisation at 55°C, 4-min elongation at 60°C and 7-min extension at 15°C. The sequencing products were purified on Séphadex® G50 5% gel and analysed with the ABI PRISM 3100 Genetic Analyser (HITACHI). The sequences were read and corrected by using the software ChromasPro version 1.34 and then aligned with BLAST to compare the sequences available in GenBank. […]

Pipeline specifications

Software tools PerlPrimer, ChromasPro
Application qPCR
Organisms Homo sapiens, Rickettsia prowazekii, Bartonella quintana
Diseases Infection