009.5 Isolation and amplification of treponemal dna for whole genome sequencing directly from the patient sample

Abstract

Introduction Syphilis, caused by Treponema pallidum ssp. pallidum (TPA), is a sexually transmitted multistage disease. Over 10 million new infections worldwide are reported every year. To date, complete genome sequences of 6 TPA strains (all passed through rabbits) have been determined. Here we present the preparation of human clinical sample for NGS without the need of TPA multiplification in rabbits. Methods The primary chancre swab was received from Department of Dermatovenereology, St. Anne’s Faculty Hospital in Brno, Czech Republic. Whole genome amplification (WGA) was carried out by multiple displacement amplification (MDA) with phi 29 polymerase after specific separation of TPA on the cell level from the human cells. Nested PCR for pol A for detection of number of TPA DNA copies was performed. Results MDA was not successful before separation of TPA from human cells through the inhibition of TPA amplification. Experimental addition of human DNA (3 ng) to the TPA DNA (10 ng) decreased the TPA amplification over 100 times. Therefore we apply MDA method after specific separation TPA on the cell level. Through this procedure we were able to prepare treponemal DNA (in concentration 1 ng/μl) for NGS isolated directly from the patient without the need of TPA propagation in rabbits. Conclusion Since all yet available whole genome sequences of TPA comes from bacteria multiplificated in rabbits, sequencing of syphilis genomic DNA isolated directly from the patient is required. Here we report, for the first time, the procedure for preparation of TPA DNA for NGS. No conflicts of interest.