Isothermal Amplification and Nanopore Sequencing as Rapid Methods for Outbreak Identification and Disease Control
Dr. Ahmed Abd El Wahed
University of Goettingen, Germany
Guest Professor at the Faculty of Veterinary Medicine, Cairo University
and the German Primate Center
The lecture discussed the use of innovative techniques like recombinase polymerase amplification assay (RPA) and nanopore sequencing to bring the diagnostic laboratory to the field. A mobile suitcase laboratory was designed and assembled for rapid, sensitive and simple detection of disease-causing pathogens in point-of-care settings. This laboratory includes a suitcase or glove-box for sample preparation and nucleic acid extraction, and a mobile suitcase for reverse transcription, amplification and detection using the RPA assay. The mobile laboratory was used for detection of Ebola virus in 928 oral swab samples in a local hospital in Matoto, Guinea. The assay has proved high sensitivity and specificity compared to real-time RT-PCR in just 30 min (15 min for RNA extraction and 15 min for RPA). The mobile laboratory was also used for identification of animal species in meat products as a tool to monitor meat adulteration. A rapid on-site detection system was developed for DNA of 6 animal species including cattle, sheep, pig, horse, chicken and turkey. The system included both monoplex and multiplex assays. Another portable system for point-of-need applications is nanopore sequencing using MinION. This system was used for serotyping of foot and mouth disease virus and differentiation of capripox viruses. The laboratory workflow of this system lasts only for 2 hours and 5 min and the entire time for sequencing does not exceed 5 hours. A bioinformatic background is not required due to the user-friendly interface of GENEIOUS as all steps of data processing are easy to execute and results are presented in a clear way as query-centric alignment. The lecture also focused on the lessons learnt from field application of these portable assays and units, which included: 1) logistics, 2) preparation of simple protocols, 3) availability of all required materials, 4) to have plan B, 5) acknowledging the people, 6) training the locals, 7) starting with 100% confident experiments, 8) avoiding much photos, 9) understanding the culture, 10) thinking globally but acting locally.