Title: Current laboratory techniques in infectious diseases: Molecular detection and cloning of pathogens and Parasitology
Keywords: Tropical medicine
Technology
Pathogens
Laboratory science (incl.. quality assurance)
Health infrastructure
Country: Germany
Institution: Germany - Center for International Health at the Ludwig-Maximilian-Universität München
Course coordinator: Martina Manhart
Arlett Heiber
Date start: 2020-01-27
Date end: 2020-01-31
About duration and dates: Preparatory pre-reading upon admission to the course (2020-01-20 – 2020-01-24) 1 week face-to-face module (2020-01-27 – 2020-01-31) Assignment submission 2 weeks after the course (2020-02-01 – 2020-02-14)
Classification: advanced optional
Mode of delivery: Face to face
Course location:
Part 1 - 2: Department for Infectious Diseases and Tropical Medicine, Leopoldstr. 5, 80802 Munich, Germany
Part 3: Institute of Medical Microbiology, Immunology and Hygiene, Trogerstr. 30, 81675 Munich, Germany
ECTS credit points: 3 ECTS credits
SIT:
90 Hours SIT

Contact hours:
Part 1: Polymerase Chain Reaction (PCR) 9 hours
Part 2: Molecular Cloning Techniques 26.5 hours
Part 3: Parasite lifecycle based immunological and diagnostic methods (Schistosomiasis)9 hours
Assesment 0.5

Self-Study:
Pre-Reading Assignment 15 hours
Part 2: Molecular Cloning Techniques 4 hours
Part 3: Parasite lifecycle based immunological and diagnostic methods (Schistosomiasis)2 hours
Post-course Protocol 24 hours
*For details, please see “Methods”.
Language: English
Description:
At the end of the course, student will be able to:
• design laboratory settings for their own research projects
• apply the basics of molecular cloning as a specific breakout-technique of molecular diagnosis
• apply the value of basic microscopical and immunological research methods in an experimental animal model
• apply, analyze and evaluate up-to-date laboratory methods and techniques in the field of direct diagnosis of infectious diseases, such as nucleic acid extractions, conventional PCR, quantitative real-time PCR, molecular cloning, nucleic acid sequence analysis, and parasite isolation in the example of Schistosoma mansoni
• appraise requirements for working in a S2-laboratory
Assessment Procedures:
Students will be assessed daily by the facilitators for their active participation on a pass/ fail basis. Day 5 will be assessed exclusively in this manner.

Day 4: Written theoretical examination (multiple choice questions, 30 minutes) in the application and evaluation of molecular methods in diagnosis of tropical infectious diseases (PCR and cloning). Grading: A - E, F.

Post-course assignment: Students will be asked to write up a post-course protocol which also requires students to actively engage in tasks set by the facilitators. Grading: A – E, F.

The final grade will be calculated as an equally weighted arithmetic mean of the two graded assessment parts.

In case of failure of the written theoretical examination, students will be subjected to an audio-visual oral online examination.
In case of failure of the post-course assignment, a revised protocol can be resubmitted within a time frame defined by the facilitator.
The maximum grade that can be awarded for resubmitted assignments can be the pass mark Grade E.
Content:
Polymerase Chain Reaction

• Students will learn the basics of molecular biology and the basic concepts of different PCR formats and techniques as well as the know how to prepare, develop and interpret a qPCR. Students will conduct a qPCR for the diagnosis of Schistosoma Mansoni infection.

Molecular Cloning Techniques

Learning and applying molecular cloning techniques. A target gene, encoding for a highly variable part of the HIV envelope protein, will be amplified and cloned into a standard plasmid vector. Competent E. coli bacteria will then be transformed with the newly generated transgene carrying plasmid. After culturing the bacteria in selective media, plasmids will be isolated and analyzed for the presence of the transgene and sent for sequencing. Sequences will be subjected to phylogenetic analysesin the context of reference strains for different HIV subtypes.
These techniques can for example be used to generate standards for Real Time PCR based quantification of pathogens.

Parasitology lifecycle based methods (Schistosomiasis)

• Familiarization of students through pre-course assignment with the etiology, clinical presentation and particularly the life cycle of schistosomiasis.

• On-campus familiarization with diagnostic parasitology laboratory facilities. Discussion of ethical aspects of animal models for basic immunological research questions in the field of host-parasite interaction (schistosome mouse model). Presentation of the different life cycle steps of schistosomiasis in two hosts (mouse and molluscs). Perform methods for qualitative and quantitative analysis of schistosome infestation. Demonstration of methods to assess the hosts immune responses at given infection timepoints.
Methods:
Pre-reading: 2 weeks prior to the course, pre-reading materials will be disseminated

Day 1: PCR I, all-day
1) 30 minutes laboratory safety instructions
2) 90 minutes lecture to introduce the basics of PCR
3) 90 minutes practical exercise (laboratory introduction and practice)
4) 2 hours practical exercise (qPCR)1 hour lecture to introduce PCR and LAMP techniques
5) 1 hour lecture to introduce diagnostic qPCR development and analysis
6) 1 hour practical exercise (Analysis, presentation and discussion of results)

Day 2: Molecular Cloning techniques I, all day

1) 30 minutes introduction of course contents and schedule
2) 1.5 hours lecture: Basic techniques in molecular cloning
3) 2 hours practical bench work (without incubation times):
• Amplification of target gene using PCR
• Analyses of PCR amplicons on agarose gels and DNA extraction
• Ligation of amplicon and plasmid vector (TopoTA cloning)
• Transformation of competent E. coli bacteria
• Plating out bacteria on ampicillin agar plates

4) 15 minutes group discussion and first day summary

Day 3: Molecular Cloning techniques II, all-day

1) 1 hour lecture on molecular cloning for vaccine development
2) 5 hours practical bench work: Picking and growing transformed bacteria in selective media. Plasmid extraction and preparation for Sanger sequencing.
3) 2 hours lecture; restriction enzymes; expression of recombinant proteins; selection of resistant bacteria; eukaryotic expression systems
4) 15 minutes group discussion and second day summary

Day 4: Molecular Cloning techniques III, all-day

1) 2 hours of practical bench work:

• molecular analyses of extracted plasmids for transgene insert using restriction enzyme digestion and via PCR analysis
1) 5 hours analysis of sequences (sequence alignment, phylogenetic analysis)
5) 30 minutes group discussion and final summary
6) 30 minutes multiple choice exam on contents of seminar days 1-4

Day 5: Parasitology lifecycle based methods (Schistosomiasis), all-day

1) 1 hour lecture on key aspects of schistosomiasis and schistosome life cycles
2) 30 minutes introduction of participants into laboratory facilities
3) 30 minutes introduction of participants into the different life cycle models
4) 5 hours of bench work with differentiation of schistosomes in its different developmental forms

5) 1 hour tutored microscopy of prepared histological samples in liver tissue for immunological granuloma assessment
Prerequisites:
• Basic laboratory skills
• Intermediate level training in infectious diseases;
• Proof of English fluency: tropEd students from an accredited tropEd home institution that have passed a core course in English language will be considered sufficiently fluent in English language. Also students that can provide proof of academic education passed entirely in English language will be considered sufficiently fluent. Applicants not being able to provide either of these criteria will be asked to proof fluency by a TOEFL (iBT score >= 79) or IELTS (score >=6.0)
Attendance:
Max. number of students 8
Up to 5 tropEd Master’s students are admitted
Selection:
Students are selected on a first-come, first-serve basis
Fees:
600 Euro.
Scholarships:
None
Major changes since initial accreditation:
Major changes since initial accreditation:
1 ECTS = 30 SIT added
Currently accredited with 2 ECTS; submitted for re-accreditation during tropEd GA in London (Sep 2019)
Student evaluation:
Students highly commend the course for the practical approach and laboratory work, but found the course title did not advertise this adequately.
Lessons learned:
Course title and some contents of the course were adapted to further improve the quality of the course.
tropEd accreditation:
Accredited in October 2015; reaccredited September 2019 in London. This course is valid until September 2024.
Remarks:
Upon completion of the one-week on-campus course, all participants are required to deliver a brief report on their personal reception and retained contents of the course, to be submitted within 14 days after the last on-campus course day. Reporting templates will be provided by CIH Office. The reports are mandatory but will not be graded.
Email Address: ttu@lrz.uni-muenchen.de
Date Of Record Creation: 2015-10-10 23:40:52 (W3C-DTF)
Date Of Record Release: 2015-10-11 03:52:14 (W3C-DTF)
Date Record Checked: 2017-10-24 (W3C-DTF)
Date Last Modified: 2020-11-23 11:28:49 (W3C-DTF)

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