CRISPR locus sequencing for Mycobacterium detection and differentiation

• Main Author: Zhen, Yashu
• Other Authors: Yap Peng Huat Eric
• Format: Final Year Project
• Language: English
• Published: 2016
• Subjects: DRNTU::Science::Biological sciences::Genetics
• Online Access: http://hdl.handle.net/10356/68041
• Institution: Nanyang Technological University

Tuberculosis has been a severe infectious disease all over the world. It is mainly transmitted by air, but also possible by food as tuberculosis not only occurs on humans, but also on other organisms, such as bovine. Mycobacterium tuberculosis Complex (MTBC) refers to a group of Mycobacterium species that sharing same ancestor and all can cause tuberculosis. As each MTBC specie also has lots of different strains, which related to drug-resistance and outbreak prevention, it is important to keep detecting and differentiating them clearly. Current method is spoligotyping, which uses primers to target the direct repeats on the CRISPR locus, amplifying the spacers which are detected by hybridizing with prepared spacer probes. However, as there is already long range sequencing method developed, it is possible to sequencing the entire CRISPR locus and get spacers data directly. Therefore, this project is focusing on designing primer to amplifying the whole CRISPR region and then sequencing the amplified products to detect and differentiate MTBC. Finally, Minion sequencing results show this method can detect MTBC; and Sanger sequencing results prove strain differentiation can also be achieved.