Muaz Khalil - MSPH Technical Report Final Defense - April 28, 2006
| April 26, 2006 | |
| Muaz Khalil presents his MSPH Technical Report Final Defense on Friday, April 28th at 9:00AM in 2301 McGavran-Greenberg. Full details follow.
Genomic Sequencing of F+RNA as a Foundation for Development of Real-Time RT-PCR Assays Male-specific RNA (F+RNA) coliphages, members of the family Leviviridae, have been studied for their potential as indicators of enteric virus pollution in environmental water samples. In addition, subtyping of F+RNA coliphages may provide information on the source of contamination as different genetic groups have been associated with either human or animal fecal pollution. To identify conserved regions across the genome of each individual genogroup for the development of genogroup-specific real-time RT-PCR assays, we compiled a panel of genetically, temporally and geographically different strains including strains belonging to genogroup I, II, III and IV as well as strains from a recently published novel subgroup (called JS). All strains were first plaque-purified and enriched and viral RNA was purified. Of 7 strains (2 gp III, 2 gp IV and 3 JS) coliphage viral RNA was 3′-end poly-adenylated using RNA polymerase and cDNA was generated using an oligo-dT oligonucleotide primer. Using partial replicase sequences of each strain for the design of strain-specific oligonucleotide primers, we generated a ~1 kbp PCR fragment encompassing the 3′-end of the viral genome. An attempt was made to amplify the 2.4 kbp fragments (3.1 kbp for alloleviviruses) of the 5′-end of each strain by first preparing cDNA followed by poly-A tail addition at the 3′-end using terminal transferase. Phylogenetic analysis of the ~1 kbp F+RNA field strains, including the prototype strains, confirmed the genetic grouping of F+RNA into 5 distinct genogroups and possibly two different subgroups into genogroup III. This study is the first attempt to generate a comprehensive genomic sequence database of F+RNA coliphages. This sequence database will serve as a framework to study the ecology of F+RNA coliphages as well as provide a rationale to design robust real-time RT-PCR assays for the sensitive detection and differentiation of F+RNA subgroups in environmental water samples. Committee Members: Dr. Jan Vinji, Advisor For further information please contact Rebecca Riggsbee Lloyd by email at Rebecca_Lloyd@unc.edu |
