Development of recombinant micobacteriophage for rapid detection of tubercle bacillus

Author(s)

Joás Lucas da Silva

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2011-11-28

Examining board members

Mario Hiroyuki Hirata; Elisabete Aparecida de Almeida; Rosilene Fressatti Cardoso; Sylvia Luisa Pincherle Cardoso Leão; Jorge Luiz Mello Sampaio

• Advisor: Mario Hiroyuki Hirata

Abstract

Classical biochemical methods for Mycobacterium tuberculosis identification are lengthy and time-consuming. On the other hand, molecular assays are rapid but expensive for developing countries. This project aimed to insert into the mycobacteriophage D29, the gene coding for the green fluorescent protein (eGFP) and use the recombineered phage to detect Mycobacterium tuberculosis rapidly and less costly. For that, the Hsp-eGFP cassette was inserted into D29 genome. Recombineered mycobacteriophages was purified and used to infect M. smegmatis mc2 155 and M. tuberculosis H37Rv from 1-6 hs at 30°C, 37°C and 42°C. Observation of fluorescent bacteria was difficult and only a small number of them were seen at 2 hs of infection. This indicated that recombineered bacteriophages were lysing cells rapidly. Deletion of LysA gene, was carried out to increase the time needed for bacterial lysing. it was not possible to purify mutant mycobacteriophages due to the low concentration of recombinant phages. We conclude that might be necessary the deletion of other genes such as LysB, a gene also involved in cell lysis and reduction LysA activity to increase the concentration of eGFP inside cells. (AU)

• FAPESP's process: 08/05082-7 - Construction and evaluation of mycobacteriophage D29 expressing green fluorecent protein to detect Mycobacterium tuberculosis in sputum
• Grantee: Joás Lucas da Silva
• Support Opportunities: Scholarships in Brazil - Doctorate