(http://www.nih.gov/researchmatters/march2009/03092009tuberculosis.htm ) (http://www.biochem.wisc.edu/faculty/rayment/lab/gallery.aspx). Luciferase Reporter Mycobacteriophage (LRP) Assays for Diagnosis of Antibiotic Resistant Mycobacterium tuberculosis. Clare Bruggeman.
(http://www.biochem.wisc.edu/faculty/rayment/lab/gallery.aspx)Luciferase Reporter Mycobacteriophage (LRP) Assays for Diagnosis of Antibiotic Resistant Mycobacterium tuberculosis
Primary Infection in the Lungs
TB in Other Organs
Fails to identify 30-50% of active cases
BACTEC and MGIT
1. Find a mycobacteriophage that infects M. tuberculosis
2. Genetically engineer the mycobacteriophage to contain luciferase.
3. Infect the cultured sputum sample with the phage.
4. If viable mycobacteria are present, light is produced in the presence of D-luciferin.
5. If the light does not diminish when the mycobacteria are treated with an antibiotic, then the mycobacteria are resistant.
6. Measure Relative Light Units (RLU) with a luminometer.
Banaiee et al. (2001)
Schematic of phAE39 and phAE40 DNA. Constructed in the E. coli cosmid pYUB216, then inserted into TM4 mycobacteriophage.
ColE1 = ORI Ap = Amp (selectable marker for E. coli).
Phsp60 = promoter Fflux = firefly luciferase (Jacobs 1993)
NTM = Nontuberculosis mycobacterium
MTC = M. tuberculosis complex
Only really changing the detection method and maybe the culture media.
“The LRP assay appears to be the most consistently accurate test” (Minion and Pai, 2010).
But the method is still slow, requires non-contaminated samples, and uses up antibiotics during susceptibility tests.
Minion, J., and M. Pai. 2010. “Bacteriophage assays for rifampicin resistance detection in Mycobacterium tuberculosis: updated meta-analysis.” Int J Tuberc Lung Dis14 (8): 941-951.
WHO.int. 2010. The World Health Organization. 29 October 2010. <http://www.who.int/en >.
CDC.gov. 2010. The Center for Disease Control and Prevention. 29 October 2010. <http://www.cdc.gov >.
“Hypersensitivity.” 2010. Practical Pathology Class Website. Department of Pathology, University of Cambridge. 29 October 2010. <http://www.path.cam.ac.uk/partIB_pract/P09/ >.
Kumar, V., P. Loganathan, G. Sivaramakrishnan, J. Kriakov, A. Dusthakeer, B. Subramanyam, J. Chan, W. Jacobs Jr. and N. Paranji Rama. 2008. “Characterization of temperate phage Che 12 and construction of a new tool for diagnosis of tuberculosis.” Tuberculosis88, 616-623.
Banaiee, N., M. Bobadilla-del-Valle, S. Bardarov Jr., P. F. Riska, P. M. Small, A. Ponce-de-Leon, W. Jacobs Jr., G. F Hatfull and J. Sifuentes-Osornio. 2001. “Luciferase Reporter Mycobacteriophages for Detection, Identification, and Antibiotic Susceptibility Testing of Mycobacterium tuberculosis in Mexico.” J ClinMicrobiol39 (11), 3883-3888.
Gali, N. J. Dominquez, S. Blanco, C. Prat, F. Alcaide, P. Coll, V. Ausina and the Mycobacteria Research Group of Barcelona. 2006. “Use of a mycobacteriophage-based assay for rapid assessment of susceptibilities of Mycobacterium tubersulosis isolates to isoniazid and influence of resistance level on assay performance.” J ClinMicrobiol44(1) 201-205.
Jacobs, W. R. Jr, R. G. Barletta, R. Udani, J. Chan, G. Kalkut, G. Sosne, T. Kieser, G. Sarkis, G. Hatfull, B. Bloom. 1993. “Rapid Assessment of Drug Susceptibilites of Mycobacterium tuberculosis by Means of Luciferase Reporter Gene.” Science, New Series 260 (5109) 819-22.