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Mycobacterium Detector

From our policy and practice work during the summer we learned of the growing need for a detector to quickly analyse whether or not a patient was infected with a mycobacterium like M.Tuberculosis which is still a major cause of death in many third world countries. TB is second to aids/HIV as the most deadly disease caused by an infectious pathogen. We have known of the existence of the pathogen behind TB since the beginning of the 20th century , however to this day every person with active TB will go on to infect 10 to 15 more people ( World health organisation 2010 ). Mycobacterial culture on solid Lowenstein-Jensen (LJ) medium is considered the gold standard isolation method (Conde et al 2009). Although the limitation of this method is the long incubation period (2-8 weeks), it is used by most developing countries because of its low cost. Techniques such as nucleic acid amplification and automated liquid culture systems are costly and depend on sophisticated tools, which prevents their routine use in poor countries. TB is diagnosed currently using acid fast stains which is based on the binding of mycobacterium like M.Tuberculosis to fuchsin which is very selective. Our detector serves as a novel way to amplify DNA in ecoli and the results of the amplification are evident in the number of colonies that form on the plate.In saying that statistics show that over 37 million lives are saved every year due to effective diagnosis. We constructed a detector which would act as a negative screening device for TB. We decided to this based on the reported growing need which we identified in our policies and practices throughout the summer. However proper treatment is available and we decided to construct our detector to allow for a cheap diagnosis of the M. Tuberculosis pathogen.

Construction & Detection of Mycobacterium Detector

Mycobacterium are unique among bacteria in that they possess several unique proteins involved in fatty acid synthesis and metabolism. By identifying such proteins which are unique to mycobacterium tuberculosis (Beile Gao et al 2006), we were able to obtain the gene sequence for the protein and establish a detector target from this sequence. One protein which contained a 24 base pair target flanked by Sma1 restrictions sites was identified, ML0319 is a lipoprotein uniquely found in Mycobacteria and also present in Norcardia. The detector construct designed to detect this target is shown in the figure below. The nucleotide base sequence for this gene has already been sequence and we hoped to be able to diagnose TB using our detector. Due to the high GC nature of the 24 base pair target sequence, we identified that BamHI and KpnI would be the restriction sites to be used in the actual detector. The proposed target nucleotide was checked to see if it would have a likely chance to form hairpin structures or if we would experience any problems with annealing or dimerization. Even though the target has a high GC content ( 68 %), it will likely to not form hairpin structures spontaneously.

Mycobacterium detector sequence indicating the biobrick prefix and suffix, the left and right target sequences and the nicking and restriction enzyme cut sites used to generate the “activated” detector. The oligonucleotides used for cloing the detector between the SpeI and PstI sites of the prefix and suffix are indicated as are the primers that can be used to generate and activated detector by the PCR amplification method

Results

The TB detector showed promise as a diagnostic tool as a negative screen. The detector was tested using a complementary top strand oligonucleotide ordered from IDT. [1]

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