Difference between revisions of "Team:Birkbeck/Discussion"
Srcraig1987 (Talk | contribs) |
Srcraig1987 (Talk | contribs) |
||
| Line 9: | Line 9: | ||
<!--<h3><b><u>Signal Detection</u></b></h3> | <!--<h3><b><u>Signal Detection</u></b></h3> | ||
<p><b><u></u></b></p> | <p><b><u></u></b></p> | ||
| − | |||
| − | <p> | + | where is this going? |
| + | <p>30 cfu/mL of sputum was the detection limit when targeting the 16s rRNA of <i>Mycobacterium</i> by TCR-2 (Drouillon <i>et al</i>., 2009 [they did not show this data]). | ||
| + | This is clearly a low detection limit. Considering the components of sputum & localisation of cells, it is unclear the efficiency of transduction our product will have on the samples. <a href="https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/TBPrepKit.htm">Kits</a> are available for decontamination of sputum samples and degrading components of the sputum. Adding this step prior to exposure of the recombinant phage to samples may be a means of improving transduction efficiency and therefore reducing the detection limit. However, some bacteria (such as <i>Staphylococcus aureus</i>) are resistant to the decontamination kits and therefore appropriate controls need to be set up. a key point to note here is that the decontamination step must not be carried out for <em>longer than 15 minutes</em> due to the reduction in cell viability of <i>Mycobacterium</i> cell (refer to <a href="https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/TBPrepKit.htm">the kit</a>).</p> | ||
| − | <p>Considering culture time at 20 minutes, there is a significant signal for P1-<i>gfp</i> (P=0.002) with no difference between the OD<sub>600</sub> growth curves at this point (P=1). This is significantly less than the hours taken for results to be generated by TCR-2 (Drouillon <i>et al</i>., 2009). Cells cannot be quantified at the 20 minutes time point as no viable count was carried out, therefore the detection limit of the <i>E. coli</i> DH5α with regards to fluorescence cannot be determined.</p> | + | <p>Considering our results, at time 0 minutes, there is a significant signal for P1-<i>gfp</i> expression device compared to the <i>E. coli</i> DH5α (P=0.005, with reference to <b>Fig. 9</b> in results section). This result cannot be regarded as this is probably carry over fluorescence from sub-culturing. Photo-bleaching prior to the first reading may yield more accurate readings when considering the expression of <i>gfp</i> from promoters. Also the error bars in each of the expression devices do appear excessive. A means of reducing this may be to sub-culture the cells & grow to an OD<sub>600</sub> of 0.2 and sub-culturing into experimental cultures in order to minimise the error bars.</p> |
| + | |||
| + | <br> | ||
| + | |||
| + | <p>Considering culture time at 20 minutes, there is a significant signal for P1-<i>gfp</i> (P=0.002) with no difference between the OD<sub>600</sub> growth curves at this point (P=1). This is significantly less than the hours taken for results to be generated by TCR-2 (Drouillon <i>et al</i>., 2009). Cells cannot be quantified at the 20 minutes time point as no viable count was carried out, therefore the detection limit of the <i>E. coli</i> DH5α with regards to fluorescence cannot be determined. With regards to <a href="https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/TBPrepKit.htm">Decontamination Kits</a>, samples would take just under one hour to process. As transduction was not characterised in this study, it is difficult to assess exactly how long this step may take.</p> | ||
| + | |||
| + | <p>Considering the application of D29 <i>Mycobacteriophage</i> could be spotted directly onto soft agar plates giving rise to plaques on both <i>M. smegmatis</i> & <i>M. tuberculosis</i> (Sampson <i>et al</i>., 2009). A <i>Mycobacteriophage</i> assay for detection of live <i>Mycobacterium</i> cells has previously been carried out using a plaque assay (Alcaide <i>et al</i>., 2003). A 1 mL decontaminated sample was washed and grown overnight in the standard <i>Mycobacterium</i> media <b>Middlebrook 7H9</b> supplemented with 10% (vol/vol) oleic acid-albumin-dextrose catalase (OADC) (Alcaide <i>et al</i>., 2003). 100 μL of <i>Mycobacteriophage</i> was added to the overnight cultures and incubated at 37<sup>o</sup>C for 1 hour before adding 100 μL of viricidal solution (Alcaide <i>et al</i>., 2003). A soft agar lawn was poured and incubated for 24 hours before counting plaques.</p> | ||
| + | |||
| + | <p>Considering the presorption phase of the assay developed by Alcaide <i>et al</i>, this would add an hour onto the protocol. Given that transduction is 100% efficient (unlikely) and that the kinetics of <i>gfp</i> expression behaves the same in <i>E. coli</i> DH5α, we are looking at from sputum sample collection until signal detection at approximately 2 hours & 30 minutes.</p> | ||
| − | <p>Considering our results, at 1 hour of culturing <i>E. coli</i> DH5α, there is a significant signal observed from the culture expressing the P1-<i>gfp</i> expression device (P<0.001). Considering the growth, there was no significant difference between the OD<sub>600</sub> of the P1-gfp expression device & the E. coli without plasmid (P=1 for both 50 mL cultures & 200 μL, with reference to <b>Fig. 1</b> & <b>Fig. 5</b> respectively). At the 1 hour time point, there is a significant difference between the 50 mL & 200 μL cultures (P=0.017) with the 200 μL cultures showing a higher OD<sub>600</sub>. This entails that the fluorescence of the 200 μL cultures at 1 hour corresponds to a higher culture density than 2.57×10<sup>6</sup> cfu/mL. Considering the TCR-2 method of detection, 2.57×10<sup>6</sup> cfu/mL is an increase in detection limit by a magnitude of 10^5 (30 cfu of | + | <br> |
| + | |||
| + | <p>Considering our results, at 1 hour of culturing <i>E. coli</i> DH5α, there is a significant signal observed from the culture expressing the P1-<i>gfp</i> expression device (P<0.001). Considering the growth, there was no significant difference between the OD<sub>600</sub> of the P1-<i>gfp</i> expression device & the <i>E. coli</i> without plasmid (P=1 for both 50 mL cultures & 200 μL, with reference to <b>Fig. 1</b> & <b>Fig. 5</b> respectively). At the 1 hour time point, there is a significant difference between the 50 mL & 200 μL cultures (P=0.017) with the 200 μL cultures showing a higher OD<sub>600</sub>. This entails that the fluorescence of the 200 μL cultures at 1 hour corresponds to a higher culture density than 2.57×10<sup>6</sup> cfu/mL.</p> | ||
| + | <p>Considering the TCR-2 method of detection, 2.57×10<sup>6</sup> cfu/mL is an increase in detection limit by a magnitude of 10^5 (30 cfu of <i>M. tuberculosis</i>/mL of sputum [Drouillon <i>et al</i>., 2009]) which is clearly an undesirable due to the slow growth of <i>M. tuberculosis</i>. The detection of a signal at the 20 minute time point </p> | ||
<p>As transduction was not characterised in this study, it is difficult to assess the potential limitations of this product might be.</p>--> | <p>As transduction was not characterised in this study, it is difficult to assess the potential limitations of this product might be.</p>--> | ||
Revision as of 17:27, 16 September 2015
Discussion
Under construction
Sean Ross Craig (data analysis, cloning, restriction diagnostics, measurements & uploading content to the wiki), Elliott Parris (measurements & restriction diagnostics), Rachel Wellman (restriction diagnostics & measurements) & Ariana Mirzarafie-Ahi (cloning).
With thanks to Dr. Vitor Pinheiro, Dr. Jane Nicklin, Bilkis Kazi, Barbara "Babz" Steijl, Luba "Aunty" Prout.