What can be done to improve the sensitivity and specificity of the tests, aside from using proper controls?
What primers are best for Toxoplasma detection?
Real-time PCR?
Analyse cDNA to confirm the presence of living Toxoplasma - is this a viable idea?
Is the detection of antibodies considered mandatory to prove Toxoplasma infection, or will PCR results suffice if done properly? Or are there some problems that I don't know about?
Will be most grateful for any ideas on the topic.
Hey,
I think improving specificity might be possible by sequencing PCR products you get.
If you are going to do such diagnostical PCRs on a daily basis be sure to always have negative controls and follow some simple lab rules such as never open the PCR tube after running the reaction in the same room as you set up the reactions, use a different lab coat for mixing the reaction and running a gel, etc...
Well these answers I actually know. Sequencing is probably not an option because in this particular case because it will be very expensive to sequences a few thousand samples or so (that is the hypothetical task in question at the moment). But would you say that PCR evidence is enough if it is done with negative controls?
I can't answer with 100% confidence about Toxoplasma, but for many infectious diseases PCR is the main diagnostic method. Though often for positive cases people tend to validate the result with a different method, such as ELISA. If you are going to have a huge screening it might be worth to compare the results of PCR and ELISA for some number of samples, and if they are consistent, just go for PCR.
And I didn't say anything about real-time PCR, but it actually could be useful if using TaqMan - it will increase specificity and in case of contamination you can often understand what is going on: the cycles when you get the signal are usually much higher. And at the same time after the validation of the primer set and a probe you don't need to run a gel for, so there is less chance of contamination (and you get your answer sooner).
Hey, its all depend what is your target. For diagnostic purposes ELISA is best but some time it gives false positive results. I always prefer PCR specially nested-PCR for toxoplasma, followed by RFLP that gives fast and accurate results to identified T.gondii infection and determine the toxo genotypes
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