Talk:Risk of Missed PRRS PCR Detection

Untitled

Under Hazard to detection, did you mean for the indentation to occur that way or was that by accident? -Vic 570vca (talk) 17:45, 3 November 2011 (UTC)

I went ahead and removed the bullet points and added arrows and spaces instead to try to make it a bit more readable. However, I'm not sure I'm happy with how it turned out. Since you said you wanted a flow chart type effect, and since there appears to be only one line of succession in the "flowchart", would just a numbered list would be better?? Let me know what you think. -Vic 570vca (talk) 13:06, 4 November 2011 (UTC)

Maybe a little info on the implications of PRRS would be helpful in the opening paragraph. Also, I really liked your pathway format. Do you mind if I borrow your idea. - Dan (570ddt (talk) 01:30, 5 November 2011 (UTC))

Cleaned up the references and some minor spelling errors. Added a sentence to clarify what a false-negative result is in the context of this article. -Vic 570vca (talk) 18:26, 6 November 2011 (UTC)

Hi Wayne! This is nicely written. One item I would recommend: In the discussion of virus mutation and evolution, I would add a few sentences about the role of genetic recombination in increasing diversity. This appears to be a major contributor to the speed of evolution and there are plausible mechanisms and computational models. I do understand why you are choosing to focus on single nucleotide substitution since this may be more difficult to detect. Cheers! 570mpp (talk) 17:45, 7 November 2011 (UTC)

As someone ignorant in this area I have a couple questions/suggestions to make the topic more approachable - it would be nice if the first paragraph had better defined problem formulation. Also could you include a sentence or link on how the virus is spread among pigs? The background on types of PCR seems excessive if users can find that information in another wiki article. Also (and you are not alone), I truncated some of your headings - wikis should be laid out with short, non-superfluous titles and headers. Very interesting topic and definitely did a nice job explaining the 'exposure scenario.' I also changed your intro so that an introduction is before the table of contents. (570ajk (talk) 18:55, 11 November 2011 (UTC))

AJK - the PCR information was to try to show the evolution of testing that has lead to this point and it is not described for this virus anywhere in Wiki. The move to the latest technology has brough much improved speed and sensitivity, but at the cost of these occaisional false-negatives. Maybe I didn't really lay that out well enough to follow my train of thought..... (570wac (talk) 22:34, 11 November 2011 (UTC))

PCR questions

Hi! I have been doing some work with using real time PCR for detection of Salmonella in environmental samples, so I know just enough to be dangerous. Can you explain to me why working with the amplified PCR product from nested PCR could produce false positives? I am not sure if it is necessary to add that to your page, but I would like to know. (in response to 570ceh question) Nested PCR involves an extra handling step mid-test.....after the first round of amplification is done (potentially already creating thousands / millions of copies of cDNA), those tubes are opened and transferred to another tube where another round of amplification is done on a smaller region within the larger cDNA. Basically, when you have to open / close tubes that likely have tons of amplified DNA, you run the risk of that material getting into places it's not supposed to go. Now, contrast that with real-time PCR in which you add only template DNA or RNA (as in the material you extracted from a clinical sample) and close the tube and let amplification go....then never open the tube again while the results are read through light emitted through the tube. 570wac (talk) 22:53, 7 December 2011 (UTC)

Also, it was my understanding that the Taqman probes used for real-time PCR bind to double stranded DNA which results from other steps in the amplification process, but that the probes were not specific to a certain sequence themselves. I am a geologist trying to learn microbiology without a strong grasp of the fundementals, so forgive me if this is a stupid question! 570ceh (talk) 16:52, 8 November 2011 (UTC) (in response to 570ceh question) Not quite....although their are variations of real-time chemistry BESIDES taq-man which fit what you are saying. But for taq-man probes, they target a specific sequence within the overall amplicon (fancy term for the part of the DNA you amplified with primers in the PCR). Anyway, taq-man probes bind to the single-stranded DNA during the extension step and are cleaved (torn apart) by taq-polymerase as it makes a new copy of DNA. After being cleaved they can be detected by real-time PCR equipment. Again, there are other forms of real-time chemistry out there....but for PRRS it's currently done with Taq-man probes. SYBR Green I, for example, is non-specific 570wac (talk) 22:55, 7 December 2011 (UTC)

I am trying to get my head around the size of the problem, not something I am very familiar with. What are the false negative error rates? What is the 'cost' of a misdiagnosis in economic, herd health, and human health terms? Do labs have their own proprietary assay probes or are they shared across industry -- by this I am trying to understand if the degree of concern for false-negative results in a given instance are lab specific.570jdw (talk) 19:52, 8 November 2011 (UTC)

(in response to 570djw question) I have only anecdotally heard about a false-negative rate from a lab of "1 every 6 months"....that is to say the test has to be re-designed to handle new isolates that often, but I don't know out of how many tests/samples that is. Some labs have their own tests and some use commercially sold tests, it varies, but sending samples to another lab is routinely done when trying to check for false-negatives. This whole phenomenon can also happen in other disease agents for any lab, but the hazard is much higher for RNA viruses than for any other target given their mutation rate. (570wac (talk) 22:49, 7 December 2011 (UTC))

Nice topic and article. I enjoyed reading it. Beyond that I don't have much to add other than possibly helping find info for false-positive numbers. I have experience with real-time PCR, but not PRRS so I have no idea about how often this happens. I suppose that makes it good information to include if it is available. I found a synopsis of a Balkan PRRS symposium...they gave some false-positive numbers for a few different commercial kits. Not sure if you will find it useful, but maybe...I just skimmed through it. Here is the link http://europrrs.net/events/split-2011/abstract-book-split-2011.pdf 570jby (talk) 18:00, 10 November 2011 (UTC)

Thanks UTC, I posted replies on your Talk. As for false-positive numbers, it is really a function of the lab more than anything - so quoting a rate for a particular test, while commonly done, is really just a conservative estimation for the lab user. Each lab controls false-positive results primarily through the way they handle testing.....and sterile technique. At any rate, though, great questions and suggestions (by everyone!).

Thanks, (570wac (talk) 22:32, 11 November 2011 (UTC))

I think it has all been said - a very interesting topic.570nlh (talk) 16:08, 13 November 2011 (UTC)