How prone is PCR to contamination?

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
velvetmagnetta
Posts: 469
Joined: Sun 23 Feb 2014 22:47

How prone is PCR to contamination?

Post by velvetmagnetta » Sun 30 Nov 2014 1:42

There have been several "Opinion Letters" from Dr. Wormser and other IDSA members and CDC staff, like Dr. Barbara Johnson, accusing researchers of experimental Polymerase Chain-Reaction (PCR) DNA contamination.

Does anyone have any facts, figures, opinions, or just thoughts about the how prone the (PCR) method of DNA detection by amplification is to contamination?

How difficult is it to keep the samples sterile? How often would you say contamination occurs in these types of experiments, and in which step or steps?

Margherita
Posts: 192
Joined: Thu 27 Sep 2012 18:22

Re: How prone is PCR to contamination?

Post by Margherita » Sun 30 Nov 2014 15:55

Hi velvetmagnetta,
Does anyone have any facts, figures, opinions, or just thoughts about the how prone the (PCR) method of DNA detection by amplification is to contamination?
Here's my opinion which, at the same time, is also a question:

Isn't it remarkable that when it comes to Lyme disease this contamination issue seems to be so important while, when using it for detection and diagnosis of other diseases, apparently it doesn't seem to be that big deal?!

hv808ct
Posts: 256
Joined: Wed 30 Jul 2008 4:11

Re: How prone is PCR to contamination?

Post by hv808ct » Sun 30 Nov 2014 21:32

Re: How prone is PCR to contamination?
Post by Margherita » Sun 30 Nov 2014 15:55

Here's my opinion which, at the same time, is also a question:
Isn't it remarkable that when it comes to Lyme disease this contamination issue seems to be so important while, when using it for detection and diagnosis of other diseases, apparently it doesn't seem to be that big deal?!
PCR contamination is always important in diagnosis (when it’s used for such) and in non-medical issues. See, for example, the following online summaries about the problems of contamination:
http://faculty.plattsburgh.edu/donald.slish/pcr.html
http://tinyurl.com/le7qee4

PCR is not a “big deal” in the diagnosis of other bacterial diseases because either serologies are perfectly adequate or identification of the particular pathogen is unnecessary to guide treatment as for example in empiric treatment of community-acquired pneumonia, or in the case of something dramatic and unusual like suspected exposure to plague or anthrax.

PCR may be useful in those rare cases where it is important to know if Mtb, for example, is carrying certain antibiotic-resistance genes, the presence of which definitely would affect treatment options.

Lymees like to compare Bb to T. pallidum, the agent of syphilis so they should probably read this review article on syphilis diagnostics (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095002/), which describes the utility and sensitivity/specificity of various assays, and the utter lack of hysteria and conspiracy theorizing associated with the results of those assays.

The fault in LD lies not in the diagnostics, but in the people who will not believe the results. Many hundreds of people in the US believe they have LD and seek objective evidence to re-enforce that belief. If they can’t get it from physicians and ID experts, and from commercial diagnostic labs, they denounce the experts and the results, insisting that some devious scheme is afoot.

Paranoid and conspiracy are well-documented features of American culture. Unfortunately, the medical component of that behavior costs the country millions of dollars each year in silly legislative efforts to subvert science-based medicine, line-item appropriations for more research and clinical trials (none of which LD activists ever approve of), hearings, meetings, the occasional lawsuit, threats, protests, quackery, and redundant or incremental research efforts to tweak something that doesn’t need tweaking.

The rational person, when repeatedly told by competent experts that he or she does not have X, usually moves on to other possibilities or to coping mechanisms. But there is little evidence of rational behavior among the denizens of Lymeland. There is instead regular evidence of psychiatric co-morbidities associated with many people insisting they have LD or chronic LD. See a list of such articles here: http://tinyurl.com/q28znn2.

duncan
Posts: 1370
Joined: Wed 5 Sep 2012 18:48

Re: How prone is PCR to contamination?

Post by duncan » Sun 30 Nov 2014 22:57

hv808ct, you may wish to disentangle yourself from all the canned rhetoric so as to avoid tripping over your logic flow. Paranoia and conspiracy would be more appropriate - I would imagine - in your neck of the woods these days what with the push for a formal investigation into the ALDF, IDSA and CDC.

The fault lies not in the Science of Lyme, but in the scientists and their flawed products, metrics and inferences. As any rational person would know, you cannot outrace that inescapable truth. You can deny it, and you can deride thousands of patients, and you can dodge it for a while with faulty and misleading studies, but it's already gaining ground. Eventually, truth will win out.

It's just a function of time.

And time is not on your side. Tick tock.

hv808ct
Posts: 256
Joined: Wed 30 Jul 2008 4:11

Re: How prone is PCR to contamination?

Post by hv808ct » Sun 30 Nov 2014 23:15

... win out. It's just a function of time. And time is not on your side. Tick tock.
by duncan
Sun 30 Nov 2014 22:57

... into the IDSA, ALDF and CDC has surpassed the 25,000 signature mark! Tick Tock.
by duncan
Wed 26 Nov 2014 14:17

Re: Petition calling for investigation of CDC, IDSA and ALDF
Tick tock...
by duncan
Tue 18 Nov 2014 15:23

... we've been forced to endure for decades. It's just a function of time. Tick tock.
by duncan
Sun 16 Nov 2014 17:39
As I said...

duncan
Posts: 1370
Joined: Wed 5 Sep 2012 18:48

Re: How prone is PCR to contamination?

Post by duncan » Sun 30 Nov 2014 23:25

Wow. You seem almost...obsessed with me. Should I feel flattered, or paranoid? Let me guess...

Henry
Posts: 1108
Joined: Thu 10 Nov 2011 18:49

Re: How prone is PCR to contamination?

Post by Henry » Mon 1 Dec 2014 0:07

Duncan: Hv808ct said -- much more eloquently that I could-- just exactly what I have told you many times:

"The fault in LD lies not in the diagnostics, but in the people who will not believe the results. Many hundreds of people in the US believe they have LD and seek objective evidence to re-enforce that belief. If they can’t get it from physicians and ID experts, and from commercial diagnostic labs, they denounce the experts and the results, insisting that some devious scheme is afoot. "

If you wish to talk about ticking clocks, just tell me how much time has been wasted -- and opportunities lost-- being treated for a disease (Lyme disease) that one doesn't have, just because one refuses to accept the results of a negative test result. Such irrational behavior has retarded a search for what is really causing of the symptoms that you and others experience. But, it is difficult to reason with someone who doesn't realize the harm that he is doing to himself. Within the LNE echo chamber, it is the blind leading the blind, re-enforcing their unsupported views ad infinitum . Tick, tock....
Last edited by Henry on Mon 1 Dec 2014 0:28, edited 1 time in total.

duncan
Posts: 1370
Joined: Wed 5 Sep 2012 18:48

Re: How prone is PCR to contamination?

Post by duncan » Mon 1 Dec 2014 0:27

Henry, your vantage is all wrong. You are approaching this incorrectly, and I cannot tell if you are just that naive or misinformed, or if it is deliberate. I try to give you the benefit of the doubt time and time again, but it seems each time I do, you respond with boilerplate.

No one would disagree with the idea that some people are being treated for Lyme that do not have Lyme. I, and most who share my perspective, have no quarrel with that. The problem is there are many people who have Lyme who are not being treated. They are not being treated because of poor inferences made from poor studies. They are not being treated because of inherent limitations in the current diagnostic arsenal. They are not getting treatment because of dangerous policies that virtually mandate they should not. They are not seeing new treatments developed because that need is being dismissed and denied.

These deficits are not just in the Science being practiced, but in basic human core values of some of the scientists practicing. One should never place dogma before patients - it will always be wrong.

Henry
Posts: 1108
Joined: Thu 10 Nov 2011 18:49

Re: How prone is PCR to contamination?

Post by Henry » Mon 1 Dec 2014 3:33

They are not being treated for Lyme disease simply because there is no evidence to indicate that they actually have Lyme disease. If anyone has failed, it is they who have failed to accept this reality. This has nothing to do with the way science is being practiced. It has more to do with the way some people solve their problems.

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LHCTom
Posts: 341
Joined: Mon 22 Oct 2012 4:18

Re: How prone is PCR to contamination?

Post by LHCTom » Mon 1 Dec 2014 5:01

There is no simple answer to this question. There are a variety of PCR methods ( RT-PCR, nested PCR, Quantitative, Real Time, semi-nested PCR...etc..) and many implemetations. There are also a variety of ways contamination can occur and multiple strategies for minimizing contamination. I would say the only accurate answer is it depends on the person performing it, the type and liklihood of sources. There are also techniques for checking the results for the liklihood its a contaminant or real.

Same thing is true of most microbiology tests. The high amplification of PCR makes it riskier than most given all else equal.

Polymerase Chain Reaction (PCR)
http://www.ncbi.nlm.nih.gov/probe/docs/techpcr/

Real-Time qRT-PCR (Real-Time Quantitative Reverse Transcription PCR)
http://www.ncbi.nlm.nih.gov/probe/docs/techqpcr/

http://www.academia.edu/8191548/How_to_ ... al_ecology

http://scholarsresearchlibrary.com/aasr ... 77-179.pdf
Paranoid and conspiracy are well-documented features of American culture.
Among the clearest examples are Bloggers like http://rel-risk.blogspot.de/
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

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