How prone is PCR to contamination?

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
phyfe
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Re: How prone is PCR to contamination?

Post by phyfe » Mon 1 Dec 2014 5:12

hv808ct wrote:
... win out. It's just a function of time. And time is not on your side. Tick tock.
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Tick tock...
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admin
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Re: How prone is PCR to contamination?

Post by admin » Mon 1 Dec 2014 13:44

Please keep the information and discussion substantial and science-based. The first two posts of this topic pose some interesting questions that can and should be answered in a scientific way without all the "noise".

velvetmagnetta
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Re: How prone is PCR to contamination?

Post by velvetmagnetta » Mon 1 Dec 2014 20:39

Thank you, admin. I was getting a little frustrated there. It was beginning to remind me of the famous "Battle of Wits":

https://www.youtube.com/watch?v=i6TQ7ljcsjk

velvetmagnetta
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Re: How prone is PCR to contamination?

Post by velvetmagnetta » Mon 1 Dec 2014 22:31

hv808ct, you wrote:
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
Thank you for these. The first link seems to be for a laboratory school experiment. It tells me how to set up and perform a PCR, but does not say much about the rates of contamination for all the variations of PCR that LHCTom mentioned.

I cannot see the second link at all. It looks like it is specifically about PCR contamination, so I would very much like to read it. Is there a .pdf version or an online version you can post? Or if not, can you cut and paste some of the relevant material from it here, in this thread?

Thanks!

Since PCR involves the amplification, or the rabid* multiplication of DNA molecules, I would imagine it may be very prone to contamination. I also imagine that most researchers know this and take appropriate precautions. I would like to know what precautions there are for each type of PCR to limit contamination? And how well do those precautions work? How well can we rely on experimental results if those contamination protocols have been met?



LHCTom - Thank you for all those links! It will take me some time to get through them. But get through them, I will! There have been too many accusations of contamination thrown around lately not to give this issue a proper investigation.

*whoops! rabid was supposed to read rapid!
Last edited by velvetmagnetta on Thu 4 Dec 2014 1:31, edited 1 time in total.

velvetmagnetta
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Re: How prone is PCR to contamination?

Post by velvetmagnetta » Mon 1 Dec 2014 22:59

OK. This is going to take quite a bit longer than I thought. I will have to go on a Wikipedia surf to educate myself on many of the terms in LHCTom's 3rd link:

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

velvetmagnetta
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Re: How prone is PCR to contamination?

Post by velvetmagnetta » Mon 1 Dec 2014 23:08

From Wikipedia, it says:

https://en.wikipedia.org/wiki/DNA_repli ... ation_fork
The polymerase chain reaction (PCR), a common laboratory technique, cyclically applies such artificial synthesis [through the application of DNA primers] to amplify a specific target DNA fragment from a pool of DNA.
Wouldn't this be ind of a fool-proof way of amplifying only the DNA you are looking for?

Henry
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Re: How prone is PCR to contamination?

Post by Henry » Tue 2 Dec 2014 15:31

Contamination can happen to anyone who is not careful. Note this recent report:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203928/

Martian
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Location: Friesland, the Netherlands

Re: How prone is PCR to contamination?

Post by Martian » Thu 4 Dec 2014 21:46

velvetmagnetta wrote: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?
I have some study material for you. The following are the references for the section ‘Polymerase chain reaction’ (PCR) under chapter LABORATORY DIAGNOSTICS in the Dutch Lyme disease guidelines version July 2013. The section discusses the sensitivity, specificity and contamination problems of PCR.
Literature
- Aberer, E., A. R. Bergmann, A. M. Derler, and B. Schmidt. 2007. Course of Borrelia burgdorferi
DNA shedding in urine after treatment. Acta Derm. Venereol. 87:39-42.
- Avery, R. A., G. Frank, and S. C. Eppes. 2005. Diagnostic utility of Borrelia burgdorferi
cerebrospinal fluid polymerase chain reaction in children with Lyme meningitis. Pediatr Infect. Dis.
J 24:705-708.
- Bayer, M. E., L. Zhang, and M. H. Bayer. 1996. Borrelia burgdorferi DNA in the urine of treated
patients with chronic Lyme disease symptoms. A PCR study of 97 cases. Infection 24:347-353.
- Bergmann, A. R., B. L. Schmidt, A. M. Derler, and E. Aberer. 2002. Importance of sample
preparation for molecular diagnosis of lyme borreliosis from urine. J Clin Microbiol. 40:4581-4584.
- Brettschneider, S., H. Bruckbauer, N. Klugbauer, and H. Hofmann. 1998. Diagnostic value of PCR
for detection of Borrelia burgdorferi in skin biopsy and urine samples from patients with skin
borreliosis. J Clin Microbiol. 36:2658-2665.
- Cerar, T., K. Ogrinc, J. Cimperman, S. Lotric-Furlan, F. Strle, and E. Ruzic-Sabljic. 2008.
Validation of cultivation and PCR methods for diagnosis of Lyme neuroborreliosis. J Clin
Microbiol. 46:3375-3379.
- Cerar, T., E. Ruzic-Sabljic, U. Glinsek, A. Zore, and F. Strle. 2008. Comparison of PCR methods
and culture for the detection of Borrelia spp. in patients with erythema migrans. Clin Microbiol.
Infect. 14:653-658.
- Gooskens, J., K. E. Templeton, E. C. Claas, and A. P. van Dam. 2006. Evaluation of an internally
controlled real-time PCR targeting the ospA gene for detection of Borrelia burgdorferi sensu lato
DNA in cerebrospinal fluid. Clin Microbiol. Infect. 12:894-900.
- Issakainen, J., H. E. Gnehm, G. M. Lucchini, and R. Zbinden. 1996. Value of clinical symptoms,
intrathecal specific antibody production and PCR in CSF in the diagnosis of childhood Lyme
neuroborreliosis. Klin Padiatr. 208:106-109.
- Jaulhac, B., I. Chary-Valckenaere, J. Sibilia, R. M. Javier, Y. Piemont, J. L. Kuntz, H. Monteil, and
J. Pourel. 1996. Detection of Borrelia burgdorferi by DNA amplification in synovial tissue samples
from patients with Lyme arthritis. Arthritis Rheum. 39:736-745.
- Karch, H., H. I. Huppertz, M. Bohme, H. Schmidt, D. Wiebecke, and A. Schwarzkopf. 1994.
Demonstration of Borrelia burgdorferi DNA in urine samples from healthy humans whose sera
contain B. burgdorferi-specific antibodies. J Clin Microbiol. 32:2312-2314.
- Lebech, A. M. and K. Hansen. 1992. Detection of Borrelia burgdorferi DNA in urine samples and
cerebrospinal fluid samples from patients with early and late Lyme neuroborreliosis by
polymerase chain reaction. J Clin Microbiol. 30:1646-1653.
- Lebech, A. M., K. Hansen, F. Brandrup, O. Clemmensen, and L. Halkier-Sorensen. 2000.
Diagnostic value of PCR for detection of Borrelia burgdorferi DNA in clinical specimens from
patients with erythema migrans and Lyme neuroborreliosis. Mol Diagn. 5:139-150.
- Luft, B. J., C. R. Steinman, H. C. Neimark, B. Muralidhar, T. Rush, M. F. Finkel, M. Kunkel, and R.
J. Dattwyler. 1992. Invasion of the central nervous system by Borrelia burgdorferi in acute
disseminated infection. JAMA 267:1364-1367.
- Maiwald, M., C. Stockinger, D. Hassler, D. M. von Knebel, and H. G. Sonntag. 1995. Evaluation of
the detection of Borrelia burgdorferi DNA in urine samples by polymerase chain reaction. Infection
23:173-179.
- McKenna, R. B. Nadelman, L. F. Cavaliere, and G. P. Wormser. 2001. Laboratory diagnostic
techniques for patients with early Lyme disease associated with erythema migrans: a comparison
of different techniques. Clin Infect. Dis. 33:2023-2027.
- Melchers, W., J. Meis, P. Rosa, E. Claas, L. Nohlmans, R. Koopman, A. Horrevorts, and J.
Galama. 1991. Amplification of Borrelia burgdorferi DNA in skin biopsies from patients with Lyme
disease. J Clin Microbiol. 29:2401-2406.
- Nocton, J. J., F. Dressler, B. J. Rutledge, P. N. Rys, D. H. Persing, and A. C. Steere. 1994.
Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients
with Lyme arthritis. N Engl. J Med 330:229-234.
- Nowakowski, J., I. Schwartz, D. Liveris, G. Wang, M. E. Aguero-Rosenfeld, G. Girao, D.
- Palacek T., P. Kuchynka, D. Hulinska, J.Schramlova, H.Hrbackova, I.Vitkova, S.Simek, J.Horal,
W.E.Louch, A.Linhart, 2010. Presence of Borrelia burgdorferi in endocardomyocardial biopsies in
patients with new-onset unexplained dilated cardiomyopathy. Med Microbiol Immunol 199:139-
143
- Priem, S., G. R. Burmester, T. Kamradt, K. Wolbart, M. G. Rittig, and A. Krause. 1998. Detection
of Borrelia burgdorferi by polymerase chain reaction in synovial membrane, but not in synovial
fluid from patients with persisting Lyme arthritis after antibiotic therapy. Ann. Rheum. Dis. 57:118-
121.
- Priem, S., M. G. Rittig, T. Kamradt, G. R. Burmester, and A. Krause. 1997. An optimized PCR
leads to rapid and highly sensitive detection of Borrelia burgdorferi in patients with Lyme
borreliosis. J Clin Microbiol. 35:685-690.
- Rauter, C., M. Mueller, I. Diterich, S. Zeller, D. Hassler, T. Meergans, and T. Hartung. 2005.
Critical evaluation of urine-based PCR assay for diagnosis of Lyme borreliosis. Clin Diagn. Lab
Immunol. 12:910-917.
- Rijpkema, S. G., D. J. Tazelaar, M. J. Molkenboer, G. T. Noordhoek, G. Plantinga, L. M. Schouls,
and J. F. Schellekens. 1997. Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto,
Borrelia garinii and group VS116 by PCR in skin biopsies of patients with erythema migrans and
acrodermatitis chronica atrophicans. Clin Microbiol. Infect. 3:109-116.
- Roux, F., E. Boyer, B. Jaulhac, E. Dernis, F. Closs-Prophette, and X. Puechal. 2007. Lyme
meningoradiculitis: prospective evaluation of biological diagnosis methods. Eur J Clin Microbiol.
Infect. Dis. 26:685-693.
- Schmidt, B., R. R. Muellegger, C. Stockenhuber, H. P. Soyer, S. Hoedl, A. Luger, and H. Kerl.
1996. Detection of Borrelia burgdorferi-specific DNA in urine specimens from patients with
erythema migrans before and after antibiotic therapy. J Clin Microbiol. 34:1359-1363.
- Schmidt, B. L., E. Aberer, C. Stockenhuber, H. Klade, F. Breier, and A. Luger. 1995. Detection of
Borrelia burgdorferi DNA by polymerase chain reaction in the urine and breast milk of patients
with Lyme borreliosis. Diagn. Microbiol. Infect. Dis. 21:121-128.
- Schwaiger, M., O. Peter, and P. Cassinotti. 2001. Routine diagnosis of Borrelia burgdorferi (sensu
lato) infections using a real-time PCR assay. Clin Microbiol. Infect. 7:461-469.
- van Dam, A. P. 2001. Recent advances in the diagnosis of Lyme disease. Expert. Rev Mol Diagn.
1:413-427.
- von Stedingk, L. V., I. Olsson, H. S. Hanson, E. Asbrink, and A. Hovmark. 1995. Polymerase
chain reaction for detection of Borrelia burgdorferi DNA in skin lesions of early and late Lyme
borreliosis. Eur J Clin Microbiol. Infect. Dis. 14:1-5.
- Wagner, E. M., B. L. Schmidt, A. R. Bergmann, A. M. Derler, and E. Aberer. 2004. Inability of onestep
real-time PCR to detect Borrelia burgdorferi DNA in urine. J Clin Microbiol. 42:938.
One of the conclusions in the section is that "The diagnostic value of PCR on blood, serum and plasma is yet unclear." The references that are given for this conclusion are the two studies of Cerar et al. (2008).

velvetmagnetta
Posts: 469
Joined: Sun 23 Feb 2014 22:47

Re: How prone is PCR to contamination?

Post by velvetmagnetta » Thu 4 Dec 2014 21:53

Oh wow. Thank you, Martian!

I'm going to have to "geek-out" for a while on these. :geek:

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