FISH method for detection of Borrelia in blood smears

Medical topics with questions, information and discussion related to Lyme disease and other tick-borne diseases.
User avatar
inmacdonald
Posts: 977
Joined: Fri 13 Jan 2012 22:32

FISH method for detection of Borrelia in blood smears

Post by inmacdonald » Wed 11 Feb 2015 17:10

FISH = Fluorescence In Situ DNA Hybridization : Detection of Borrelia in Human blood without Culture

An Epifluorescent Microscope is required to do this testing.
Method:
Borrelia specific Molecular Beacon type DNA PROBES are manufactured, purified, and
The Validation of the Probes is carried out by multiple research laboratories
Which receive the PROBES, AND glass slides with POSITIVE and NEGATIVE
MICROBES, HUMAN CELLS, AND HUMAN TISSUES.
FISH Method: Standardized FISH ACCORDING TO TEXTBOOK descriptions of the FISH PROCEDURE.
Briefly: Heating to 100 deg C., cool to annealing temperature - specific temp for each probes,
Hybridization temperatures for the specimen flooded with Probe- locked in for 10 minutes,
Cool the slides to 24 deg centigrade, Multiple wash steps in Distilled H2O, air dry the slides
Examine- uncoverslipped - at 1000x - using single wavelength of exciting light which
Is specific for the Fluorochrome that is built into the
Molecular Beacon DNA Probe
Document any Positive images with a high resolution 18 megapixel Camera
Also photograph microscope fields which contain no fluorescence ( background)

Link:

Your poster is now live at: http://f1000.com/posters/browse/summary/1097535

Respectfully,
Alan B. MacDonald, MD, FCAP
February 11, 2015

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

Re: FISH method for detection of Borrelia in blood smears

Post by hv808ct » Thu 12 Feb 2015 20:22

FISH method for detection of Borrelia in blood smears
Post by inmacdonald » Wed 11 Feb 2015 17:10

Is FISH DNA probe methodology a Practical Test Method ????
Apparently not. Check PubMed.
Department of Research, The Dr Paul H Duray MD Research Endowment Trust, Inc, Naples, FL, USA
Isn’t this a house—sans basement—in a Naples retirement community?

User avatar
inmacdonald
Posts: 977
Joined: Fri 13 Jan 2012 22:32

Re: FISH method for detection of Borrelia in blood smears

Post by inmacdonald » Fri 13 Feb 2015 16:52

Comments on the F1000 Website

______________________________________________________
Comments f1000 poster.jpg
Comments on F1000 poster website
Comments f1000 poster.jpg (231.4 KiB) Viewed 2935 times

-------------------------------------------------------------------------

Link :
http://f1000.com/posters/Browse/summary/1097535


___________________________________________________

Comments are invited

Respectfully,
Alan B. MacDonald,MD, FCAP
Feb 13,2015

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

Re: FISH method for detection of Borrelia in blood smears

Post by hv808ct » Sun 22 Feb 2015 18:45

FISH method for detection of Borrelia in blood smears
Post by inmacdonald » Wed 11 Feb 2015 17:10
fluoresence in situ hybridization – FISH
http://f1000.com/posters/browse/summary/1097535
Just a reminder that FISH dates back to about 1996 and is essentially PCR with colored lights. The specificity and sensitivity is only as good as the probe itself.

Please also remember that 1) “The Dr Paul H Duray MD Research Endowment Trust, Inc,” is a private home in a retirement community in Naples, FL., 2) this poster was “not presented at a conference,” and 3) “…most posters on this site present work that is preliminary in nature and has not been peer reviewed.”

Of those “peers” who have commented on this poster, one is Elena Cook, a former mental patient in the UK who seems to think this work is further proof that B. burgdorferi is some kind of bioweapon*; R. Stricker who was banned from NIH funding, fired by UCSF, and spent some time at a penis enlargement clinic—as a clinician and not as a patient…I think; Peter Mayne, Lyme quack with a clinic in Australia where there is no LD, but where there are patients with money wanting to be treated for LD, and E. Sapi who seems to have a contamination problem in her lab and therefore see Bb here, there, and everywhere**.

*http://flash.lymenet.org/ubb/ultimatebb ... 198#000003

** http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6315a4.htm and
http://jcm.asm.org/content/52/3/721.long

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

Re: FISH method for detection of Borrelia in blood smears

Post by LHCTom » Sun 1 Mar 2015 0:48

hv808ct
The specificity and sensitivity is only as good as the probe itself.
So.

The probe is not unlike PCR primers and hybridize specific sections of nucleotides and fluoresce upon binding. I'm an amateur and I could go to the NCBI and other nucleotide databases and find 25 or so nucleotide strings with 100% specificity to every Borrelia in the database and not to anything else.

So Mr. big brain, specifically explain why the technique would not work if there were Borrelia in the test sample. If one probe hybridized ( bound ) and fluoresced, why would it not be able to be seen under the appropriate microscope. Of course if there is no Borrelia in the sample, there is nothing to bind to. If by chance the probe nucleotide sequence did not match because it was never seen before, it would not work. It is certainly susceptible to contamination but it can be managed with good quality control and redundancy.

Instead of playing the same bullshit conspiracy game, why don't you explain why the technique has problems. You and your conspiracy approach is as bad as the people you criticize. You actually sound like Wacko Elena.

These molecular beacons are commonly used in many fields effectively. Many use them like http://www.sigmaaldrich.com/technical-d ... acons.html utilize them for:

SNP detection
Allele discrimination
Pathogen detection
Multiplexing
Viral load quantification
Gene expression analysis
Gene copy determination
Endpoint genotyping
in vitro quantification or detection

Maybe these people knoiw more than your silly references... If you looked beyond your nose you would see the world... not just hatred toward the ill....

http://www.sigmaaldrich.com/technical-d ... 0EMVO.dpuf

http://www.molecular-beacons.org/

http://www.genelink.com/newsite/products/mbintro.asp

https://www.idtdna.com/pages/products/g ... ar-beacons



Just a reminder, ELISA and Western Blot and PCR predate 1996. Duh... So what does that mean.

Ever considered Flow Cytometry for much more reliable antibody detection not nearly as genotype or temporally sensitive? Oh its not FDA approved even though the people who really know what they are doing use it all the time.

http://cvi.asm.org/content/15/6/981.full


And by the way, there are 14 tick borne diseases acknowledged by the CDC. Of those, only Lyme disease has FDA approved tests. That means to diagnose any of the other 13 tick borne diseases, only "Homebrew" tests are available. Many of these 13 diseases are more serious than Lyme. So the oh my god these are not FDA approved is a bunch of BS. Being FDA approved does not mean any more than the same conspiracy crap you promote.

Are you actually able to explain something sensible or are you just the other side of Elena making wild accusations that have no bearing on reality? How about showing a brain or above average IQ or creativity...
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

User avatar
ChronicLyme19
Posts: 564
Joined: Mon 11 Aug 2014 17:42
Location: NY, USA

Re: FISH method for detection of Borrelia in blood smears

Post by ChronicLyme19 » Sun 1 Mar 2015 2:21

Wait, clarification, I thought there were no FDA "approved" tests, only ones that were "cleared"?
Half of what you are taught is incorrect, but which half? What if there's another half missing?

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

Re: FISH method for detection of Borrelia in blood smears

Post by LHCTom » Sun 1 Mar 2015 21:52

I think the words approved and cleared are both used.

Under FDA Medical Devices, it says both clearances and approvals. An in-vitro test like Lyme tests are cleared or approved once they get their 510(k). The words appear to be used somewhat interchangeably but its the 510(k).

Here is the list of 510(k) approved or cleared in-vitro tests for Borrelia

http://www.accessdata.fda.gov/scripts/c ... /index.cfm

Here is an example of fairly new Zeus Borrelia Vlse-1/PEPC10 510(k) http://www.accessdata.fda.gov/cdrh_docs ... 100728.pdf

The 510(k) essentially means this new device is considered "Substantially Equivalent" to previously approved devices.

So you need to convince the FDA, your test is as good as previously approved tests. I wonder what happens when you are the first and there is no previous test to be equivelant to?

A liitle on the process...

http://www.fda.gov/medicaldevices/produ ... efault.htm

You can search for tests with 510(k) approval or clearences here

http://www.accessdata.fda.gov/scripts/c ... /index.cfm

Turns out I was wrong, there are 2 510(k) tests for Tularemia

So 2 out 0f 14 TBDs http://www.cdc.gov/ticks/diseases/ have 510(k) cleared/approved tests...

The FDA gets 100% of the data from the company for a 510(k). As far as I can tell, the FDA doesn't look into the reality of what the supplier tells them. So there is nothing to prevent a supplier from fudging the data to get their 510(k). They won't get caught until the market discovers they aren't actually equivalent.
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

User avatar
ChronicLyme19
Posts: 564
Joined: Mon 11 Aug 2014 17:42
Location: NY, USA

Re: FISH method for detection of Borrelia in blood smears

Post by ChronicLyme19 » Mon 2 Mar 2015 1:53

Hmmm...

http://www.fda.gov/AboutFDA/Transparenc ... 194468.htm
Cleared medical devices: These medical devices are ones that FDA has determined to be substantially equivalent to another legally marketed device. A premarket notification, referred to as a 510(k), must be submitted to FDA for clearance. A 510(k) is most often submitted by the medical device manufacturer.

Approved medical devices: Approved medical devices are those devices for which FDA has approved a premarket approval (PMA) application prior to marketing. This approval process is generally reserved for high-risk medical devices and involves a more rigorous premarket review than the 510(k) pathway.
Half of what you are taught is incorrect, but which half? What if there's another half missing?

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

Re: FISH method for detection of Borrelia in blood smears

Post by velvetmagnetta » Mon 2 Mar 2015 8:21

hv808ct wrote:
FISH method for detection of Borrelia in blood smears
Post by inmacdonald » Wed 11 Feb 2015 17:10

Is FISH DNA probe methodology a Practical Test Method ????
Apparently not. Check PubMed.
Department of Research, The Dr Paul H Duray MD Research Endowment Trust, Inc, Naples, FL, USA
Isn’t this a house—sans basement—in a Naples retirement community?
Oh yes. Nothing good can come from basement science. Wait...wasn't Borrelia burgdorferi, you know, the spirochete that causes Lyme disease, discovered in a basement laboratory?

Lorima
Posts: 914
Joined: Mon 29 Oct 2007 20:47

Re: FISH method for detection of Borrelia in blood smears

Post by Lorima » Mon 2 Mar 2015 14:54

Maybe hv808ct had traumatic childhood experience(s) in a basement, like running into a nest of spiders, or being abused. That's the only reason I can come up with, for his basement problem. Unless it is symbolic, and represents the personal or collective unconscious, which seems likely to be pretty raw and scary, for someone as hostile as hv808ct. But that is straying into the humanities, which probably isn't productive here, though I think it is where some of the root causes of the "Lyme Wars" probably lie.

Careful critical analysis existing published science is sufficient, to discredit the views of hv808ct's faction. Too bad they aren't capable of looking directly at the physical aspects of the science, and ignoring all the unwarranted spin.

My father was an engineer and an inventor, and had a great workshop in our basement, where we spent a lot of time together. Maybe that is where I got my bias toward dealing with physical things, rather than social phenomena (although I think it may be somewhat genetic: my father's two brothers were also engineers, and as far as I can tell there wasn't any particular social pressure to do that.) Also, my scout troop had our meetings in the basement, and that is where I practiced music and painted. I have a lot of positive, creative associations with basements.
"I have to understand the world, you see."
Richard Feynman

Post Reply