inmacdonald schreef:The FISH DNA hybridization. Will settle the heretofore contentious debates about the REALITY OF
CHRONIC BORRELIA ILLNESSES.
I WILL PROVIDE THE METHODOLOGY AND THE DETAILS OF MANUFACTURE OF THE
MOLECULAR beacon DNA PROBES IN AN UPCOMING PEER REVIEWED PUBLICATION.
AN Epifluorescent microscope is required to accomplish the microscopic exams.
Alan B. MacDonald, MD,FCAP
JANUARY 28, 2015
Bron: LymeNetEurope
inmacdonald schreef:DNA probes are used commonly in Pathology and in Microbiology.....
In Situ Hybridization. ( ISH ) is standardized!.
Entire textbooks are published !!!
ISH
AND
FISH
DO AN AMAZON BOOKS SEARCH TO VERIFY.!!
THE TECHNIQUE OF in Situ Hybridization has been around in research for Decades.
It works
It makes hybridized target VISIBLE UNDER THE MICROSCOPE.
THE FDA HAS NO JURISDICTION OVER in situ hybridization. Or FISH either
Alan B. MacDonald, MD, FCAP
JANUARY 29, 2015
Bron: LymeNetEurope
Exploring the association between Morgellons disease and Lyme disease: identification of Borrelia burgdorferi in Morgellons disease patients
Marianne J Middelveen, Cheryl Bandoski, Jennie Burke, Eva Sapi, Katherine R Filush, Yean Wang, Agustin Franco, Peter J Mayne and Raphael B Stricker
12 February 2015 BMC Dermatology 2015, 15:1 | doi:10.1186/s12895-015-0023-0
Bb molecular beacons
Dr. Alan MacDonald designed the DNA sequences and generously donated the Bb molecular beacon DNA probes. Probe FlaB, a sequence of 23 nucleotides, was derived from the Bb open reading frame (ORF) BB0147 of the flagellin B gene that contains more than 1000 nucleotides. A nucleotide BLAST search of the 23 nucleotide probe sequence disclosed no matches other than that of Bb BB0147. Probe 740 was derived from the Bb ORF BB740 representing a Bb inner cell membrane protein, and a nucleotide BLAST search disclosed no matches other than that of the Bb ORF BB740.
Bb DNA staining and detection with molecular beacons was performed by the following protocol, as previously described [12]. Paraffin sections of dermatological specimens and culture pellets were completely dewaxed by baking at 60°C then immersed in serial 100% xylene baths, followed by serial immersion through 100% ethanol, 90% ethanol, 80% ethanol and distilled H2O, then air-dried. Fixed sections were immersed in 20 μl of working DNA beacon solution. Sectioned specimens were covered with plastic cut from a Ziploc® freezer bag then were heated at 90°C for 10 minutes to denature all DNA and RNA. Heat was reduced to 80°C for 10 minutes, then samples cooled gradually to room temperature. The stained slides were washed in PBS, and covered with 30% glycerol and a glass coverslip, then examined under an EPI Fluor microscope. Staining of research specimens was performed alongside staining of positive and negative controls. Positive controls consisted of a known Bb strain embedded in agarose, formalin-fixed and sectioned, as well as experimentally Bb-infected mouse liver sections. The negative control consisted of uninfected mouse liver sections.
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