[Leish-l] PCR and Leish detection in sand flies

[Hanafi, Hanafi CTR EG NAMRU3]

Dear dr. Hamarsheh,

Thank you for your detailed explanations and for your advise. However we
tested hundreds of SF in pools and individuals using the same primers and
same protocols and the results were perfects. 

Thank you 
Regards

Hanafi

Hanafi  A.  Hanafi, Ph. D.
Medical Research Scientist
Vector Biology Research Program
U.S. Naval Medical Research Unit No.3
Cairo, Egypt
Tel & Fax:  ++2-02-2342-3090
E-mail: hanafi.hanafi.ctr.eg at med.navy.mil
        yahanafi at yahoo.com


-----Original Message-----
From: Dr Omar Hamarsheh [mailto:ohamarsheh at gmail.com] 
Sent: Tuesday, July 12, 2011 1:56 PM
To: Hanafi, Hanafi CTR EG NAMRU3 ; 'Leish-L'
Cc: Obenauer, Peter J. LCDR; Kaldas, Rania M CIV EG NAMRU3
Subject: RE: [Leish-l] PCR and Leish detection in sand flies

Dear Dr Hanafi,

I would suggest the following:

1. the phenol-chloroform DNA extraction method generates small DNA amount
but of high quality
2. I personally do not believe in the use of sand fly pools, I prefer to use
single flies (more work but quality results). DNA of single flies may pooled
afterwards.
3. if DNA of blood fed sand flies is used in the PCR, it will generate
streaks, probably from blood or any other agents. I remember few years ago
when we tried to amplify DNA from blood fed sand flies we got all the time
streaks. Sand flies without blood in their guts or blood is already digested
and passed will be amplified very nicely if DNA is extracted with
phenol-chloroform method.
4. For smears: I think it is related to one of these problems:1) poor
quality of the DNA (2) The primers used may targeted nonspecifically both
sand fly DNA and Leishmania DNA if existed, since ribosomal genes of both
Leishmania and sand flies are multi copy. I think, if quality of DNA is
excluded; ITS1 primers used to characterize Leishmania species will amplify
nonspecifically sand fly DNA and this may mask the actual bands of
Leishmania if existed.