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

[narayan raj bhattarai]

Dear Hanafi

As I am also using the same Qiagen DNA minikit for the DNA extraction from pools of P. argentipes in Nepal but I don't have any problem in DNA extraction and amplification however I am not using the same primers whatever you have mentioned. In my lab, the extraction protocol works properly. The smearing of the amplicons might be related to other parameters like concentration of different reagents and cycling conditions which can be solved by extensive optimization in your set up.
thanks
Narayan

===========
Dr. Narayan Raj Bhattarai
Assistant Professor
Department of Microbiology
B. P. Koirala Institute of Health Sciences, Dharan, Nepal


--- On Tue, 7/12/11, Hanafi, Hanafi  CTR EG NAMRU3 <hanafi.hanafi.ctr.eg at med.navy.mil> wrote:

From: Hanafi, Hanafi  CTR EG NAMRU3 <hanafi.hanafi.ctr.eg at med.navy.mil>
Subject: [Leish-l] PCR and Leish detection in sand flies
To: "Leish-L" <Leish-l at lineu.icb.usp.br>
Cc: "Obenauer, Peter J. LCDR" <Peter.Obenauer at med.navy.mil>, "Kaldas, Rania M CIV EG NAMRU3" <Rania.Kaldas.eg at med.navy.mil>
Date: Tuesday, July 12, 2011, 2:17 AM

Dear Leish-L

Does anyone can help explain what happened to our PCR run to test some sand 
flies for Leish.

We tested 3 pools of SF collected from endemic CL foci in Libya. Details were 
as follow:

1- DNA extraction using Qiagen DNA mini Kit
2- Amplification using Leishmania GENUS primer/probe set
3- Ct for 3 pools were: A) P. papatasi (Ct: 36.0621)
                        B) P. papatasi (Ct: 36.046)
                        C) P. longicuspis (Ct: 35.7944)

4- Amplification using Internal Transcribed spacer 1 primers according to 
Schonian 2003 (L5.8S and LITS.R) showed faint bands.
5- Reamplified ITS1 region using the same primers and PCR conditions, and by 
running the samples on 3% Agarose gel, is gave a smeary band that stuck in the 
well, didn't run like the controls.
6- Step 4 & 5 were repeated but the same outcome appeared.
7- Using Leishmania major specific primer/probe sets, samples were NEGATIVE 
for L. major, controls were excellent.
8- Tried Nested ITS1 according to Schonian 2003, using the same primers and 
PCR conditions.
9- Same smeary gel appeared again and we don't know why. Please advise

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



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