[Leish-l] PCR and Leish detection in sand flies
Hanafi, Hanafi CTR EG NAMRU3
hanafi.hanafi.ctr.eg at med.navy.mil
Tue Jul 12 06:17:07 BRT 2011
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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