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

[Kaldas, Rania M CIV EG NAMRU3]

Good morning dear professors,

I am so thankful for all your input in troubleshooting our controversial
samples of sand flies, I have tried to amplify another fragment and am
planning in going further with sequencing to confirm the product I am
getting. My internal controls were excellent in all of my assays, but they
are cultures of different strains of Leishmania, and had too much DNA. The
confusing part here is that the ITS.1 product show smears on the Agarose gel
even with 3 % concentration, which is a first to me. There is another
thought of trying to amplify a specific target for all sand flies (e.g. 18 s
ribosomal RNA) to make sure there is actually sand fly DNA, also our
extraction kit is still good for use (far away from being expired).

Thanks again for the information, and am looking forward to sharing with all
of you solving this mystery.

Have a wonderful day

Respectfully,
  
  Rania M. Kaldas
  
  Medical Research Technician
  Vector Biology Research Program
  U.S. Naval Medical Research Unit No. 3 (NAMRU-3) 
  Tel.: ++2-02-23480507
  Fax:  ++2-02-23423090
  E-mail: rania.kaldas.eg at med.navy.mil 
  



 

-----Original Message-----
From: Hanafi, Hanafi CTR EG NAMRU3 
Sent: Thursday, July 21, 2011 7:47 AM
To: Kaldas, Rania M CIV EG NAMRU3
Subject: FW: [Leish-l] PCR and Leish detection in sand flies



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. rer. nat. Gabriele Schönian"
[mailto:gabriele.schoenian at charite.de] 
Sent: Wednesday, July 20, 2011 6:13 PM
To: Hanafi, Hanafi CTR EG NAMRU3 
Subject: Re: [Leish-l] PCR and Leish detection in sand flies


Dear Dr. Hanafi
as already suggested by Omar and Narayan something might have got wrong
with your DNA extraction. I do not think that using a kit instead of
classical phenol-chloroform extraction or working with sand fly pools were
the problem.

Did you include any controls in your experiment (DNA extraction control or
inhibition controls in the PCR assay)? Have you ever tried to amplify sand
fly targets with your DNA extracts? I think you should do troubleshooting
for understanding what went wrong with your PCR assay.

Do not hesitate to contact me for further questions.

Sincerely yours,
Gabriele Schönian


> 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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> Leish-l mailing list
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>


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