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Pepsin-HCl digests and PCR

Greg Appleyard greg.appleyard at usask.ca
Mon Jun 7 10:44:11 EST 1999

Hi John,

I worked on a project to identify the species/biotype of Trichinella 
isolates collected a variety of wildlife species. Samples of muscle 
(tongue and other bits) were collected and stored frozen (some for many 
months) prior to shipping to the lab. The meat samples were trimmed and 
then subjected to pepsin-HCL digests. Larvea were collected by 
differential sedimentation in pilsner flasks. 

In prepration for PCR, the larvae were either simply boiled in water or 
boiled and then subjected to a typical phenol/chloroform extaction 
protocol. Extraction of nucleic acids simplt by boiling is not as 
sensitive as phenol/cloroform extractions but when many larvae (30+) were 
available either method worked well.

My guess is that if the larave of the parasites you are looking for in 
mucosa will survive the pepsin/HCL digest intact, then they will be in 
good shape for DNA extraction and PCR. The key is that they need to 
survive the digest intact.

If you need more information about our protocol you can contact me;
greg.appleyard at usask.ca

Greg Appleyard
University of Saskatchewan

In article <3751149D.3018637B at abdn.ac.uk>, j.dallas at abdn.ac.uk says...
>I am seeking advice on how to prepare larvae of
>intestinal nematodes from field-collected samples in a
>suitable state for PCR assays.

>John Dallas
>Dr J.F. Dallas
>Senior Research Fellow
>NERC Molecular Genetics in Ecology Initiative
>Department of Zoology
>University of Aberdeen
>Tillydrone Avenue
>Aberdeen AB24 2TZ
>Tel.  +1224 272 273
>FAX   +1224 272 396
>Email j.dallas at abdn.ac.uk

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