purifying Parvovirus B19 DNA from plasma
Dr. J.P. Clewley
jclewley at hgmp.mrc.ac.uk
Wed Nov 13 13:11:54 EST 1996
David Herns wrote:
>I would appreciate any suggestion on how best to get Parvovirus B19
>out of plasma, for eventual PCR. I am hoping to amplify a rather
>larger PCR product (2 - 3 KB), and would appreciate any tips. The
>plasma is from a person with known Parvovirus B19 infection.
>In addition, any tips on how best to store plasma with Parvo B19 ...
>i.e. in the refrigerator, -20C, -70C, etc ...
This lab uses a Silica/GuSCN extraction method, the Boom method, as described in
the 2 papers by Boom et al below (for interest, 2 papers on variations of the
method are also given):
Rapid & simple method for purification of nucleic acids. Boom et al, J Clin
Micro 28: 495-503, 1990.
Rapid purification of hepatitis B virus DNA from serum. Boom et al, J Clin Micro
29: 1804-1811, 1991.
(An inexpensive and simple method for DNA purifications on silica particles,
Carter & Milton, NAR 21: 1044, 1993.DNA extraction from Pleistocene bones by a
silica-based purification method. Hoss & Paabo NAR 21: 3913-3914, 1993.)
Our lab B19 PCR methods are described in:
Clewley, J. P. 1993. Polymerase chain reaction detection of parvovirus B19, p.
367-373. In D. H. Pershing and T. F. Smith and F. C. Tenover and T. J. White
(ed.), Diagnostic Molecular Microbiology: Principles and Applications. ASM
Press, Washington D.C. Clewley, J. P., and B. J. Cohen. 1994. Investigation of
human parvovirus B19 infection using PCR, p. 205-215. In J. P. Clewley (ed.),
The Polymerase Chain Reaction for Human Viral Diagnosis. CRC Press, Boca Raton.
You can amplify almost the whole genome (5kb of 5.5kb) by standard PCR,
but use a long PCR kit (Boehringer, Perkin Elmer) if you have any problems.
B19 is a parvovirus and hence very stable. Sera/plasma can be kept at -20 C, but
perhaps you should always be a little paranoid and keep precious specimens in
aliquots in (separate) -70 C freezers, but who does that?
Jon Clewley, Virus Reference Division, Central Public Health Laboratory
61 Colindale Avenue, London NW9 5HT, United Kingdom
Phone: + 44 (0) 181 200 4400 ext 3245 Fax: + 44 (0) 181 200 1569
Email:jclewley at hgmp.mrc.ac.uk or jclewley at phls.co.uk
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