DNA isolation from BCG

Ulrich Maier maier_hml at muenster.netsurf.de
Wed Nov 24 10:38:12 EST 1999


On Sun, 14 Nov 1999 16:50:27 +0100, Mick <drag at eskulap.am.lublin.pl>
wrote:

>Few weeks ago I started PCR test detecion of  Mycobacterium
>tuberculosis  in tuberculosis patients,
>but in electrophoresis I got only bands of positiv control.
>I  have investigate about 20 samples obtained from patients with
> suspipitons of pulmonary and extra-pulmonary tuberculosis 
>(sputum, pleural effusion, bronchaial washing).
>I suspect there must be sth wrong with isolation protocol or
>concentration of BCG.
>I wil be grateful for any help.
>
> Michal Fik
>
Hope that this is not a commercial investigation of specimen, you are
far away from a suitable validated method (;-)).

- single step PCR and gel electrophoresis is not sensitive enough for
the detection of MTB-positive staining-negative specimen. 

-spike a sputum with cultured MTB-bacteria and prove your
DNA-isolation method.

- use decontaminated specimen

- always check for PCR-inhibitors in the DNA-isolate by spiking the
amplification mix with MTB DNA.

- check sensitivity of your amplification and detection by using a
diluted positive amplicon or culture DNA isolate as template.

- mycobacteria are difficult to crack, boiling or lysozyme digestion
works fine,  imho.

-Try PCR-ELISA or nested PCR to increase your sensitivity.

- Not every specimen from a tb-positive patient is PCR positive.

Feel free to ask via E-mail, if there are still questions.

Yours,

Uli Maier






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