DNA isolation from BCG
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>
>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
- 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.
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