Determination of polymerase chain reaction efficiency for diagnosis of tuberculous meningitis in Chelex-100 extracted DNA samples.
METHODS
Polymerase chain reaction (PCR) offers great promise for the rapid, sensitive and specific diagnosis of tuberculous meningitis (TBM). However, the isolation of DNA of high quantity and quality from cerebrospinal fluid (CSF) samples is critical for successful PCR assays.


OBJECTIVE
To develop and use a single-tube method for the isolation of PCR-compatible DNA from Mycobacterium tuberculosis using Chelex-100 chelating resin, which does not require organic solvents or detergents.


METHODS
The study focused on the standardisation of a suitable Chelex protocol and its evaluation in 32 CSF samples from TBM and non-TBM subjects. A simultaneous comparison was made with the conventional phenol/chloroform extraction method.


RESULTS
PCR was found to be more sensitive, more rapid and less technically demanding with the Chelex protocol than the conventional phenol/chloroform extraction method (sensitivity 84.2% vs. 73.6%).


CONCLUSIONS
The single-tube method and the simplicity of the procedure permits early and reliable diagnosis of TBM and makes it an attractive method for routine laboratory assays.
Nagdev
K J
KJ
Biochemistry Research Laboratory, Central India Institute of Medical Sciences, Nagpur, India.
Kashyap
R S
RS
Deshpande
P S
PS
Purohit
H J
HJ
Taori
G M
GM
Daginawala
H F
HF
D000293:Adolescent; D000328:Adult; D002555:Cerebrospinal Fluid / Q000382:microbiology*; D002614:Chelating Agents; D002648:Child; D004269:DNA, Bacterial / Q000032:analysis*; D003937:Diagnosis, Differential; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012117:Resins, Synthetic; D012680:Sensitivity and Specificity; D014390:Tuberculosis, Meningeal / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000382:microbiology; D055815:Young Adult
D002614:Chelating Agents; D004269:DNA, Bacterial; D012117:Resins, Synthetic; C024997:Chelex 100