Evaluation of a nested PCR targeting IS6110 of Mycobacterium tuberculosis for detection of the organism in the leukocyte fraction of blood samples.
OBJECTIVE
Tuberculosis poses a serious health problem in resource-poor settings such as India. Polymerase chain reaction (PCR) is presently seen as a promising alternative to conventional smear microscopy and culture techniques. Undiagnosed fever is a condition where the aetiology could include tuberculosis in a significant percentage. This paper evaluates a nested PCR (nPCR) using Hotstar Taq for the detection of M. tuberculosis in patients with febrile illness using insertion element, IS6110 as a target.


METHODS
A total of 355 samples (301 HIV status unknown and 54 HIV seropositives) from patients primarily with febrile illness were tested for the presence of M. tuberculosis. Blood culture was done in a commercial automated blood culture system and nPCR in DNA extracts from buffy coat samples. Hotstar Taq polymerase was used to enhance the sensitivity of nPCR and the lower limit of detection was determined by using cloned plasmid.


RESULTS
Among the patients tested, 2% were positive by automated culture system and 6.8% of patients were positive by nPCR. Majority of the positives were from HIV seropositive individuals. The sensitivity of the nPCR was 100% and the specificity was 95.1%. The lower limit of detection was less than 1 genome copy per microlitre. Among the nPCR positives, patients from rural community were significantly higher than from the peri-urban community.


CONCLUSIONS
The nPCR had a high sensitivity and specificity on buffy coat samples using Hotstar Taq polymerase in the reaction mix. Thus the technique is a valuable tool in the diagnosis of tuberculosis.
Nandagopal
B
B
Sri Narayani Hospital & Research Centre, Thirumalaikodi, Sripuram, Vellore 632 055, India.
Sankar
S
S
Lingesan
K
K
Appu
K C
KC
Sridharan
G
G
Gopinathan
A K
AK
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001431:Bacteriological Techniques / Q000379:methods*; D001769:Blood / Q000382:microbiology*; D002648:Child; D002675:Child, Preschool; D004251:DNA Transposable Elements / Q000235:genetics*; D005260:Female; D006801:Humans; D007194:India; D007962:Leukocytes / Q000382:microbiology*; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis*; D055815:Young Adult
D004251:DNA Transposable Elements