Evaluation of a nested PCR targeting IS6110 of Mycobacterium tuberculosis for detection of the organism in the leukocyte fraction of blood samples
Autor: | Balaji Nandagopal, Karthikeyan Lingesan, Anilkumar Gopinathan, Gopalan Sridharan, Sathish Sankar, KC Appu |
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Rok vydání: | 2010 |
Předmět: |
Adult
Male Microbiology (medical) Tuberculosis Adolescent lcsh:QR1-502 India Buffy coat Polymerase Chain Reaction Sensitivity and Specificity lcsh:Microbiology law.invention Mycobacterium tuberculosis Young Adult chemistry.chemical_compound Tuberculosis diagnosis law Leukocytes medicine Humans Blood culture Child IS6110 Polymerase chain reaction Aged Aged 80 and over Bacteriological Techniques medicine.diagnostic_test biology Middle Aged medicine.disease biology.organism_classification Virology nested PCR Blood chemistry Child Preschool Immunology DNA Transposable Elements Female rural Nested polymerase chain reaction Taq polymerase |
Zdroj: | Indian Journal of Medical Microbiology, Vol 28, Iss 3, Pp 227-232 (2010) |
ISSN: | 0255-0857 |
Popis: | Purpose: 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. Material and 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. |
Databáze: | OpenAIRE |
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