Improvement of Mycobacterium tuberculosis detection in clinical samples using DNA purified by glass matrix

• Published in: Journal of microbiological methods Vol. 28; no. 2; pp. 139 - 146
• Main Authors: Rossetti, Maria L.R., B. Jardim, Susana, Rodrigues, Vivian de F.S., Moura, Andréia R., Oliveira, Hugo, Zaha, Arnaldo
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01.02.1997; Elsevier Science
• Subjects: Bacterial diseases; Biological and medical sciences; Diagnosis; Human bacterial diseases; Infectious diseases; Medical sciences; Mycobacterium tuberculosis; Polymerase chain reaction; Tuberculosis and atypical mycobacterial infections; Polymerase chain reaction; Mycobacterium tuberculosis; Diagnosis; Performance evaluation; Human; Purification; Respiratory disease; Mycobacterial infection; Method; Infection; Tuberculosis; Mycobacteriales; DNA; Bacteriosis; Mycobacteriaceae; Bacteria; Actinomycetes; Detection
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(97)00978-0

A glass matrix was used to purify Mycobacterium tuberculosis DNA from clinical samples before amplification by polymerase chain reaction (PCR). The procedure was established by analysing a subset of 130 clinical samples (70 from patients with tuberculosis and 60 from patients without tuberculosis). The clinical samples included sputum, urine, cerebrospinal fluid, blood and serum. The total number of samples positive by PCR before the glass matrix step was 33 and after the glass matrix step was 58, showing that this purification step improved markedly the detection of M. tuberculosis DNA. The detection limit of the amplified DNA in agarose gels stained with ethidium bromide was 10 CFU for serum and cerebrospinal fluid and 50 CFU for sputum. Once established, the procedure was used in the analysis of 179 additional samples from 133 patients suspected of tuberculosis. In general there was an agreement between the results of PCR and clinical diagnosis for tuberculosis. In the case of sputum samples it was possible to compare the PCR and culture results. When considering all the sputum samples analysed, in general the PCR was slightly less sensitive than the culture test. However, when applied to the analysis of samples from patients suspected of tuberculosis, six samples that were culture negative were positive by PCR. Considering the confirmed results of all clinical samples analysed, the sensitivity of the method described in this work (97.4%) was much higher than culture test (45.5%). This difference was mainly due to the low sensitivity of culture test in cerebrospinal fluid, serum and blood samples. The results presented in this work indicate that the inclusion of the glass matrix step in the preparation of DNA improves the detection of M. tuberculosis in clinical samples.