Differentiation between Mycobacterium tuberculosis and Mycobacterium avium by Amplification of the 16S-23S Ribosomal DNA Spacer

Abstract

Differentiation between Mycobacterium tuberculosis andM. avium is helpful for the treatment of disseminated mycobacterial infection in AIDS patients. This can traditionally be done by time-consuming biochemical tests or with Accuprobe. Previously, PCR restriction enzyme analysis (PCR-REA) of the 16S-23S rRNA gene spacer was shown to be able to identify a limited number of strains ofMycobacterium. In this study the method was improved by using more specific primers and was tested with 50 clinical isolates ofM. tuberculosis and 65 clinical isolates of M. avium complex. Probes specific to the spacers ofM. tuberculosis and M. avium were also tested. Both M. tuberculosis and M. avium could be reliably identified either by PCR-REA or by PCR-hybridization, with the results completely agreeing with those obtained by biochemical tests and with the Accuprobe, respectively. The method may therefore be useful as an alternative in-house method for identification of the bacteria.

Immune Reconstitution Syndrome From Nontuberculous Mycobacterial Infection After Initiation of Antiretroviral Therapy in Children With Hiv Infection

“…was carried out by using the polymerase chain reaction and restriction enzyme analysis technique as previously described. 16 Determination of CD4 cell count and plasma HIV RNA titer was performed at weeks 0, 8, 24 and 48 of ART and whenever clinical syndrome of IRS was noted. CD4 cell counts were performed by a FACSCount apparatus (Becton Dickinson, Mountain View, CA).…”

Rapid differentiation of mycobacteria by simplex real-time PCR with melting temperature calling analysis

“…A great effort is being made towards the development of tools for the differentiation of Mycobacterium species and molecular biology methods have shown great promise. Among these methods, amplification of species-specific sequences, PCR amplification and restriction enzyme analysis, hybridization with species-specific oligonucleotide probes with or without prior DNA amplification, nucleic acid sequence determination and MALDI-TOF-MS are time-saving and accurate but expensive and laborious (Sansila et al 1998). In contrast, real-time PCR with fluorogenic probes is rapid, simple and cost-effective for differentiating Mycobacterium species (Shrestha et al 2003;Tobler et al 2006;Lim et al 2008;Lee et al 2009).…”

Identification and Semiquantitation of Mycobacterium avium Using a Competitive PCR Method

“…To our knowledge, the present study is the first report on the rapid detection and quantitation of M. avium by direct PCR. Some PCR methods reported to date amplify all mycobacterial species in the first step, and subsequently identify M. avium by hybridization or restriction enzyme reactions (21,22). Therefore, the M. avium genome would not be amplified if much of the genome from other mycobacterium, such as M. tuberculosis, was present simultaneously in the sample (23), as is sometimes the case for AIDS.…”

“…Several approaches have been described for the identification of M. avium by polymerase chain reaction (PCR) (2,3,9,14,15,21,22). By those approaches, determination of the presence or absence of M. avium DNA is feasible, but quantitative information cannot be obtained.…”