Evaluation of the accuracy and reproducibility of a practical PCR panel assay for rapid detection and differentiation of Mycobacterium avium subspecies

J. L E Ellingson, J. R. Stabel, William Ramses Bishai, R. Frothingham, J. M. Miller

Research output: Contribution to journalArticle

Abstract

The Mycobacterium avium subspecies (MAs) include the closely related MAs avium and MAs paratuberculosis. This study was conducted to evaluate the performance of a PCR panel assay as a diagnostic tool to detect and differentiate MAs avium and MAs paratuberculosis infection. Specific oligonucleotides primers derived from the 16 S rRNA (MAs) sequence, insertion elements IS901 (MAs avium), IS1245 Mycobacterium avium complex (MAC), IS900 (MAs paratuberculosis), and the hspX (MAs paratuberculosis) gene sequences were synthesized and used in preassembled PCR reaction mixtures. These five primer sets made up the PCR panel assay. To determine the accuracy of the PCR panel assay for MAs avium and MAs paratuberculosis strain detection and differentiation, lysates of mycobacterial DNA from 120 (n = 120) strains were tested with the PCR panel assay by one laboratory (1). The PCR panel assay specifically detected and differentiated 91/91 (100%) of MAs avium and MAs paratuberculosis strains tested in this study. The PCR panel assay also specifically differentiated all MAs avium and MAs paratuberculosis strains from all but one (M. intracellulare, serovar 23) of the other mycobacterial strains tested. To confirm the accuracy and evaluate the reproducibility of the PCR panel assay, samples were numbered and given to a different laboratory (2) as 'unknowns' for identification by the PCR panel assay. In this study, the overall accuracy for strain identification using the PCR panel assay was 99.2% (119/120). The reproducibility of the PCR panel assay when comparing data from laboratory 1 with laboratory 2 was found to be 100% (120/120). These results indicate that this 'easy-to-use', rapid PCR method can accurately and reliably detect and differentiate closely related MAs avium and MAs paratuberculosis from each other and from other mycobacterial species. The PCR panel assay can also differentiate mixed cultures of MAs. The simplicity of this PCR method could be beneficial to laboratories that test for members of MAs. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)153-161
Number of pages9
JournalMolecular and Cellular Probes
Volume14
Issue number3
DOIs
StatePublished - Jun 2000

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Mycobacterium avium
Paratuberculosis
Polymerase Chain Reaction
Mycobacterium avium Complex
DNA Transposable Elements
DNA Primers

Keywords

  • Insertion elements
  • Mycobacterium avium subspecies (MAs)
  • PCR panel assay
  • Single-copy genes

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

Cite this

Evaluation of the accuracy and reproducibility of a practical PCR panel assay for rapid detection and differentiation of Mycobacterium avium subspecies. / Ellingson, J. L E; Stabel, J. R.; Bishai, William Ramses; Frothingham, R.; Miller, J. M.

In: Molecular and Cellular Probes, Vol. 14, No. 3, 06.2000, p. 153-161.

Research output: Contribution to journalArticle

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abstract = "The Mycobacterium avium subspecies (MAs) include the closely related MAs avium and MAs paratuberculosis. This study was conducted to evaluate the performance of a PCR panel assay as a diagnostic tool to detect and differentiate MAs avium and MAs paratuberculosis infection. Specific oligonucleotides primers derived from the 16 S rRNA (MAs) sequence, insertion elements IS901 (MAs avium), IS1245 Mycobacterium avium complex (MAC), IS900 (MAs paratuberculosis), and the hspX (MAs paratuberculosis) gene sequences were synthesized and used in preassembled PCR reaction mixtures. These five primer sets made up the PCR panel assay. To determine the accuracy of the PCR panel assay for MAs avium and MAs paratuberculosis strain detection and differentiation, lysates of mycobacterial DNA from 120 (n = 120) strains were tested with the PCR panel assay by one laboratory (1). The PCR panel assay specifically detected and differentiated 91/91 (100{\%}) of MAs avium and MAs paratuberculosis strains tested in this study. The PCR panel assay also specifically differentiated all MAs avium and MAs paratuberculosis strains from all but one (M. intracellulare, serovar 23) of the other mycobacterial strains tested. To confirm the accuracy and evaluate the reproducibility of the PCR panel assay, samples were numbered and given to a different laboratory (2) as 'unknowns' for identification by the PCR panel assay. In this study, the overall accuracy for strain identification using the PCR panel assay was 99.2{\%} (119/120). The reproducibility of the PCR panel assay when comparing data from laboratory 1 with laboratory 2 was found to be 100{\%} (120/120). These results indicate that this 'easy-to-use', rapid PCR method can accurately and reliably detect and differentiate closely related MAs avium and MAs paratuberculosis from each other and from other mycobacterial species. The PCR panel assay can also differentiate mixed cultures of MAs. The simplicity of this PCR method could be beneficial to laboratories that test for members of MAs. (C) 2000 Academic Press.",
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