Use of conventional and real-time polymerase chain reaction for confirmation of Mycobacterium avium subsp. paratuberculosis in a broth-based culture system ESP II

Sung G. Kim, Eun H. Kim, Caroline J. Lafferty, Loretta J. Miller, Hye J. Koo, Susan M. Stehman, SungJae Shin

Research output: Contribution to journalArticle

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Abstract

The ESP II Culture System (ESP II), a broth-based culture system, has been modified and optimized for culturing Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in animal feces since 2000. Conventional and real-time polymerase chain reaction (PCR) assays based on the IS900 sequence were performed as confirmatory tests for M. paratuberculosis in ESP II liquid culture medium. There were no differences between test results of conventional and real-time PCR assays. During the 5-week incubation period, if acid-fast bacilli (AFB) were detected in ESP culture-positive samples, IS900 PCR assays were performed to confirm whether those AFB were M. paratuberculosis. At the end of the 5-week incubation, AF staining was performed on all ESP II-negative cultures to screen any false-negative cultures; IS900 PCR assays were performed on AFB-positive cultures. During a period of 1 year, of a total of 18,499 ESP II cultures, 2,814 (15.2%) PCR confirmation assays were performed. Of those, 2,259 (80%) were both ESP and PCR positive; 104 (4%) were ESP positive and PCR negative; 423 (15%) were ESP negative and PCR positive; 28 (1%) were both ESP and PCR negative. The AF-staining step after the 5-week incubation produced 423 (15%) more PCR-positive cultures. Of a total of 2,814 AFB-positive cultures, 132 (5%) were not confirmed as M. paratuberculosis. Further studies are needed for speciation of non-M. paratuberculosis isolates.

Original languageEnglish
Pages (from-to)448-453
Number of pages6
JournalJournal of Veterinary Diagnostic Investigation
Volume16
Issue number5
DOIs
Publication statusPublished - 2004 Jan 1

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Paratuberculosis
Mycobacterium avium
Mycobacterium avium subsp. paratuberculosis
Real-Time Polymerase Chain Reaction
quantitative polymerase chain reaction
polymerase chain reaction
Polymerase Chain Reaction
Bacillus
assays
Acids
acids
Staining and Labeling
paratuberculosis
Feces
Culture Media
culture media
feces
testing
liquids
Bacilli

All Science Journal Classification (ASJC) codes

  • veterinary(all)

Cite this

Kim, Sung G. ; Kim, Eun H. ; Lafferty, Caroline J. ; Miller, Loretta J. ; Koo, Hye J. ; Stehman, Susan M. ; Shin, SungJae. / Use of conventional and real-time polymerase chain reaction for confirmation of Mycobacterium avium subsp. paratuberculosis in a broth-based culture system ESP II. In: Journal of Veterinary Diagnostic Investigation. 2004 ; Vol. 16, No. 5. pp. 448-453.
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abstract = "The ESP II Culture System (ESP II), a broth-based culture system, has been modified and optimized for culturing Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in animal feces since 2000. Conventional and real-time polymerase chain reaction (PCR) assays based on the IS900 sequence were performed as confirmatory tests for M. paratuberculosis in ESP II liquid culture medium. There were no differences between test results of conventional and real-time PCR assays. During the 5-week incubation period, if acid-fast bacilli (AFB) were detected in ESP culture-positive samples, IS900 PCR assays were performed to confirm whether those AFB were M. paratuberculosis. At the end of the 5-week incubation, AF staining was performed on all ESP II-negative cultures to screen any false-negative cultures; IS900 PCR assays were performed on AFB-positive cultures. During a period of 1 year, of a total of 18,499 ESP II cultures, 2,814 (15.2{\%}) PCR confirmation assays were performed. Of those, 2,259 (80{\%}) were both ESP and PCR positive; 104 (4{\%}) were ESP positive and PCR negative; 423 (15{\%}) were ESP negative and PCR positive; 28 (1{\%}) were both ESP and PCR negative. The AF-staining step after the 5-week incubation produced 423 (15{\%}) more PCR-positive cultures. Of a total of 2,814 AFB-positive cultures, 132 (5{\%}) were not confirmed as M. paratuberculosis. Further studies are needed for speciation of non-M. paratuberculosis isolates.",
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Use of conventional and real-time polymerase chain reaction for confirmation of Mycobacterium avium subsp. paratuberculosis in a broth-based culture system ESP II. / Kim, Sung G.; Kim, Eun H.; Lafferty, Caroline J.; Miller, Loretta J.; Koo, Hye J.; Stehman, Susan M.; Shin, SungJae.

In: Journal of Veterinary Diagnostic Investigation, Vol. 16, No. 5, 01.01.2004, p. 448-453.

Research output: Contribution to journalArticle

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