Korean J Clin Microbiol.  2011 Sep;14(3):97-102. 10.5145/KJCM.2011.14.3.97.

Removal of PCR Inhibitors in Real-time PCR for Mycobacterium tuberculosis

• Yun HY
• Kim HS
• Lee YK
• Kang HJ
• Kim JS
• Song W
• Lee KM

• KMID: 1449479
• DOI: http://doi.org/10.5145/KJCM.2011.14.3.97

Abstract

BACKGROUND
The inhibition rates for nucleic acid tests of Mycobacterium tuberculosis have been reported to range from less than 1% to more than 10%. Specimen dilution, boiling, addition of bovine serum albumin (BSA), and a silica membrane can be used to override amplification inhibitors in nucleic acid tests of M. tuberculosis. The inhibition rate for real-time PCR of M. tuberculosis (COBAS TaqMan MTB test; Roche Diagnostics, Manheim, Germany) and effective strategies to override PCR inhibitors were investigated in this study.
METHODS
The inhibition rate for COBAS TaqMan MTB test was investigated in 980 clinical specimens. The effectiveness of PCR inhibitor removal by repeated run, dilution, boiling, addition of BSA, and use of silica membrane were evaluated in the inhibited specimens.
RESULTS
Inhibitory substances were present in 4.1% of specimens (40/980). Among 40 inhibited specimens, inhibitory substances were removed in 12 (30%), 30 (75%), 27 (67.5%), 25 (62.5%) and 12 (30%) specimens with repeated run, dilution, addition of RBS, boiling and use of silica membrane, respectively.
CONCLUSION
The overall inhibition rate for the COBAS TaqMan MTB test was 4.1%. Dilution, boiling and addition of BSA were shown to be more effective than repeated run and use of silica membrane for removal of PCR inhibitors. A combination of two methods might be useful and should be studied in the future.

Keyword

Mycobacterium tuberculosis; Real-time PCR; Inhibitors

MeSH Terms

Membranes
Mycobacterium
Mycobacterium tuberculosis
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Serum Albumin, Bovine
Silicon Dioxide
Tuberculosis
Serum Albumin, Bovine
Silicon Dioxide

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