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Yun, Kim, Lee, Kang, Kim, Song, and Lee: Removal of PCR Inhibitors in Real-time PCR for Mycobacterium tuberculosis
Original Article

Korean J Clin Microbiol 2011;14(3):97-102.

Published online: 21 September 2011

DOI: https://doi.org/10.5145/KJCM.2011.14.3.97

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

Hye Young Yun1, Han-Sung Kim1, 2, Young Kyung Lee1, 2, Hee Jung Kang1, 2, Jae-Seok Kim2, Wonkeun Song2, Kyu Man Lee2

1Department of Laboratory Medicine, Hallym University Sacred Heart Hospital, Chuncheon, Korea

2Hallym University College of Medicine, Chuncheon, Korea

Correspondence: Han-Sung Kim, Department of Laboratory Medicine, Hallym University Sacred Heart Hospital, 896 Pyeongchon-dong, Anyang 431-070, Korea. (Tel) 82-31-380-3932, (Fax) 82-31-380-3934, (E-mail) kimhan@hallym.ac.kr

Received 10 March 2011       Revised 1 June 2011       Accepted 3 August 2011

Copyright © 2011 Korean Journal of Clinical Microbiology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 realtime 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.

Keywords: Mycobacterium tuberculosis, Real-time PCR, Inhibitors

REFERENCES

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Table 1.
PCR inhibition rates according to specimen types
Specimen group No. of specimen No. (%) with inhibition
Respiratory tract∗ 561 18 (3.2)
Pleural fluid 191 17 (8.9)
Urine 74 5 (6.8)
CSF 58 0 (0.0)
Other Total 96 980 0 (0.0) 40 (4.1)

∗ Includes sputa (n=459), bronchial washing (n=1), and tracheal aspirates (n=1).

Table 2.
Resolution rate of PCR inhibition among 40 inhibited specimens by the methods to remove inhibitors
Specimen type No. of specimen No. (%) of resolution at repeat with the method
None Dilution∗ BSA† Boiling Silica membrane
Sputum 18 7 (38.9) 12 (66.7) 10 (55.6) 11 (61.1) 5 (27.8)
Pleural fluid 17 3 (17.6) 14 (83.5) 14 (83.5) 14 (83.5) 7 (41.2)
Urine 5 2 (40.0) 4 (80.0) 3 (60.0) 0 (0.0) 0 (0.0)
Total 40 12 (30.0) 30 (75.0) 27 (67.5) 25 (62.5) 12 (30)

∗ 1: 10 dilution

† 0.025% bovine serum albumin.

Table 3.
Repeated PCR results of 40 inhibited specimens with strategies to remove inhibitors
Specimen type None Dilution∗ BSA† Boiling Silica membrane
Sputum I I I I I
Sputum I I I I I
Sputum I I I I I
Sputum I I I I I
Sputum I N I I I
Sputum I N I I I
Sputum N N N I I
Sputum N N I N I
Sputum I N N N I
Sputum I N N N I
Sputum I N N N I
Sputum N N N N I
Sputum N N N N I
Sputum I I I N N
Sputum I I N N N
Sputum N N N N N
Sputum N N N N N
Sputum P P P P P
Pleural fluid I I N N N
Pleural fluid I N I I I
Pleural fluid N N I I I
Pleural fluid I I N N I
Pleural fluid I N N N I
Pleural fluid I N N N I
Pleural fluid I N N N I
Pleural fluid I N N N I
Pleural fluid I N N N I
Pleural fluid N N N N I
Pleural fluid I I I I N
Pleural fluid I N N N N
Pleural fluid I N N N N
Pleural fluid I N N N N
Pleural fluid I N N N N
Pleural fluid N N N N N
Pleural fluid I N N N I
Urine I I I I I
Urine I N I I I
Urine I N N I I
Urine N N N I I
Urine N N N I I

∗ 1: 10 dilution

† 0.025% bovine serum albumin. Abbreviations: I, inhibition; N, negative; P, positive.

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