Korean J Lab Med.  2007 Feb;27(1):40-45. 10.3343/kjlm.2007.27.1.40.

Usefulness of Multiplex Real-Time PCR and Melting Curve Analysis in Identification of Nontuberculous Mycobacteria

• Kang SH
• Yoo KC
• Park KU
• Song J
• Kim EC

• KMID: 1121658
• DOI: http://doi.org/10.3343/kjlm.2007.27.1.40

Abstract

BACKGROUND: Nontuberculous mycobacteria (NTM) should be correctly identified to the species level, because of different treatment plans among NTM species. This study was performed to assess the usefulness of real-time PCR and melting curve analysis in the identification of NTM. METHODS: One hundred fifty-two clinical NTM isolates were identified to the species level by PCR-restriction fragment length polymorphism analysis (PRA). Those strains were then identified by multiplex real-time PCR and melting curve analysis on the 16S rRNA gene and hsp65 gene. RESULTS: In the 16S rRNA gene fragment analysis, M. abscessus-M. chelonae group showed melting point at temperatures above 65 degrees C and M. avium complex (MAC; M. avium and M. intracelluare) below 48 degrees C, which differentiated M. abscessus-M. chelonae group and MAC from other NTM. In the hsp65 gene fragment analysis, M. abscessus-M. chelonae group was clearly divided into M. abscessus type I, M. abscessus type II, and M. chelonae according to the melting points at 61.25 degrees C, 66.06 degrees C, and 57.58 degrees C, respectively. CONCLUSIONS: With the multiplex real-time PCR and melting curve analysis of 16S rRNA and hsp65 genes, M. abscessus and M. chelonae were readily identified and MAC were differentiated from other NTM. Especially, M. abscessus and M. chelonae, which were not differentiated from each other with the 16S rRNA gene fragment analysis, were identified with hsp65 gene fragment analysis.

Keyword

Nontuberculous mycobacteria; Species identification; Real-time PCR; Melting curve analysis

MeSH Terms

Bacterial Proteins/genetics
Chaperonins/genetics
Computer Systems
DNA, Bacterial/chemistry
Mycobacteria, Atypical/genetics/*isolation & purification
Nucleic Acid Denaturation
Polymerase Chain Reaction/*methods
RNA, Ribosomal, 16S/genetics

Reference

References

1. Portaels F. Epidemiology of mycobacterial diseases. Clin Dermatol. 1995; 13:207–22.

2. Prince DS, Peterson DD, Steiner RM, Gottlieb JE, Scott R, Israel HL, et al. Infection with Mycobacterium avium complex in patients without predisposing conditions. N Engl J Med. 1989; 321:863–8.

3. Scientific Committee in Korean Academy of Tuberculosis and Respiratory Dieseases. National survey of mycobacterial diseases other than tuberculosis in Korea. Tuberc Respir Dis. 1995; 42:277–94.

4. Koh WJ, Kwon OJ, Lee KS. Diagnosis and treatment of nontuberculous mycobacterial pulmonary diseases: a Korean perspective. J Korean Med Sci. 2005; 20:913–25.

5. Jeong J, Lee SH, Jeong US, Chang CH, Kim SR. Identification of mycobacteria using high performance liquid chromatography in clinical specimens. Korean J Clin Micorbiol. 2004; 7:148–55.

6. Kim HK, Kim YR, Park JP, Kim NH, OK CH, Jung MH, et al. Isolation of nontuberculous mycobacteria by DNA probe and clincal characteristics of patients with NTM pulmonary disease. Tuberc Respir Dis. 2005; 58:248–56.

7. Lee H, Park HJ, Cho SN, Bai GH, Kim SJ. Species identification of mycobacteria by PCR-restriction fragment length polymorphism of the rpoB gene. J Clin Microbiol. 2000; 38:2966–71.

8. Kim H, Kim SH, Shim TS, Kim MN, Bai GH, Park YG, et al. PCR restriction fragment length polymorphism analysis (PRA)-algorithm targeting 644 bp Heat Shock Protein 65 (hsp65) gene for differentiation of Mycobacterium spp. J Microbiol Methods. 2005; 62:199–209.

9. Lachnik J, Ackermann B, Bohrssen A, Maass S, Diephaus C, Puncken A, et al. Rapid-cycle PCR and fluorimetry for detection of mycobacteria. J Clin Microbiol. 2002; 40:3364–73.

10. Shrestha NK, Tuohy MJ, Hall GS, Reischl U, Gordon SM, Procop GW. Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR. J Clin Microbiol. 2003; 41:5121–6.

11. Telenti A, Marchesi F, Balz M, Bally F, Bottger EC, Bodmer T. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol. 1993; 31:175–8.

12. Shinnick TM. The 65-kilodalton antigen of Mycobacterium tuberculosis. J Bacteriol. 1987; 169:1080–8.

13. Sedlacek L, Rifai M, Feldmann K, Bange FC. LightCycler-based differentiation of Mycobacterium abscessus and Mycobacterium chelonae. J Clin Microbiol. 2004; 42:3284–7.

14. Kusunoki S, Ezaki T. Proposal of Mycobacterium peregrinum sp. nov., nom. rev., and elevation of Mycobacterium chelonae subsp. abscessus (Kubica et al.) to species status: Mycobacterium abscessus comb. nov. Int J Syst Bacteriol. 1992; 42:240–5.

15. Ringuet H, Akoua-Koffi C, Honore S, Varnerot A, Vincent V, Berche P, et al. hsp65 sequencing for identification of rapidly growing mycobacteria. J Clin Microbiol. 1999; 37:852–7.

16. Koh WJ, Kwon OJ, Yu CM, Jeon K, Suh GY, Chung MP, et al. Recovery rate of nontuberculous mycobacteria from acid-fast-bacilli smear-positive sputum speciment. Tuberc Respir Dis. 2003; 54:22–32.

17. Park CM, Heo SR, Park KU, Song JH, Lee JH, Lee CT, et al. Isolation of nontuberculous mycobacteria using polymerase chain reaction-restriction fragment length polymorphism. Korean J Lab Med. 2006; 26:161–7.

18. Lee JY, Choi HJ, Lee HY, Joung EY, Huh JW, Oh YM, et al. Recovery rate and characteristics of nontuberculous mycobacterial isolates in a university hospital in Korea. Tuberc Respir Dis. 2005; 58:385–91.

19. Wagner D, Young LS. Nontuberculous mycobacterial infections: a clinical review. Infection. 2004; 32:257–70.

20. Griffith DE, Girard WM, Wallace RJ Jr. Clinical features of pulmonary disease caused by rapidly growing mycobacteria. An analysis of 154 patients. Am Rev Respir Dis. 1993; 147:1271–8.

21. Yang SC, Hsueh PR, Lai HC, Teng LJ, Huang LM, Chen JM, et al. High prevalence of antimicrobial resistance in rapidly growing mycobacteria in Taiwan. Antimicrob Agents Chemother. 2003; 47:1958–62.

22. British Thoracic Society. Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Subcommittee of the Joint Tuberculosis Committee of the British Thoracic Society. Thorax. 2000; 55:210–8.