Skip to main content
SpringerLink
Log in

An Appraisal of PCR-Based Technology in the Detection of Mycobacterium tuberculosis

  • Review Article
  • Published:
Molecular Diagnosis & Therapy Aims and scope Submit manuscript

Abstract

Tuberculosis is an under-recognized yet catastrophic health problem, particularly in developing countries. The HIV pandemic has served to increase the number of susceptible individuals, and multidrug-resistance and poor socioeconomic conditions also augment the prevalence and the consequences of the disease. To control the disease and its spread, it is vital that tuberculosis diagnostics are accurate and rapid. Whereas microscopy and culture have several limitations (low sensitivity is a problem for the former, while the latter has a delayed turnaround time), PCR-based techniques targeting regions of the Mycobacterium tuberculosis genome such as IS6110 have proved to be useful.

The purpose of this review is to assess the use of PCR-RFLP, nested PCR and real-time PCR protocols and the choice of target regions for the detection of M. tuberculosis. Real-time PCR for the detection of M. tuberculosis target genes in clinical specimens has contributed to improving diagnosis and epidemiologic surveillance in the past decade. However, targeting one genome sequence such as IS6110 may not by itself be sufficiently sensitive to reach 100% diagnosis, especially in the case of pulmonary tuberculosis. Additional testing for target genome sequences such as hsp65 seems encouraging. An interesting approach would be a multiplex real-time PCR targeting both IS6110 and hsp65 to achieve comprehensive and specific molecular diagnosis. This technology needs development and adequate field testing before it becomes the acceptable gold standard for diagnosis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Table I
Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. MacNeil JR, McRill C, Steinhauser G, et al. Jails, a neglected opportunity for tuberculosis prevention. Am J Prev Med 2005 Feb; 28(2): 225–8

    Article  PubMed  Google Scholar 

  2. World Health Organization. Global tuberculosis control: a short update to the 2009 report. Geneva: WHO, 2009 [online]. Available from URL: http://www.who.int/tb/publications/global_report/2009/update/en/index.html [Accessed 2010 Nov 11]

  3. Pantoja A, Floyd K, Unnikrishnan KP, et al. Economic evaluation of publicprivate mix for tuberculosis care and control, India: part I. Socio-economic profile and costs among tuberculosis patients. Int J Tuberc Lung Dis 2009 Jun; 13(6): 698–704

    PubMed  Google Scholar 

  4. Pratt RJ. Extensively drug-resistant (XDR) tuberculosis: a new threat to global public health. Br J Infect Control 2007 Apr; 8(2): 20–2

    Article  Google Scholar 

  5. Chakravorty S, Sen MK, Tyagi JS. Diagnosis of extrapulmonary tuberculosis by smear, culture, and PCR using universal sample processing technology. J Clin Microbiol 2005 Jun; 43(6): 4357–62

    Article  PubMed  CAS  Google Scholar 

  6. Singh NP, Parija SC. The value of fluorescence microscopy of auramine stained sputum smears for the diagnosis of pulmonary tuberculosis. Southeast Asian J Trop Med Public Health 1998 Dec; 29(4): 860–3

    PubMed  CAS  Google Scholar 

  7. Gennaro ML. Immunologic diagnosis of tuberculosis. Clin Infect Dis 2000; 30 Suppl. 3: S243–6

    Article  PubMed  Google Scholar 

  8. Bhatia AS, Kumar S, Harinath BC. Immunodiagnosis of tuberculosis: an update. Indian J Clin Biochem 2003; 18(2): 1–5

    Article  PubMed  CAS  Google Scholar 

  9. Steingart KR, Henry M, Laal S, et al. A systematic review of commercial serological antibody detection tests for the diagnosis of extrapulmonary tuberculosis. Postgrad Med J 2007 Nov; 83(985): 705–12

    Article  PubMed  Google Scholar 

  10. Lyamuya E, Bredberg-R0dén U, Massawe A, et al. Performance of a modified HIV-1 p24 antigen assay for early diagnosis of HIV-1 infection in infants and prediction of mother-to-infant transmission of HIV-1 in Dar es Salaam, Tanzania. J Acquir Immune Defic Syndr Hum Retrovirol 1996 Aug 1; 12(4): 421–6

    Article  PubMed  CAS  Google Scholar 

  11. Seremba E, Ocama P, Opio CK, et al. Poor performance of hepatitis C antibody tests in hospital patients in Uganda. J Med Virol 2010 Aug; 82(8): 1371–8

    Article  PubMed  CAS  Google Scholar 

  12. Savelkoul PH, Catsburg A, Mulder S, et al. Detection of Mycobacterium tuberculosis complex with real-time PCR: comparison of different primer-probe sets based on the IS6110 element. J Microbiol Methods 2006; 66(1): 177–80

    Article  PubMed  CAS  Google Scholar 

  13. Tortoli E, Marcelli F. Use of the INNO LiPA Rif. TB for detection of Mycobacterium tuberculosis DNA directly in clinical specimens and for simultaneous determination of rifampin susceptibility. Eur J Clin Microbiol Infect Dis 2007 Jan; 26(1): 51–5

    Article  PubMed  CAS  Google Scholar 

  14. Takahashi T, Tamura M, Asami Y, et al. Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology. J Clin Microbiol 2008 May; 46(5): 1708–15

    Article  PubMed  CAS  Google Scholar 

  15. Nandagopal B, Sankar S, Lingesan K, et al. Evaluation of a nested PCR targeting IS6110 of Mycobacterium tuberculosis for detection of the organism in the leukocyte fraction of blood samples. Indian J Med Microbiol 2010 Jul; 28(3): 227–32

    Article  PubMed  CAS  Google Scholar 

  16. Pinsky BA, Banaei N. Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level. J Clin Microbiol 2008 Jul; 46(7): 2241–6

    Article  PubMed  CAS  Google Scholar 

  17. Shah DH, Verma R, Bakshi CS, et al. A multiplex-PCR for the differentiation of Mycobacterium bovis and Mycobacterium tuberculosis. FEMS Microbiol Lett 2002; 214(1): 39–43

    Article  PubMed  CAS  Google Scholar 

  18. Greco S, Rulli M, Girardi E, et al. Diagnostic accuracy of in-house PCR for pulmonary tuberculosis in smear-positive patients: meta-analysis and meta-regression. J Clin Microbiol 2009 Mar; 47: 569–76

    Article  PubMed  CAS  Google Scholar 

  19. Gunson RN, Collins TC, Carman WF. Practical experience of high throughput real-time PCR in the routine diagnostic virology setting. J Clin Virol 2006 Apr; 35(4): 355–67

    Article  PubMed  CAS  Google Scholar 

  20. Herthnek D, Bolske G. New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis. BMC Microbiol 2006 Jan; 6: 87

    Article  PubMed  Google Scholar 

  21. Mackay IM, Arden KE, Nitsche A. Real-time PCR in virology. Nucleic Acids Res 2002 Mar; 30: 1292–305

    CAS  Google Scholar 

  22. Stuyver L, Rossau R, Wyseur A, et al. Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay. J Gen Virol 1993; 74: 1093–102

    Article  PubMed  CAS  Google Scholar 

  23. Mijs W, De Vreese K, Devos A, et al. Evaluation of a commercial line probe assay for identification of mycobacterium species from liquid and solid culture. Eur J Clin Microbiol Infect Dis 2002 Nov; 21(11): 794–802

    Article  PubMed  CAS  Google Scholar 

  24. Yoshida S, Suzuki K, Iwamoto T, et al. Comparison of rifabutin susceptibility and rpoB mutations in multi-drug-resistant Mycobacterium tuberculosis strains by DNA sequencing and the line probe assay. J Infect Chemother 2010 Oct; 16(5): 360–3

    Article  PubMed  CAS  Google Scholar 

  25. Gopinath K, Kumar S, Singh S. Prevalence of mycobacteremia in Indian HIV-infected patients detected by the MB/BacT automated culture system. Eur J Clin Microbiol Infect Dis 2008 Jun; 27(6): 423–31

    Article  PubMed  CAS  Google Scholar 

  26. Ganguly KC, Hiron MM, Mridha ZU, et al. Comparison of sputum induction with broncho-alveolar lavage in the diagnosis of smear-negative pulmonary tuberculosis. Mymensingh Med J 2008 Jul; 17(2): 115–23

    PubMed  CAS  Google Scholar 

  27. Ani A, Okpe S, Akambi M, et al. Comparison of a DNA based PCR method with conventional methods for the detection of M. tuberculosis in Jos, Nigeria. J Infect Dev Countries 2009 Jul 1; 3(6): 470–5

    CAS  Google Scholar 

  28. James P, Gupta R, Christopher DJ, et al. Evaluation of the diagnostic yield and safety of closed pleural biopsy in the diagnosis of pleural effusion. Indian J Tuberc 2010 Jan; 57(1): 19–24

    PubMed  Google Scholar 

  29. Oberoi A, Kaur H. Comparison of rapid colorimetric method with conventional method in the isolation of Mycobacterium tuberculosis. Indian J Med Microbiol 2004 Jan; 22(1): 44–6

    PubMed  CAS  Google Scholar 

  30. Peres RL, Maciel EL, Morais CG, et al. Comparison of two concentrations of NALC-NaOH for decontamination of sputum for mycobacterial culture. Int J Tuberc Lung Dis 2009 Dec; 13(12): 1572–5

    PubMed  CAS  Google Scholar 

  31. Sankar S, Balakrishnan B, Nandagopal B, et al. Comparative evaluation of nested PCR and conventional smear methods for the detection of Mycobacterium tuberculosis in sputum samples. Mol Diagn Ther 2010; 14(4): 223–7

    Article  PubMed  CAS  Google Scholar 

  32. Narayanan S, Parandaman V, Narayanan PR, et al. Evaluation of PCR using TRC4 and IS6110 primers in detection of tuberculous meningitis. J Clin Microbiol 2001 May; 39: 2006–8

    Article  PubMed  CAS  Google Scholar 

  33. Kocagöz T, Yilmaz E, Ozkara S, et al. Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplified procedure. J Clin Microbiol 1993 Jun; 31: 1435–8

    PubMed  Google Scholar 

  34. Stefano CR, Vago L, Bonetto S, et al. Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR. Mol Pathol 1999 Jun; 52: 158–60

    Article  Google Scholar 

  35. Boddinghaus B, Wichelhaus TA, Brade V, et al. Removal of PCR inhibitors by silica membranes: evaluating the Amplicor Mycobacterium tuberculosis kit. J Clin Microbiol 2001 Oct; 39: 3750–2

    Article  PubMed  CAS  Google Scholar 

  36. Amaro A, Duarte E, Amado A, et al. Comparison of three DNA extraction methods for Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium. Lett Appl Microbiol 2008 Jul; 47(1): 8–11

    Article  PubMed  CAS  Google Scholar 

  37. Elbir H, Muhsin A, Muhsin A, et al. A one-step DNA PCR-based method for the detection of Mycobacterium tuberculosis complex grown on Lowenstein-Jensen media. Am J Trop Med Hyg 2008 Feb; 78: 316–7

    PubMed  CAS  Google Scholar 

  38. van Embden JD, Cave MD, Crawford JT, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 1993 Feb; 31(2): 406–9

    PubMed  Google Scholar 

  39. Kamerbeek J, Schouls L, Kolk A, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997 Apr; 35(4): 907–14

    PubMed  CAS  Google Scholar 

  40. Supply P, Lesjean S, Savine E, et al. Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J Clin Microbiol 2001 Oct; 39(10): 3563–71

    Article  PubMed  CAS  Google Scholar 

  41. Supply P, Allix C, Lesjean S, et al. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 2006 Dec; 44(12): 4498–510

    Article  PubMed  CAS  Google Scholar 

  42. Williams KJ, Ling CL, Jenkins C, et al. A paradigm for the molecular identification of Mycobacterium species in a routine diagnostic laboratory. J Med Microbiol 2007 May; 56: 598–602

    Article  PubMed  CAS  Google Scholar 

  43. Foongladda S, Pholwat S, Eampokalap B, et al. Multi-probe real-time PCR identification of common Mycobacterium species in blood culture broth. J Mol Diagn 2009; 11(1): 42–8

    Article  PubMed  CAS  Google Scholar 

  44. Richardson ET, Samson D, Banaei N. Rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria by multiplex, real-time PCR. J Clin Microbiol 2009 May; 47(5): 1497–502

    Article  PubMed  CAS  Google Scholar 

  45. Patel JB, Leonard DG, Pan X, et al. Sequence-based identification of Mycobacterium species using the MicroSeq 500 16S rDNA bacterial identification system. J Clin Microbiol 2000 Jan; 38(1): 246–51

    PubMed  CAS  Google Scholar 

  46. Wilson KH, Blitchington RB, Greene RC. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J Clin Microbiol 1990 Sep; 28: 1942–6

    PubMed  CAS  Google Scholar 

  47. Kirschner P, Springer B, Vogel U, et al. Genotypic identification of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 1993 Nov; 31(11): 2882–9

    PubMed  CAS  Google Scholar 

  48. Tumwasorn S, Kwanlertjit S, Mokmued S, et al. Comparison of DNA targets for amplification by polymerase chain reaction for detection of Mycobacterium tuberculosis in sputum. J Med Assoc Thai 1996; 79 Suppl. 1: 113–8

    Google Scholar 

  49. Wei CY, Lee CN, Chu CH, et al. Determination of the sensitivity and specificity of PCR assays using different target DNAs for the detection of Mycobacterium tuberculosis. Kaohsiung J Med Sci 1999; 15(7): 396–405

    PubMed  CAS  Google Scholar 

  50. Gengvinij N, Pattanakitsakul SN, Chierakul N, et al. Detection of Mycobacterium tuberculosis from sputum specimens using one-tube nested PCR. Southeast Asian J Trop Med Public Health 2001 Mar; 32: 114–25

    PubMed  CAS  Google Scholar 

  51. Bannalikar AS, Verma R. Detection of Mycobacterium avium & M. tuberculosis from human sputum cultures by PCR-RFLP analysis of hsp65 gene and pncA PCR. Indian J Med Res 2006 Feb; 123(2): 165–72

    PubMed  CAS  Google Scholar 

  52. Mathema B, Kurepina NE, Bifani PJ, et al. Molecular epidemiology of tuberculosis: current insights. Clin Microbiol Rev 2006 Oct; 19(4): 658–85

    Article  PubMed  CAS  Google Scholar 

  53. Hanekom M, van der Spuy GD, Gey van Pittius NC, et al. Discordance between mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing and IS6110 restriction fragment length polymorphism enotyping for analysis of Mycobacterium tuberculosis Beijing strains in a setting of high incidence of tuberculosis. J Clin Microbiol 2008 Oct; 46(10): 3338–45

    Article  PubMed  CAS  Google Scholar 

  54. Narayanan S, Das S, Garg R, et al. Molecular epidemiology of tuberculosis in a rural area of high prevalence in South India: implications for disease control and prevention. J Clin Microbiol 2002 Dec; 40(12): 4785–8

    Article  PubMed  CAS  Google Scholar 

  55. Sankar S, Balakrishnan B, Nandagopal B, et al. Comparative evaluation of two polymerase chain reactions targeting different genomic regions to detect Mycobacterium tuberculosis in sputum. Indian J Med Microbiol 2010 Oct–Dec; 28(4): 303–7

    Article  PubMed  CAS  Google Scholar 

  56. Balamurugan R, Venkataraman S, John KR, et al. PCR amplification of the IS6110 insertion element of Mycobacterium tuberculosis in fecal samples from patients with intestinal tuberculosis. J Clin Microbiol 2006 May; 44(5): 1884–6

    Article  PubMed  CAS  Google Scholar 

  57. Querol JM, Farga MA, Granda D, et al. The utility of polymerase chain reaction (PCR) in the diagnosis of pulmonary tuberculosis. Chest 1995 Jun; 107(6): 1631–5

    Article  PubMed  CAS  Google Scholar 

  58. Borun M, Sajduda A, Pawlowska I, et al. Detection of Mycobacterium tuberculosis in clinical samples using insertion sequences IS6110 and IS990. Tuberculosis (Edinb) 2001 Jan; 81(4): 271–8

    Article  CAS  Google Scholar 

  59. Ben Kahla I, Ben Selma W, Marzouk M, et al. Evaluation of a simplified IS6110 PCR for the rapid diagnosis of Mycobacterium tuberculosis in an area with high tuberculosis incidence. Pathol Biol (Paris). Epub 2009 May 22

  60. Basil MV, Pathak R, Singh K, et al. Direct early identification of Mycobacterium tuberculosis by PCR-restriction fragment length polymorphism analysis from clinical samples. Jpn J Infect Dis 2010; 63: 55–7

    Google Scholar 

  61. Hidaka E, Honda T, Ueno I, et al. Sensitive identification of mycobacterial species using PCR-RFLP on bronchial washings. Am J Respir Crit Care Med 2000; 161: 930–4

    PubMed  CAS  Google Scholar 

  62. Broccolo F, Scarpellini P, Locatelli G, et al. Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by two realtime calibrated PCR assays. J Clin Microbiol 2003 Oct; 41(10): 4565–72

    Article  PubMed  CAS  Google Scholar 

  63. Halse TA, Edwards J, Cunningham PL, et al. Combined real-time PCR and rpoB gene pyrosequencing for rapid identification of Mycobacterium tuberculosis and determination of rifampin resistance directly in clinical specimens. J Clin Microbiol 2010 Apr; 48(4): 1182–8

    Article  PubMed  CAS  Google Scholar 

  64. Mathema B, Bifani PJ, Driscoll J, et al. Identification and evolution of an IS6110 low-copy-number Mycobacterium tuberculosis cluster. J Infect Dis 2002; 185: 641–9

    Article  PubMed  CAS  Google Scholar 

  65. Devallois A, Goh KS, Rastogi N. Rapid identification of mycobacteria to species level by PCR-restriction fragment length polymorphism analysis of the hsp65 gene and proposition of an algorithm to differentiate 34 myco-bacterial species. J Clin Microbiol 1997 Nov; 35(11): 2969–73

    PubMed  CAS  Google Scholar 

  66. Taylor TB, Patterson C, Hale Y, et al. Routine use of PCR-restriction fragment length polymorphism analysis for identification of mycobacteria growing in liquid media. J Clin Microbiol 1997 Jan; 35(1): 79–85

    PubMed  CAS  Google Scholar 

  67. Kim H, Kim SH, Shim TS, et al. Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65). Int J Syst Evol Microbiol 2005 Jul; 55: 1649–56

    Article  PubMed  CAS  Google Scholar 

  68. Khosravi AD, Seghatoleslami S, Hashemzadeh M. Application of PCR-based fingerprinting for detection of nontuberculous mycobacteria among patients referred to tuberculosis reference center of Khuzestan province, Iran. Res J Microbiol 2009; 4(4): 143–9

    Article  CAS  Google Scholar 

  69. Centers for Disease Control and Prevention (CDC). Human tuberculosis caused by Mycobacterium bovis — New York City, 2001-2004. MMWR Morb Mortal Wkly Rep 2005; 54(24): 605–8

    Google Scholar 

  70. Prasad HK, Singhal A, Mishra A, et al. Bovine tuberculosis in India: potential basis for zoonosis. Tuberculosis (Edinb) 2005 Sep–Nov; 85(5-6): 421–8

    Article  CAS  Google Scholar 

  71. da Silva Rocha A, Barreto AMW, Campos CED, et al. Novel allelic variants of Mycobacteria isolated in Brazil as determined by PCR-restriction enzyme analysis of hsp65. J Clin Microbiol 2002 Nov; 40(11): 4191–6

    Article  PubMed  Google Scholar 

  72. Kim K, Lee H, Lee MK, et al. Development and application of multiprobe realtime PCR method targeting the hsp65 gene for differentiation of Mycobacterium species from isolates and sputum specimens. J Clin Microbiol 2010 Sep; 48(9): 3073–80

    Article  PubMed  CAS  Google Scholar 

  73. Dubaniewicz A, Wybieralska MD, Sternau A, et al. Mycobacterium tuberculosis complex and mycobacterial heat shock proteins in lymph node tissue from patients with pulmonary sarcoidosis. J Clin Microbiol 2006 Sep; 44(9): 3448–51

    Article  PubMed  Google Scholar 

  74. König B, Tammer I, Sollich V, et al. Intra- and interpatient variability of the hsp65 and 16S-23S intergenic gene region in Mycobacterium abscessus strains from patients with cystic fibrosis. J Clin Microbiol 2005 Jul; 43(7): 3500–3

    Article  PubMed  Google Scholar 

  75. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molec Biol Evolution 2007; 24: 1596–9

    Article  CAS  Google Scholar 

  76. Hillemann D, Warren R, Kubica T, et al. Rapid detection of Mycobacterium tuberculosis Beijing genotype strains by real-time PCR. J Clin Microbiol 2006 Feb; 44(2): 302–6

    Article  PubMed  CAS  Google Scholar 

  77. Haldar S, Chakravorty S, Bhalla M, et al. Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons. J Med Microbiol 2007 Oct; 56: 1356–62

    Article  PubMed  CAS  Google Scholar 

  78. Kumar P, Nath K, Rath B, et al. Visual format for detection of Mycobacterium tuberculosis and M. bovis in clinical samples using molecular beacons. J Mol Diagn 2009; 11:430–8

    Article  PubMed  CAS  Google Scholar 

  79. Park JS, Kang YA, Kwon SY, et al. Nested PCR in lung tissue for diagnosis of pulmonary tuberculosis. Eur Respir J 2010 Apr; 35(4): 851–7

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

No sources of funding were used to prepare this manuscript. The authors have no conflicts of interest that are directly relevant to the content of this review.

Author information

Authors and Affiliations

  1. Division of Biomedical Research, Sri Narayani Hospital and Research Centre, Thirumalaikodi, Sripuram, Vellore, 632 055, Tamil Nadu, India

    Sathish Sankar, Mageshbabu Ramamurthy, Balaji Nandagopal & Gopalan Sridharan

Authors
  1. Sathish Sankar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mageshbabu Ramamurthy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Balaji Nandagopal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gopalan Sridharan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sathish Sankar.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sankar, S., Ramamurthy, M., Nandagopal, B. et al. An Appraisal of PCR-Based Technology in the Detection of Mycobacterium tuberculosis . Mol Diagn Ther 15, 1–11 (2011). https://doi.org/10.1007/BF03257188

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03257188

Keywords

Search

Navigation