J Vet Sci.  2014 Jun;15(2):233-239. 10.4142/jvs.2014.15.2.233.

A novel low-cost method for Mycobacterium avium subsp. paratuberculosis DNA extraction from an automated broth culture system for real-time PCR analysis

• Salgado M
• Verdugo C
• Heuer C
• Castillo P
• Zamorano P

• KMID: 1784644
• DOI: http://doi.org/10.4142/jvs.2014.15.2.233

Abstract

PCR is a highly accurate technique for confirming the presence of Mycobacterium avium subsp. paratuberculosis (Map) in broth culture. In this study, a simple, efficient, and low-cost method of harvesting DNA from Map cultured in liquid medium was developed. The proposed protocol (Universidad Austral de Chile [UACH]) was evaluated by comparing its performance to that of two traditional techniques (a QIAamp DNA Stool Mini Kit and cethyltrimethylammonium bromide [CTAB] method). The results were statistically assessed by agreement analysis for which differences in the number of cycles to positive (CP) were compared by Student's t-test for paired samples and regression analysis. Twelve out of 104 fecal pools cultured were positive. The final PCR results for 11 samples analyzed with the QIAamp and UACH methods or ones examined with the QIAamp and CTAB methods were in agreement. Complete (100%) agreement was observed between data from the CTAB and UACH methods. CP values for the UACH and CTAB techniques were not significantly different, while the UACH method yielded significantly lower CP values compared to the QIAamp kit. The proposed extraction method combines reliability and efficiency with simplicity and lower cost.

Keyword

DNA extraction; Map; MGIT; paratuberculosis; PCR

MeSH Terms

Animals
Bacteriological Techniques/economics/*veterinary
Cattle
Cattle Diseases/diagnosis/*microbiology
DNA, Bacterial/chemistry/genetics
Female
Mycobacterium avium subsp. paratuberculosis/*genetics
Paratuberculosis/diagnosis/*microbiology
Real-Time Polymerase Chain Reaction/veterinary
Reproducibility of Results
DNA, Bacterial

Figure

• 

  Fig. 1 Bland-Altman plots for comparing all cycle positive values with mean differences and 95% limits of agreement. (A) Comparison of results for the QIAamp kit and UACH method. (B) Comparison of data for the CTAB and UACH methods. (C) Comparison of the results for the QIAamp kit and CTAB method.

Reference

1. Alinovi CA, Ward MP, Lin TL, Moore GE, Wu CC. Real-time PCR, compared to liquid and solid culture media and ELISA, for the detection of Mycobacterium avium ssp. paratuberculosis. Vet Microbiol. 2009; 136:177–179.

2. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet. 1986; 1:307–310.

3. Cook KL, Britt JS. Optimization of methods for detecting Mycobacterium avium subsp. paratuberculosis in environmental samples using quantitative, real-time PCR. J Microbiol Methods. 2007; 69:154–160.

4. Dvorska L, Bull TJ, Bartos M, Matlova L, Svastova P, Weston RT, Kintr J, Parmova I, Van Soolingen D, Pavlik I. A standardised restriction fragment length polymorphism (RFLP) method for typing Mycobacterium avium isolates links IS901 with virulence for birds. J Microbiol Methods. 2003; 55:11–27.

5. Grant IR, Kirk RB, Hitchings E, Rowe MT. Comparative evaluation of the MGIT™ BACTEC culture systems for the recovery of Mycobacterium avium subsp. paratuberculosis from milk. J Appl Microbiol. 2003; 95:196–201.

6. Gumber S, Whittington RJ. Comparison of BACTEC 460 and MGIT 960 systems for the culture of Mycobacterium avium subsp. paratuberculosis S strain and observations on the effect of inclusion of ampicillin in culture media to reduce contamination. Vet Microbiol. 2007; 119:42–52.

7. Harris NB, Barletta RG. Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine. Clin Microbiol Rev. 2001; 14:489–512.

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

9. Herthnek D, Englund S, Willemsen PTJ, Bölske G. Sensitive detection of Mycobacterium avium subsp. paratuberculosis in bovine semen by real-time PCR. J Appl Microbiol. 2006; 100:1095–1102.

10. Hosek J, Svastova P, Moravkova M, Pavlik I, Bartos M. Methods of mycobacterial DNA isolation from different biological material: a review. Vet Med (Praha). 2006; 51:180–192.

11. Käser M, Ruf MT, Hauser J, Marsollier L, Pluschke G. Optimized method for preparation of DNA from pathogenic and environmental mycobacteria. Appl Environ Microbiol. 2009; 75:414–418.

12. Kim SG, Kim EH, Lafferty CJ, Miller LJ, Koo HJ, Stehman SM, Shin SJ. Use of conventional and real-time polymerase chain reaction for confirmation of Mycobacterium avium subsp. paratuberculosis in a broth-based culture system ESP II. J Vet Diagn Invest. 2004; 16:448–453.

13. Lee A, Griffiths TA, Parab RS, King RK, Dubinsky MC, Urbanski SJ, Wrobel I, Rioux KP. Association of Mycobacterium avium subspecies paratuberculosis with Crohn disease in pediatric patients. J Pediatr Gastroenterol Nutr. 2011; 52:170–174.

14. Lombard JE. Epidemiology and economics of paratuberculosis. Vet Clin North Am Food Anim Pract. 2011; 27:525–535.

15. Motiwala AS, Strother M, Theus NE, Stich RW, Byrum B, Shulaw WP, Kapur V, Sreevatsan S. Rapid detection and typing of strains of Mycobacterium avium subsp. paratuberculosis from broth cultures. J Clin Microbiol. 2005; 43:2111–2117.

16. Nielsen SS, Toft N. Ante mortem diagnosis of paratuberculosis: a review of accuracies of ELISA, interferon-γ assay and faecal culture techniques. Vet Microbiol. 2008; 129:217–235.

17. Odumeru J, Gao A, Chen S, Raymond M, Mutharia L. Use of the bead beater for preparation of Mycobacterium paratuberculosis template DNA in milk. Can J Vet Res. 2001; 65:201–205.

18. Pavlik I, Horvathova A, Dvorska L, Bartl J, Svastova P, du Maine R, Rychlik I. Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis. J Microbiol Methods. 1999; 38:155–167.

19. Svastova P, Pavlik I, Bartos M. Rapid differentiation of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis by amplification of insertion element IS901. Vet Med (Praha). 2002; 47:117–121.

20. Sweeney RW, Whitlock RH, McAdams SC. Comparison of three DNA preparation methods for real-time polymerase chain reaction confirmation of Mycobacterium avium subsp. paratuberculosis growth in an automated broth culture system. J Vet Diagn Invest. 2006; 18:587–590.

21. Tortoli E, Nanetti A, Piersimoni C, Cichero P, Farina C, Mucignat G, Scarparo C, Bartolini L, Valentini R, Nista D, Gesu G, Tosi CP, Crovatto M, Brusarosco G. Performance assessment of new multiplex probe assay for identification of mycobacteria. J Clin Microbiol. 2001; 39:1079–1084.

22. van Soolingen D, Hermans PW, de Haas PE, Soll DR, van Embden JD. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol. 1991; 29:2578–2586.

23. Wells SJ, Collins MT, Faaberg KS, Wees C, Tavornpanich S, Petrini KR, Collins JE, Cernicchiaro N, Whitlock RH. Evaluation of a rapid fecal PCR test for detection of Mycobacterium avium subsp. paratuberculosis in dairy cattle. Clin Vaccine Immunol. 2006; 13:1125–1130.

24. Whittington RJ, Marsh I, Turner MJ, McAllister S, Choy E, Eamens GJ, Marshall DJ, Ottaway S. Rapid detection of Mycobacterium paratuberculosis in clinical samples from ruminants and in spiked environmental samples by modified BACTEC 12B radiometric culture and direct confirmation by IS900 PCR. J Clin Microbiol. 1998; 36:701–707.

25. Whittington RJ, Marsh I, McAllister S, Turner MJ, Marshall DJ, Fraser CA. Evaluation of modified BACTEC 12B radiometric medium and solid media for culture of Mycobacterium avium subsp. paratuberculosis from sheep. J Clin Microbiol. 1999; 37:1077–1083.