مقایسه کیفی و کمی نتایج مربوط به چند روش استخراج DNA از ژنوم مایکوباکتریوم‌های بیماری‌زا و ساپروفیت

محورهای موضوعی : آسیب شناسی درمانگاهی دامپزشکی

علی نصیری ¹ , یونس انزابی ² , محمد رضا مشایخی ³

1 - دانش‌آموخته کارشناسی ارشد زیست‌شناسی- ژنتیک، دانشکده علوم‌پایه، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.
2 - استادیار گروه پاتوبیولوژی، دانشکده دامپزشکی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.
3 - استادیار گروه زیست شناسی، دانشکده علوم‌پایه، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.

تاریخ دریافت : 1396/04/30 تاریخ پذیرش : 1397/02/15 تاریخ انتشار : 1397/02/01

کلید واژه: ژنوم, استخراج DNA, مایکوباکتریوم‌, بیماری‌زا, ساپروفیت,

چکیده مقاله :

با توجه به اینکهنخستینمرحلهجهتانجامآزمایش هایمولکولیمانند واکنش زنجیره ای پلی مراز (Polymerase Chain Reaction; PCR)داشتن DNA کافیوباکیفیتمی باشد، هدفازانجاماینمطالعهمقایسهپنج روشمختلفاستخراج DNAژنومی مایکوباکتریوم هابه منظور معرفی بهترین روش بود. بدین منظور از پرگنه های خالص مایکوباکتریوم اویوم پاراتوبرکلوزیس به عنوان مایکوباکتریوم بیماری زا و نیز سویه ای از یک مایکوباکتریوم ساپروفیت به نام مایکوباکتریوم فلئی استفاده شد. کمیت و کیفیتDNAهای به دست آمده توسط هریکاز روش ها، به ترتیب بر اساس غلظت DNAاستخراجی (نانوگرم در میکرولیتر) در طول موج 260 نانومتر و نیز بر مبنای نسبت OD در طول موج های 280/260 با استفاده از دستگاه نانودراپ محاسبه و موردارزیابی قرارگرفت. استخراج DNA از ژنوم مایکوباکتریوم هابااستفادهازروش CTAB (Cetyltrimethylammonium bromide) بیشترین میزان و روشمبتنی بر جوشانیدن کمترینمیزانDNAرا حاصل کرد. همچنین مقایسهمیزانخلوصDNAاستخراج شدهدراینروش ها نیزنشانداد که استخراج DNA بااستفادهاز روشCTABبیشترینمیزان وروشمبتنی بر جوشانیدن بازهم کمترین میزانخلوصراداشتهاست. همچنین بین دو نوع باکتری از نظر کیفیت DNAهای استخراج‌شده در هیچ یک از روش های استفاده شده برای استخراج DNA اختلاف معنی داری وجود نداشت. با توجه به نتایج مطالعه حاضر به نظر می رسد که استفادهازروش CTAB یکی از مناسب ترین روش ها جهت استخراج DNAژنومیاز مایکوباکتریوم هاجهتتشخیص مولکولی آنها در نمونه های مختلفمی باشد.

چکیده انگلیسی:

Given that the first step in performing molecular techniques such as PCR (Polymerase Chain Reaction (is having enough high quality DNA, the aim of this study was to compare five different extraction methods of genomic DNA of mycobacteria in order to introduce the best method. For this purpose, pure colonies of Mycobacterium avium paratuberculosis were used as pathogenic mycobacterium and also a saprophytic strain called Mycobacterium phelei. The quality and quantity of the obtained DNA by each method was calculated, measured and evaluated respectively; based on extracted DNA concentrations (nanograms per microliter) at a wavelength of 260 nm and also optical density (OD) ratio based on the 260/280 wavelength by using the Nanodrop technique. The highest and lowest amounts of mycobacterial genome DNA was extracted using the CTAB (Cetyltrimethylammonium bromide) and boiling methods respectively. Comparison of the purity of extracted DNA also indicated that the highest purity was obtained using the CTAB method and the lowest using the boiling method. Also no significant differences were observed regarding the quality of extracted DNA from pathogenic and saprophytic mycobacteria among the evaluated extraction methods. Thus, according to the results of this study it appears that using the CTAB method is the most suitable way to extract genomic DNA for molecular detection of mycobacteria in different samples.

منابع و مأخذ:

Aghazade, R., Zali, M.R., Bahrami, A., Amin, K., Ghahghaie, F., and Firouzi, F. (2004). Inflammatory bowel disease in IRAN: A review of 448 cases. Archives of Iranian Medicine, 7(3): 210-216.

• Anzabi, Y, Tabatabayi, A.H. and Asgharzade, M. (2006). A Survey on the infection status of Mycobacterium avium paratuberculosis in dairy cattles using PCR & culture of Tabriz region. Journal of Comprative Pathobiology, 2(4): 297-310. [In Persian]

• Anzabi, Y.,  Farashi Bonab, S. and Moggaddam, Gh.A. (2009). Efficiency of direct microbial diagnosis, IS900 PCR and microbial culture for detection of Mycobacterium avium subsp. paratuberculosis in the feces of apparently healthy cattle. Veterinary Journal of Islamic Azad University-Tabriz Branch, 2(4): 309-317. [In Persian]

• Anzabi, Y. and Hanifian, S. (2012). Detection of Mycobacterium avium subspecies paratuberculosis in pasteurized milk by IS900 PCR and culture method. African Journal Microbiology Research, 6(7):1453-1456.

• Barrington, GM., Gay, J.M. and Eriks, I.S., (2003). Temporal Patterns of diagnostic results in serial samples from Cattle with advanced paratuberculosis infections. Journal of Veterinary  Diagnosis, 15(2):195-200.

• Bauerfeind, R., Benazzi, S., Weiss, R., Schliesser, T., Willems, H. and Baljer, G. (1996). Molecular Characterization of Mycobacterium paratuberculosis Isolates from Sheep, Goats and Cattle by Hybrization with a DNA probe to Insertion Element IS900. Journal of Clinical Microbiology, 34(7):1617-1621.

• Bono, T., Jemmi, T. and Bernascconi, C. (1995). Genotupic characterization of Mycobacterium avium strains recovered from animals and their comparison to human strains. Applied and Enviromental Microbilogy, 61(1): 371-373.

• Butcher, P.D., Hutchinson, N.A., Doran, T.J. and Dale, J.W. (1996). The application of molecular techniques to the diagnosis and epidemiology of mycobacterial disease. Journal of Applied  Bacteriology, 81(25): 53-71.

• Chan, K.C., Yeung, S.W., Lui, W.B., Rainer, T.H. and Lo, Y.M. (2005). Effects of preanalytical factors on the molecular size of cell-free DNA in blood. Clinical Chemistry, 51(4): 781-784.

• Chiodini, R.J. and Rossiter, C.A. (1996). Paratuberculosis: A potential zoonosis? Veterinary Clinics of North America: Food Animal Practice, 12(2): 457-467.

 •Fang, Y.,   Wu, W.H.,   Pepper, J.L.,   Larsen, J.L., Marras, S.A., Nelson, E.A., et al. (2002). Comparison of Real-Time, Quantitative PCR with Molecular Beacons to Nested PCR and Culture Methods for Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Fecal Samples. Journal of Clinical Microbiology, 40(1):287-291.

• Collins, D.M., Hilbink, F. and West, D.M. (1998). Investigation of Mycobacterium paratuberclosis in sheep by faecal culture, DNA characterization and the Polymerase Chain Reaction. Veterinary  Record, 133(24): 566-600.

• Corti, S. and Stephan, R. (2002). Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland. BMC Microbiology, 2(15): 1-14.

• Cousins, D.V., Whittington, R., Marsh, I., Masters, A., Evans, R.J. and Kluver, P. (1999). Mycobacteria distinct from Mycobacterium avium subsp. paratuberculosis isolated from the faeces of ruminants possess IS900-like sequences detectable IS900 polymerase chain reaction: implications for diagnosis. Molecular and Cellular Probes, 13(6): 431-442.

• Dell’isola, B., Poyart, C., Goulet, O., Mougenot, J.F., Sadoun-Journo, E., Brousse, N., et al. (1994) Detection of Mycobacterium paratuberculosis by polymerase chain reaction in children with Crohn’s disease. Journal of Infectious Disease, 169(2): 449-451.

• Eltholth, M.M., Marsh, V.R., Van Winden, S. and Guitian, F.J. (2009). Contamination of food products with Mycobacterium avium paratuberculosis: a systematic review. Journal of Applied and Environmental Microbiology, 107: 1061-1071.

• Elphinstone, M.S, Hinten, G.N, Anderson, M.J. and Nock, C.J. (2003). An inexpensive and high-throughput procedure to extract and purify total genomic DNA for population studies. Molecular Ecology Notes, 3(2): 317-320.

• Englund, S., Ballagi-Pordány, A. and Bölske, G. (1999). Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis. Diagnostic microbiology and infectious disease, 33(3): 163-171.

• Englund, S. (2003). IS900/ERIC-PCR as a tool to distinguish Mycobacterium avium subsp.paratuberculosis from closely related mycobacteria. Veterinary Microbiology, 96(3): 277-287.

• Fathi, R., Sarkarati, F., Eslami, M., Rezavand, B. and Nourizadeh, A. (2011). Detection of Mycobacterium avium subsp. paratuberculosis in cow milk using culture and PCR methods. Archives of Razi Institute, 66(2): 95-100.

• Khakpoor, M., Fardin, M., Ahmadi, H. and Nehzati, A. (2011).The infection status of Mycobacterium avium paratuberculosis in traditional dairy cattle farms in Mogham region. Food Hygion Scientific-Research Journal, 1(4): 45-52. [In Persian]

• Klanicova, B., Slana, I., Roubal, P., Pavlik, I. and Kralik, P. (2012). Mycobacterium avium subsp. paratuberculosis survival during fermentation of soured milk products detected by culture and quantitative real time PCR methods. International Journal of Food Microbiology, 157(2): 150-155.

• Marsh, I., Whittington, R. and Cousins, D. (1999). PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311. Molecular and Cellular Probes, 13(2): 115-126.

• Millar, D., Ford, J., Sanderson, J., Withey, S., Tizard, M., Doran, T., et al. (1996). IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows, milk in England and Wales. Applied and  Enviromental Microbiology, 62(9): 3446-3452.

• Nebbia, P., Robino, P., Zoppi, S. and De Meneghi, D. (2006). Detection and excretion pattern of Mycobacterium avium subsp. paratuberculosis in milk of asymptomatic sheep and goats by Nested-PCR. Small Ruminant Research, 66(1-3): 116-120.

• Queipo-Ortuño, M.I., Colmenero, J.D.D., Macias, M., Bravo, M.J. and  Morata, P. (2008). Preparation of Bacterial DNA Template by Boiling and Effect of Immunoglobulin G as an Inhibitor in Real-Time PCR for Serum Samples from Patients with Brucellosis. Clinical Vaccine Immunology, 15(2): 293-296.    

• Rodreguez-Lazaro, D., DAgostino, M., Herrewegh, A., Pla, M., Cook, N., and Ikonomopoulos, J. (2005). Real-time PCR-based methods for detection of Mycobacterium avium subsp. paratuberculosis in water and milk. International Journal of Food Microbiology, 101(1): 93-104. 

• Sharma, G., Singh, S.V., Sevilla, I., Singh, A.V., Whittington, R.J., Just, R.A., et al. (2007). Evaluation of indigenous milk ELISA with culture and m-PCR for the diagnosis of Bovine Johnes (BJD) in lactating Indian dairy cattle. Research Veterinary Science, 84(1): 30-37.

• Singh, S.V., Singh, A.V., Singh, R., Sandhu, K.S., Singh, P.K. and Sohal, J.S. (2007). Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal PCR for the diagnosis of bovine Johne’s disease (BJD) in India. Comparative Immunology, Microbiology and Infectious Disease, 30(3):175-186.

• Sivakumar, P., Tripathi, B.N. and Nem, S. (2004). Detection of Mycobacterium avium subsp. paratuberculosis in intestinal and lymph node tissues of water buffaloes (Bubalus bubalis) by PCR and bacterial culture. Veterinary Microbiology, 108(3-4):263-270.

 • Tripathi, B.N., Periasamy, S., Paliwal, O.P. and Singh, N. (2005). Comparison of IS900 tissue PCR, bacterial culture, Johnin and serological test for diagnosis of naturally occurring paratuberculosis in goats. Veterinary Microbiology, 116(1-3): 129-137.

• Wells, S.J., Collins, M.T., Faaberg, K.S., Wees, C., Tavornpanich, S. and Petrini, K.R. (2006). Evaluation of a rapid fecal PCR test for detection of Mycobacterium avium subsp. paratuberculosis in dairy cattle. Clinical and Vaccine Immunology, 13(10):1125-1130.

• Whittington, R.J., Reddacliff, L., Marsh, I. and Saunders, V. (1999). Detection of Mycobacterium avium subsp. paratuberculosis in formalin-fixed paraffin-embedded intestinal tissue by IS900 polymerase chain reaction. Australian  Veterinary Journal, 77(6): 392-397.

• Wren, B. and Dorrell, N. (2002). Functional Microbial Genomics. Volume 33, London: England, Academic Press, pp: 3-49.