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PRINT ISSN : 2319-7692
Online ISSN : 2319-7706 Issues : 12 per year Publisher : Excellent Publishers Email : editorijcmas@gmail.com / submit@ijcmas.com Editor-in-chief: Dr.M.Prakash Index Copernicus ICV 2018: 95.39 NAAS RATING 2020: 5.38 |
Compared to conventional bacterial culture methods, PCR technology drastically alters disease diagnostics, and the use of magnetic beads in nucleic acid extraction and purification sparked a technological revolution in biological research. Therefore, this study aims to investigate the relationship between the cycle threshold (Ct) values of the real-time PCR reaction, which are employed as indicators of DNA concentration, and the magnetic beads that are carried over during the DNA extraction of mastitis pathogens from dairy cow milk samples. The Mag MAX™ CORE Nucleic Acid Purification Kit, one of the rabid and simple extraction methods that uses magnetic separation technique, was used to extract DNA, while applied Biosystems™ 7500 PCR equipment and applied Biosystems Vet MAX™ MastiType multiplex qPCR kits are utilized in DNA amplification. Eighty-five milk samples from cows with mastitis were obtained; of these, 53 samples (62.4%) had a normal DNA yield retrieved in which 79 microorganisms were identified; in contrast, 32 samples (37.6%) had a magnetic bead carried over from which there were 34 identified microorganisms. On the other hand, there were two samples (6.3%), and supernatants were unable to be obtained. To investigate the relation between cycle threshold and magnetic beads carried over during extraction, all data were analyzed by a T-test of independence, and it showed a significant difference in DNA concentration between each other (T-test =.066). The same data was retested by the Chi Square test, which showed the same significant result (Chi square =.054). This result highlighted the relationship between DNA concentration and magnetic bead carryover and its impact on DNA yield.
DNA extraction, magnetic beads, cow mastitis, threshold cycles
Abd El Aal A, Abd Elghany N, Mohamadin A, and El Badry A. (2010): Comparative study of five methods for DNA extraction from whole blood samples. International Journal of Health Science, Vol. 3 Issue 1, p285-287.
Abdelhameed, M. F. M., et al., (2024): Magnetic beads carried over in extracted DNA elution from mastitis cow milk. International Journal of Current Microbiology and Applied Sciences, 13(01): 73-80. https://doi.org/10.20546/ijcmas.2024.1301.009.
Abdulrahman, A.; et al., (2020): Association between RT-PCR Ct values and COVID-19 new daily cases: A multicenter cross-sectional study. Le Infezioni in Medicina, n. 3, 416-426, preprint https://doi.org/10.1101/2020.12.07.20245233.
Abo-Youssef, A. M., et al., (2020): Febuxostat attenuates testosterone induced benign prostatic hyperplasia in rats via inhibiting JAK/STAT axis. Life Sci. 2020, 260, 118414. Cited from https://doi.org/10.1016/j.lfs.2020.118414
Amin, A. S., Hamouda, R., & Abdel-All, A. A. A. (2011): PCR Assays for Detecting Major Pathogens of Mastitis in Milk Samples. World Journal of Dairy & Food Sciences, 6, 199–206.
Anita, T. (2014): Bovine mastitis: an important dairy cattle disease. Indian Dairyman, 66(4), 62–65.
Carpi F M, et al., (2011) Human DNA Extraction Methods: Patents and Applications. Recent Patents on DNA & Gene Sequences 5(1): 1-7. https://doi.org/10.2174/187221511794839264
Corman Victor M., et al., (2020): Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Europe's journal on infectious disease surveillance, epidemiology, prevention, and control, Volume 25, Issue 3, 23 January. https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045
Elise Martin et al., (2018): Mastitis Diagnosis Utilizing a Turnkey Solution from Thermo Fisher Scientific’s Animal Health Group.
Hawkins T L, O’Connor-Morin T, Roy A, Santillan C., (1994): DNA purification and isolation using a solid-phase. Nucleic Acids Research; 22(21):4543–4544. https://doi.org/10.1093/nar/22.21.4543
Nakatsu, Y., et al., (2015): The xanthine oxidase inhibitor febuxostat suppresses development of non-alcoholic steatohepatitis in a rodent model. Am. J. Physiol. Gastrointest. Liver Physiol. 2015, 309, G42–G51. Cited from Rabaan, A. A., et al., (2021): Viral Dynamics and Real-Time RT-PCR Ct Values Correlation with Disease Severity in COVID-19. Diagnostics, 11, 1091. https://doi.org/10.1152/ajpgi.00443.2014
Patterson, C. (2017): Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs, and Goats. Volumes 1 and 2.
Rabaan, A. A., et al., (2021): Viral Dynamics and Real-Time RT-PCR Ct Values Correlation with Disease Severity in COVID-19. Diagnostics, 11, 1091. https://doi.org/10.3390/diagnostics11061091
Rhoads, D., et al., (2020): College of American Pathologists microbiology committee perspective: Caution must be used in interpreting the cycle threshold (Ct) value. Clinical Infectious Diseases. https://doi.org/10.1093/cid/ciaa1199.
Romero, J., Benavides, E., and Meza, C. (2018): Assessing Financial Impacts of Subclinical Mastitis on Colombian Dairy Farms. Frontiers in Veterinary Science, Volume 5, Article 273. https://doi.org/10.3389/fvets.2018.00273
Ruegg, P. L. (2017): A 100-Year Review: Mastitis Detection, Management, and Prevention, Journal of Dairy Science. https://doi.org/10.3168/jds.2017-13023.
Saiyed Z M, et al., (2006): Application of magnetic particles (Fe3O4) for isolation of genomic DNA from mammalian cells. Analytical Biochemistry;356(2):306–308. https://doi.org/10.1016/j.ab.2006.06.027
Saiyed Z M, Ramchand C N, Telang S D., (2008): Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support. Journal of Physics: Condens Matter;20(20):204153. https://doi.org/10.1088/0953-8984/20/20/204153
Saiyed Z., Ramchand C. (2007): Extraction of Genomic DNA Using Magnetic Nanoparticles (Fe3O4) as a Solid-Phase Support. American Journal of Infectious Diseases, 3(4), 225-229. https://doi.org/10.3844/ajidsp.2007.225.229
Sonja Berensmeier (2006): Magnetic particles for the separation and purification of nucleic acids, Applied Microbiol Biotechnol 73:495–504. https://doi.org/10.1007/s00253-006-0675-0
Taponen, S., et al., (2009): Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing. Journal of Dairy Science, 92(6), 2610–2617. https://doi.org/10.3168/jds.2008-1729.
Thermo Fisher, (2016): Real-time PCR: Understanding CT.
Watson R J, Blackwell B. (2000): Purification and characterization of a common soil component which inhibits the polymerase chain reaction. Canadian Journal of Microbiology 46:633-642. https://doi.org/10.1139/w00-043
Watson, J.; Whiting, P. F.; Brush, J. E. (2020): Interpreting a COVID-19 test result. BMJ, 369:m1808, https://doi.org/10.1136/bmj.m1808.
Zakary, E. M., Nassif, M. Z., & Mohammed, G. M. O. (2011). Detection of Staphylococcus aureus in Bovine Milk and Its Product by Real Time PCR Assay. In Global Journal of Biotechnology & Biochemistry (Vol. 6, Issue 4, pp. 171–177).