The Correlation between Threshold Cycle Values and Magnetic Beads Carried Over During DNA Extraction of Mastitis Milk Samples

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.