Clinical evaluation of an automated Real-Prep system for extracting nucleic acids to detect mycobacterial infection.

ABSTRACT

Real-time PCR tests have been widely used to detect mycobacterial infection. The quality of extracted DNA is crucial for obtaining accurate results of the real-time PCR tests, and automated extraction methods are faster and more effective than manual extraction. The novel Real-Prep automated extraction system has not yet been verified by direct comparisons to existing methods. In this study, we compared it with manual extraction, and the Nextractor system, an automated extraction method commonly used in Korea. From August to December 2018, 238 specimens, including sputum, bronchial washing, pericardial fluid, bronchial aspiration, pleural fluid, and closed pus samples, were collected and examined at Yeungnam University Hospital. After decontamination, smear microscopy, and culturing, DNA was extracted using the three methods. The DNA extraction efficiency (total amount of DNA [ng]/input specimen volume [µL]) and purity (A260/280 ratio), which indicates the presence of contaminants, were compared. Real-time PCR tests were conducted using the DNA extracted by each method. The cycle threshold, which is inversely related to the initial amount of mycobacterial DNA, and the percentage agreement between the PCR results of the three methods were evaluated. Our study revealed that the DNA extraction efficiency of the Real-Prep system was higher than that of manual extraction. There was no significant difference in DNA purity between the methods, and the percentage agreement for Mycobacterium tuberculosis and non-tuberculous mycobacteria among all three methods was almost perfect. The performance of the Real-Prep system was similar to that of the Nextractor system and superior to that of manual extraction. The Real-Prep system, a new automated nucleic acid extraction device, has a clear benefit because of its relative speed and low hands-on time. Therefore, the Real-Prep system is a useful substitute for manual DNA extraction, which has the potential to reduce workloads in laboratories and as a sensitive non-tuberculous mycobacteria detection method throughout the world.