Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2019

Evaluation of Nucleic Acid Extraction Methods for Detection of DNA Viruses in Urine Specimens (#116)

Hui Ling Bay 1 , Liat Hui Loo 1 2 , Matthias Maiwald 1 2 3
  1. Microbiology Laboratory, Department of Pathology and Laboratory Medicine, KK Women's and Children's Hospital, Singapore
  2. Duke-NUS Medical School, Singapore
  3. Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Urine specimens may contain inhibitory elements that interfere with real-time polymerase chain reaction (qPCR). Our laboratory has been extracting viral nucleic acids from urine specimens via a manual spin column protocol using the QIAamp Viral RNA Mini Kit. Buffer AVL in this kit can remove PCR inhibitors in urine. Automated nucleic acid extraction is performed using EZ1 Virus Mini Kit, which does not contain any reagents that removes PCR inhibition. Thus, an evaluation was performed on automated extraction and manual extraction, in terms of performance and processing time, and whether automated extraction is able to replace manual extraction.

Twenty-eight PCR-positive urine specimens were selected, 24 positive for cytomegalovirus and 4 adenovirus-positive. All specimens were retested in parallel, using automated extraction method as well as manual extraction in the same test setup, to ensure comparability of results and integrity of specimens. The specimens were retested using in-house real-time PCR assays, each with an additional reaction to which 400 copies of virus DNA was added (spiked controls) in order to monitor PCR inhibition.

All 28 samples were positive using real-time PCR after both extraction protocols. Spiked controls showed no inhibition in any of the samples. The Ct values did not differ significantly from each other (p=0.25). Automated extraction is able to process up to 14 specimens and requires approximately 45 minutes. This is advantageous for high numbers of specimens. Manual extraction of 1 specimen requires approximately 20 minutes, and is beneficial for small numbers of specimens.

The results suggest that automated extraction for urine specimen is acceptable, and has the potential of replacing manual extraction. However, due to the relatively small number of cases evaluated, further evaluation is necessary.