Abstract
Urine holds great promise as a non-invasive sampling method for molecular diagnostics. The cell-free nucleic acids of urine however are small, labile, and difficult to purify. Here an efficient method for the purification of these nucleic acids is presented. An empirically derived protocol was devised by first identifying conditions that allowed recovery of a 100 base pair (bp) DNA, followed by optimization using a quantitative polymerase chain reaction (qPCR) assay. The resulting method efficiently purifies both small sized DNAs and RNAs from urine, which when combined with quantitative reverse transcription PCR (qRTPCR), demonstrably improves detection sensitivity. Fractionation experiments reveal that nucleic acids in urine exist both in the cell-free and cellular fraction, roughly in equal proportion. Consistent with previous studies, amplicons > 180bp show a marked loss in PCR sensitivity for cell-free nucleic acids. Finally, the lysis buffer developed here also doubles as an effective preservative, protecting against nucleic acid degradation for at least two weeks under simulated field conditions. With this method, volumes of up to 25ml of whole urine can be purified in a high-throughput and cost-effective manner. Coupled with its ability to purify both DNA and RNA, the described method may have broad applicability for improving the diagnostic utility of urine, particularly for the detection of low abundant targets.
Highlights
Owing to its non-invasive nature, the use of urine for detecting human pathogens is gaining traction as a useful diagnostic tool [1,2,3,4,5,6,7,8,9,10]
The infectious agent itself need not necessarily be present in urine for detection of disease. Both tuberculosis (TB) and human papillomavirus (HPV) infection have been successfully diagnosed with PCR testing of urine samples [18,19,20,21]
A 60bp product of the human housekeeping actin gene was chosen as the target since its widespread expression and small amplicon size would minimize any bias based on the size or source of the purified nucleic acids (S6 Appendix)
Summary
Owing to its non-invasive nature, the use of urine for detecting human pathogens (via amplification of pathogen-associated nucleic acids with polymerase chain reaction, PCR) is gaining traction as a useful diagnostic tool [1,2,3,4,5,6,7,8,9,10] This utility was highlighted during the recent Zika and Ebola outbreaks where PCR testing of urine was found to be a highly effective strategy for identifying infected individuals [11,12,13,14,15,16,17]. The sensitivity and specificity of these methods are still too low and inconsistent to justify their use for routine screening, highlighting the need for further improvements
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
