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A rapid purification method for trace amounts of cell-free DNA in urine.

Author(s): Fukushima R, Ogura Y, Hosokawa C, Watanabe N, Ishikawa F, Shibanuma M, Kato M

Publication: Anal Sci, 2024, Vol. , Page

PubMed ID: 39652287 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the recovery of spiked-in DNA from urine using the QIAamp and MonoFas Kits and discussed the development of a new method to increase the detection of low-abundance DNA.  Urine collected from an unspecified number of healthy men was spiked with COVID-19 DNA and centrifuged at 600 x g for 10 min. DNA was isolated from urine supernatant using the MonoFas Kit and the QIAamp MinElute ccfDNA Kit. In order to improve detection of trace DNA, the authors developed an in-house cation sorbents protocol and tested it using four cation sorbents (MA-2 modified with tertiary amines, PSA modified with primary and tertiary amines, and SAX and SAX-2 modified with quaternary amines) and phosphate buffered saline spiked with 600 copies/mL DNA or a DNA ladder. The in-house method involved four cycles of mixing urine with cation sorbents, centrifugation (details not provided), particle washing in phosphate buffered saline, addition of sodium bicarbonate to detach the DNA, and purification of the DNA using the MonoFas Kit. DNA was quantified by real-time PCR amplification of the COVID-19 gene and confirmed by Sanger sequencing. DNA size was evaluated using the MCE-202 MultiNA DNA-500 Kit.  

   Summary of Findings:

   DNA recovery from urine specimens spiked with 6,000 or 60,000 copies/mL (5-10%) was comparable when DNA was extracted using the QIAamp or MonoFas Kit , but recovery was higher and less variable when DNA was extracted using the QIAamp rather than MonoFas Kit from urine spiked with 600,000 copies DNA/mL (approximately 15% versus 5%). The authors conclude that the two commercial extraction kits perform similarly for low-abundance DNA.  During development of an in-house method to increase DNA recovery, PSA cation sorbents had the best DNA recovery (cycle threshold, CT= 29.98), followed by the smaller particle SAX (CT =30.57) and MA-2 (CT=34.73). No amplifiable DNA was recovered using the larger particle SAX. The in-house method recovered 150% as much of the 100 bp fragment of the NDA ladder as the MonoFas Kit. However, recovery of the 500, 1,000 and 2,000 bp fragments were comparable using the in-house method and the MonoFAS Kit and recovery of the 4,000 and 10,000 bp fragments were much lower using the in-house method. The authors demonstrated they were able to effectively detect DNA in urine spiked with 6,000-600,000 copies/mL DNA using the in-house method in conjunction with the MonoFAS Kit.  The authors conclude the in-house method can detect DNA at a concentration of ≥1.2 copies/µL.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Capillary electrophoresis-MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAamp Kit MonoFas Kit In-house method
   Biospecimen Aliquots and Components	Biospecimen components	Spiked with 6,000 copies DNA /mL Spiked with 60,000 copies DNA /mL Spiked with 600,000 copies DNA /mL

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