Improved DNA typing of human urine by adding EDTA

Milde, A.; Haas-Rochholz, Hildegard; Kaatsch, Hans-Jürgen

doi:10.1007/s004140050237

Cited by 40 publications

(44 citation statements)

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“…A similar decrease in the amount of human urinary DNA in men has been found in studies undertaken in the United States (Johnson et al, 2007), Italy (Cannas et al, 2009), and Germany (Milde et al, 1999) but was not observed in Russian (Bryzgunova et al, 2006) or Zambian (Cannas et al, 2009) …”

Section: Discussionsupporting

confidence: 81%

“…EDTA preservatives for urine are commonly prepared to a final concentration of 10 mM using commercially available urine transport tubes sold specifically for downstream molecular analysis (Ingersoll et al, 2008). Milde et al (1999) found that storing urine at room temperature with sodium azide for 30 days or at -20°C with EDTA for 72 days provided the best protocol for the analysis of human DNA. This protocol did not stabilize urinary DNA in samples from Zambian subjects, however (Cannas et al, 2009).…”

Section: Populationsmentioning

confidence: 99%

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Genotyping of urinary samples stored with EDTA for forensic applications

Zhang

Zhao²,

Zhao³

et al. 2012

Genet. Mol. Res.

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ABSTRACT. Individual identification of urinary samples is necessary when sample switching or handling are suspected during a judicial process. To improve the rate of successful genotyping of urinary samples, we examined the stability of DNA in urinary samples stored for up to 30 days. Urinary samples from 20 healthy individuals (10 males and 10 females) were stored at -80°C with different concentrations of EDTA (0, 10 and 40 mM). Urinary DNA was extracted at days 0, 3, 9, and 30 after collection. The Quantifiler Human DNA Quantification Kit was used for measuring DNA concentration. Twenty STR loci were co-amplified using amelogeninspecific PCR with the Goldeneye 20A Kit. Significant differences in DNA concentration were observed between samples from females and males. In the case of female urinary DNA preservation with 10 and 40 mM EDTA the mean detection rate reached 0.95 after up to 30 days; for the male urinary samples, the mean detection rate of urinary DNA preserved with 40 mM EDTA was significantly higher than with 10 mM. We concluded that 40 mM EDTA is the best concentration for preservation of the DNA in urinary samples.

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Section: Discussionsupporting

confidence: 81%

Section: Populationsmentioning

confidence: 99%

Genotyping of urinary samples stored with EDTA for forensic applications

Zhang

Zhao²,

Zhao³

et al. 2012

Genet. Mol. Res.

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“…A urine aliquot was tested with a urinalysis reagent strip (Multistix; Bayer) and assayed for creatinine on a Roche MODULAR analyzer. To inhibit possible nuclease activities, we mixed the sample with 0.5 mol/L EDTA, pH 8.0 (Invitrogen), to a final concentration of 10 mmol/L (1,13 ). To separate the cellfree and cellular urine components, we centrifuged urine samples at 3000g at 4°C for 10 min and filtered the supernatant through a 0.45-m filter (Milex-GV; Millipore) to remove any remaining cells or cell debris.…”

Section: Phasementioning

confidence: 99%

“…Real-time quantitative PCR assays were used to quantify the LEP (leptin) and SRY (sexdetermining region Y) genes in a 50-L reaction mixture, as previously described (13 ). In female patients who received male stem cells, the SRY signal reflected donor-derived DNA, and the LEP signal reflected the total DNA from both the donor and recipient.…”

Section: Phasementioning

confidence: 99%

Presence of Donor-Derived DNA and Cells in the Urine of Sex-Mismatched Hematopoietic Stem Cell Transplant Recipients: Implication for the Transrenal Hypothesis

Hung¹,

Shing²,

Chim

et al. 2009

Clinical Chemistry

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Background: The term “transrenal DNA” was coined in 2000 to signify that DNA in urine may come from the passage of plasma DNA through the kidney barrier. Although DNA in the urine has the potential to provide a completely noninvasive source of nucleic acids for molecular diagnosis, its existence remains controversial. Methods: We obtained blood and urine samples from 22 hematopoietic stem cell transplant (HSCT) recipients and used fluorescence in situ hybridization, PCR for short tandem repeats, mass spectrometry, quantitative PCR, and immunofluorescence detection to study donor-derived DNA in the urine. Results: All HSCT recipients exhibited high amounts of donor-derived DNA in buffy coat and plasma samples. Male donor–derived DNA was detected in supernatants of urine samples from all 5 female sex-mismatched HSCT recipients. Surprisingly, the amount of DNA in urine supernatants was not correlated with the plasma value. Moreover, cell-free urine supernatants contained DNA fragments >350 bp that were absent in plasma. Donor-derived polymorphs were detected in urine by fluorescence in situ hybridization. Coincidentally, donor-derived cytokeratin-producing epithelial cells were discovered in urine samples from 3 of 10 sex-mismatched HSCT recipients as long as 14.2 years after transplantation. Conclusions: This report is the first to demonstrate the presence of donor-derived DNA in the urine of HSCT recipients; however, we show that much of this DNA originates from donor-derived cells, rather than from the transrenal passage of cell-free plasma DNA. Our discovery of donor-derived cytokeratin-producing epithelial cells raises interesting biological and therapeutic implications, e.g., the capacity of marrow stem cells to serve as an extrarenal source for renal tubule regeneration.

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“…29 DNA detection may be affected by EDTA, regardless of storage temperature. 30,31 In our review, studies that documented the addition of EDTA to urine samples 15,18 were too few to analyze on metaregression. It is unclear why centrifuge speeds of 3000-4000 rpm 9,14-18 would yield higher sensitivity than a speed of 10 000 rpm.…”

Section: Discussionmentioning

confidence: 98%

Diagnostic accuracy of nucleic acid amplification tests in urine for pulmonary tuberculosis: A meta-analysis

2015

Indian Journal of Tuberculosis

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OBJECTIVE-To determine the diagnostic accuracy of tuberculosis (TB) nucleic acid amplification tests (NAATs) in urine samples for individuals with active pulmonary tuberculosis (PTB).DESIGN-Systematic review and meta-analysis. Electronic databases and reference lists were searched without age or setting restrictions up to May 2015. Eligible articles examined Mycobacterium tuberculosis NAATs in urine samples for PTB diagnosis in patients with sputum culture as the reference standard, and reported sufficient data to separately calculate sensitivity or specificity.RESULTS-Eight studies, including 1212 participants from seven countries with a mean age ranging from 28 to 48 years, were included. Polymerase chain reaction (PCR) with insertion sequence (IS) 6110, rpoB or cfp32/hf6 as gene targets was used for NAATs. The pooled sensitivity and specificity was respectively 0.55 (95%CI 0.36-0.72) and 0.94 (95%CI 0.78-0.99), with slightly higher sensitivity in human immunodeficiency virus positive individuals, at 0.59 (95%CI 0.20-0.89). Sensitivity was higher in sputum microscopy-positive than -negative individuals. Storage temperatures below −70°C, centrifuge speed <5000 rpm and IS6110 increased sensitivity on meta-regression.Correspondence to: Diana Marangu, Department of Paediatrics and Child Health, University of Nairobi, P O Box 19676 -0202, Nairobi, Kenya. marangud@gmail.com. Conflicts of interest: none declared. HHS Public Access KeywordsNAAT; PCR; systematic review; meta-regression In 2013, it was estimated that there were 9 million incident tuberculosis (TB) cases globally, 3.3 million of whom were not diagnosed. 1 Urine nucleic acid amplification tests (NAATs) have potential for use in the diagnosis of pulmonary tuberculosis (PTB), the most common form of TB worldwide. [2][3][4] The relative simplicity of urine collection could be ideal to address diagnostic challenges of active PTB, 5 and could be exploited in point-of-care (POC) testing of active PTB at all levels of health care, and potentially in households and the community. 6,7 In addition to being safer to handle, normal urine production yields frequent and relatively large amounts, making urine specimens convenient to obtain and easier to test in comparison to sputum. Children could benefit the most from urine NAATs for PTB diagnosis due to challenges in obtaining sputum samples, particularly from young children who cannot expectorate, and the paucibacillary nature of their disease. 5,8 With the advancement of molecular techniques and wider use of the NAAT Xpert ® MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) in many high TB endemic countries, exploration of alternative samples such as urine for TB diagnosis is increasing due to its ease of collection and potential for decentralized implementation. 9,10 Cell-free nucleic acids released from dying human cells and Mycobacterium tuberculosis microorganisms may be filtered through the kidneys, resulting in the detection of trans-renal DNA fragments in urine. 6 Although the exact pathophysiological mech...

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Improved DNA typing of human urine by adding EDTA

Abstract: The effect of different EDTA concentrations on the DNA content of urine samples was examined and compared to untreated urine at various storage temperatures and times. The results indicate that adding EDTA increases the DNA stability for long time storage especially at low temperatures.

Cited by 40 publications

References 0 publications

Genotyping of urinary samples stored with EDTA for forensic applications

Genotyping of urinary samples stored with EDTA for forensic applications

Presence of Donor-Derived DNA and Cells in the Urine of Sex-Mismatched Hematopoietic Stem Cell Transplant Recipients: Implication for the Transrenal Hypothesis

Diagnostic accuracy of nucleic acid amplification tests in urine for pulmonary tuberculosis: A meta-analysis

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