DNA extraction from human urinary sediment

Yokota, Masaharu; Tatsumi, Noriyuki; Izumi, Tohru; Takubo, Takayuki; Hiyoshi, M

doi:10.1002/(sici)1098-2825(1998)12:2<88::aid-jcla3>3.0.co;2-f

Cited by 29 publications

(13 citation statements)

References 3 publications

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“…Obvious advantages of urine are that sample collection is noninvasive and large quantities can easily be obtained from individual patients. Examination of urine sediment reveals a variety of cells including phagocytes (30), in which H. capsulatum survives by avoidance of lytic digestion (31). Additionally, DNA released from dying fungal cells is known to cross the renal barrier and is subsequently excreted in urine as cell-free DNA in lengths suitable for detection using PCR (32).…”

Section: Discussionmentioning

confidence: 99%

Development of a Loop-Mediated Isothermal Amplification Method for Detection of Histoplasma capsulatum DNA in Clinical Samples

et al. 2014

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c Improved methods for the detection of Histoplasma capsulatum are needed in regions with limited resources in which the organism is endemic, where delayed diagnosis of progressive disseminated histoplasmosis (PDH) results in high mortality rates. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid inexpensive molecular diagnosis of this disease. Primers for LAMP were designed to amplify the Hcp100 locus of H. capsulatum. The sensitivity and limit of detection were evaluated using DNA extracted from 91 clinical isolates of known geographic subspecies, while the assay specificity was determined using DNA extracted from 50 other fungi and Mycobacterium tuberculosis. Urine specimens (n ‫؍‬ 6) collected from HIV-positive individuals with culture-and antigen-proven histoplasmosis were evaluated using the LAMP assay. Specimens from healthy persons (n ‫؍‬ 10) without evidence of histoplasmosis were used as assay controls. The Hcp100 LAMP assay was 100% sensitive and specific when tested with DNA extracted from culture isolates. The median limit of detection was <6 genomes (range, 1 to 300 genomes) for all except one geographic subspecies. The LAMP assay detected Hcp100 in 67% of antigen-positive urine specimens (4/6 specimens), and results were negative for Hcp100 in all healthy control urine specimens. We have shown that the Hcp100 LAMP assay is a rapid affordable assay that can be used to expedite culture confirmation of H. capsulatum in regions in which PDH is endemic. Further, our results indicate proof of the concept that the assay can be used to detect Histoplasma DNA in urine. Further evaluation of this assay using body fluid samples from a larger patient population is warranted.

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Section: Discussionmentioning

confidence: 99%

Development of a Loop-Mediated Isothermal Amplification Method for Detection of Histoplasma capsulatum DNA in Clinical Samples

et al. 2014

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“…DNA extraction from fresh urine has been described before, yielding DNA usable for PCR analysis in up to 35% of healthy males and up to 75% of healthy females [ 2 , 6 , 7 ]. Storage of fresh urine at 4°C or lower temperatures results in significant degradation of human DNA, resulting in low recovery rates during long-term storage [ 8 - 13 ].…”

Section: Discussionmentioning

confidence: 99%

“…Traditionally, for these analyses, DNA is recovered from buffy coats. Since DNA in urine has been reported to deteriorate quickly [ 2 ], urine is generally not used for the purpose of DNA analysis.…”

Section: Introductionmentioning

confidence: 99%

DNA extraction from long-term stored urine

et al. 2013

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BackgroundTraditionally, for DNA analyses, DNA is recovered from buffy coats. Since DNA in urine has been reported to deteriorate quickly, this option is often not considered. To complete our DNA database in patients with ANCA-associated vasculitis, we aimed to extract DNA from stored urine.MethodsUrine was stored at the time of kidney biopsy from patients included in our regional kidney biopsy database, who had given informed consent for further study. Urine was subsequently filtered, dialyzed, concentrated and freeze dried and finallyresolubilized and centrifuged. DNA was extracted using the high pure PCR template preparation kit (Roche Diagnostics). Next, concentration and purity were determined by Nanodrop analysis and by Quant-iT analysis.ResultsOne hundred and eighty-one patients with ANCA-associated vasculitis were included. Of 114 patients (63%), DNA was available. From 53 of the remaining 67 patients, stored urine was available. Of the 53 samples that were processed, 46 (86.8%) yielded DNA with a mean concentration of 258.7 ng/μL (range 33.2-529) with a mean purity ratio of 1.81 (λ 260/280).ConclusionDNA extraction from fresh urine has been described before, yielding DNA usable for PCR analysis in healthy subjects. Storage of fresh urine at 4°C or lower temperatures results in significant degradation of the DNA, making recovery of DNA more difficult with longer periods of storage. In the current study, we demonstrated that DNA could be retrieved from subsequently filtered, dialyzed, concentrated and freeze dried urine that was stored at room temperature. In addition, we demonstrated tthat this DNA could be used for PCR analysis. This method is useful when no other material from these patients is available.

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“…However, the use of PCR overcomes some of these restrictions and permits DNA typing in situations that were previously unthinkable. In literature, some studies on DNA typing from urine samples were performed and several variables encountered in extracting DNA from urine samples have been considered (1)(2)(3)(4)(5)(6)(7). For example Gasparini et al have reported cystic fibrosis typing using urine samples, while others, e.g., Roest et al, have demonstrated the use of urine samples for population studies.…”

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Quality and Quantity of DNA Isolated from Frozen Urine in Population-Based Research

Hel

Luijt

Mesquita

et al. 2002

Analytical Biochemistry

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In several population-based studies in the past urine samples were collected and stored for future research. We set out to determine the reliability of using such samples for genotyping DNA markers in epidemiologic research. A source of DNA extracted from exfoliated nucleated cells in urine is provided by the DOM cohort, in which specimens were collected 15-25 years ago. We have examined the quality of the DNA in 48 of these samples by measuring the amount of DNA isolated and its ability to provide an adequate PCR template for amplicons of different lengths. MTHFR polymorphism was analyzed in 644 specimens to determine the inter-and intraobserver reproducibility. Although the DNA amount was variable, 26 to 89% of the samples, depending both on the length of the PCR amplicon and on PCR conditions, yielded a visible PCR product. The intra-and interobserver agreements were comparable ( 0.86 and 0.88, respectively). Our results demonstrate that frozen urine samples can be used for DNA typing studies in women after prolonged periods of storage, but with sometimes unpredictable results. Ultimately, the genotype success rate was 89.3%. Urine collection can be considered as a useful method of obtaining DNA in large cohort studies and other circumstances when blood samples cannot be obtained or have not been stored. © 2002 Elsevier Science (USA)

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DNA extraction from human urinary sediment

Cited by 29 publications

References 3 publications

Development of a Loop-Mediated Isothermal Amplification Method for Detection of Histoplasma capsulatum DNA in Clinical Samples

Development of a Loop-Mediated Isothermal Amplification Method for Detection of Histoplasma capsulatum DNA in Clinical Samples

DNA extraction from long-term stored urine

Quality and Quantity of DNA Isolated from Frozen Urine in Population-Based Research

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