Volume 3, Issue 1 , Pages e3-e4, December 2011
UTI preventing DNA degradation of storing urinary samples for genotyping
Article Outline
- Abstract
- 1. Introduction
- 2. Materials and methods
- 3. Results and discussion
- Role of funding
- Conflict of interest
- Acknowledgement
- References
- Copyright
Abstract
In forensic practices, individual identification of urinary samples is necessary when sample switching or handling are suspected. DNA degradation with time elapsing and the low yield of extracted urinary DNA prevent its application. Storage of urine prior to analysis is increasingly advocated yet no best practice has emerged. To improve the genotyping based on storing urinary samples, we employed UTI (Urinary Trypsin Inhibitor) to prevent urinary DNA degradation. Urinary samples from 10 (5 females and 5 males) healthy volunteers from China were stored at −80
°C with different concentrations (0–0.8μg/mL) of UTI. Urinary DNA was genotyped with Identifiler Kit at days of 1, 3, 5, 7, 9, 11 and 13 after storage. The average detection rate of STR loci of urinary DNA with or without UTI were statistically significant and UTI at 0.2μg/mL level is enough for urine storing. Also, significant differences in DNA yield were noted between female and male urine samples.
Keywords: Forensic genetics, Urine, Urinary Trypsin Inhibitor (UTI), Individual identification
1. Introduction
Dopes such as ketamine and Benzedrinum were abused in China since 1990s that was spread as a serious social problem. Detection of dope metabolic products in urine is the primal method for judging drug abuse. Assessment of sample origin is necessary when sample switching or handling are suspected during the justice investigations. There are about 400 epithelial cells (i.e. renal tubular, transitional urothelial, and squamous) and macrophages per milliliter human urine, which made urine based DNA detection possible [1], [2], [3]. However, urea, uric acid, creatine, mineral salt, urokinase and bacterium made urinary DNA degraded very fast and prevented the potential utility of urine for individual identification [4], [5]. UTI is a urine derived glucoprotein that inhibits the activity of pancreatic enzymes. Due to its specific biologic activity, UTI was used in the treatment of pancreatitis, traumatic and endotoxic shock to protect the cells from proteases damage. Here we reported that UTI applied to storing urinary samples improving the stability of urinary DNA.
2. Materials and methods
2.1. Samples
Medistream urine samples (about 8–10mL/time) were collected from 10 healthy volunteers (5 females and 5 males) for 6 times totally. Blood specimens collected from the same subject served as references. Aliquots (1mL) of urinary samples with different concentrations of UTI (0.0, 0.1, 0.2, 0.4 and 0.6μg/mL) were stored at −80°C for different storage periods (1, 3, 5, 7, 9, 11 and 13 days).
Genomic DNA was extracted using the improved Chelex-100 and proteinase K protocol and concentrated to 20μL with Microcon-100 device (Gene Company Limited, Shanghai, China).
2.2. Analysis of STR loci
DNA was amplified with Indentifiler Kit (Applied Biosystems, Foster city, CA, USA) on a GeneAmp System 9700 thermal cycler. PCR amplification was performed according to the manufacturer's instructions. Capillary electrophoresis were carried out using the AB Prism 3130 Genetic Analyzer (Applied Biosystems, Foster city, CA, USA) and genotyping results were analyzed by GeneMapper V3.2.1 software.
Compared with reference samples, detection numbers of STR loci of urinary samples were counted while drop-out or drop-in were kicked out. The average detection rate of STR loci of urinary samples with different concentrations of UTI stored for different periods in different gender were calculated, respectively. CMH χ2 test of the differences of average detection rate of STR loci of urinary DNA was assessed using SAS v8.1 software. The level of significance was 0.05. Curve fitting of distributions was processed by GraphPad Prism v4.0 software.
3. Results and discussion
The differences of different concentrations of UTI to the average detection rate of STR loci of urinary DNA were statistically significant by CMH χ2 test (χ2CMH=293.37, df=5, P<0.0001). The average detection rate of STR loci of female urinary DNA with UTI appeared 100.00%±0.00% during 9 days. At the 13th day, the detection rate of urinary DNA with 0.2, 0.4 and 0.6μg/mL UTI levels are higher than urinary DNA with 0.1μg/mL (P<0.05) and no statistically significant between them (P>0.05) (shown in Fig. 1a). The average detection rate of STR loci of male urinary DNA with UTI appeared linear correlation during 13 days (R2 all exceed 0.9500) and all exceeded the rate of urinary DNA without UTI except the first day. The detection rate of urinary DNA with UTI at 0.2, 0.4 and 0.6μg/mL levels are higher than 0.1μg/mL (shown in Fig. 1b).
Fig. 1.
Average detection rate of STR loci of urine specimens stored with different concentrations of UTI during 13 days (a) female (b) male. (A) Stored without UTI; (B) stored with 0.1
μg/mL UTI; (C) stored with 0.2, 0.4 and 0.6μg/mL UTI.
In this study, we have demonstrated that urinary DNA stability can be higher variable and UTI has optimized effects to the urine storage for further identification. And UTI at 0.2μg/mL level is enough for urine storing. However, further work is required to identify the source of this variability and the case of degradation. Also, significant differences in DNA yield were noted between female and male urine samples.
Role of funding
This study was supported by grants from the National Nature Science Foundation, PR China (nos. 30901701 and 81172908).
Conflict of interest
None.
Acknowledgement
The authors would like to thank Pro Jinzhong Chen for reviewing the manuscript.
References
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- Implications of storing urinary DNA from different populations for molecular analysis. PLoS ONE. 2009;4(9):e6985
- DNA typeability in liquid urine and urine stains using AmpFISTR SGM Plus. Adv. Med. Sci. 2006;51:36–38
- . Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J. Clin. Pathol. 1991;44:360–365
PII: S1875-1768(11)00002-3
doi:10.1016/j.fsigss.2011.08.001
© 2011 Elsevier Ireland Ltd. All rights reserved.
Volume 3, Issue 1 , Pages e3-e4, December 2011
