Vu N T, Chaturvedi A K, Canfield D V
Toxicology and Accident Research Laboratory, Federal Aviation Administration, U.S. Department of Transportation, Oklahoma City, OK 73125-5066, USA.
Forensic Sci Int. 1999 May 31;102(1):23-34. doi: 10.1016/s0379-0738(99)00034-1.
Urine is often the sample of choice for drug screening in aviation/general forensic toxicology and in workplace drug testing. In some instances, the origin of the submitted samples may be challenged because of the medicolegal and socioeconomic consequences of a positive drug test. Methods for individualization of biological samples have reached a new boundary with the application of the polymerase chain reaction (PCR) in DNA profiling, but a successful characterization of the urine specimens depends on the quantity and quality of DNA present in the samples. Therefore, the present study investigated the influence of storage conditions, sample volume, concentration modes, extraction procedures, and chemical preservations on the quantity of DNA recovered, as well as the success rate of PCR-based genotyping for DQA1 and PM loci in urine. Urine specimens from male and female volunteers were divided and stored at various temperatures for up to 30 days. The results suggested that sample purification by dialfiltration, using 3000-100,000 molecular weight cut-off filters, did not enhance DNA recovery and typing rate as compared with simple centrifugation procedures. Extraction of urinary DNA by the organic method and by the resin method gave comparable typing results. Larger sample volume yielded a higher amount of DNA, but the typing rates were not affected for sample volumes between 1 and 5 ml. The quantifiable amounts of DNA present were found to be greater in female (14-200 ng/ml) than in male (4-60 ng/ml) samples and decreased with the elapsed time under both room temperature (RT) and frozen storage. Typing of the male samples also demonstrated that RT storage samples produced significantly higher success rates than that of frozen samples, while there was only marginal difference in the DNA typing rates among the conditions tested using female samples. Successful assignment of DQA1 + PM genotype was achieved for all samples of fresh urine, independent of gender, starting sample volume, or concentration method. Preservation by 0.25% sodium azide was acceptable for sample storage at 4 degrees C during a period of 30 days. For longer storage duration, freezing at -70 degrees C may be more appropriate. Thus, the applicability of the DQA1 + PM typing was clearly demonstrated for individualization of urine samples.
在航空/一般法医毒理学以及工作场所药物检测中，尿液通常是药物筛查的首选样本。在某些情况下，由于药物检测呈阳性会带来法医学和社会经济后果，送检样本的来源可能会受到质疑。随着聚合酶链反应（PCR）在DNA分型中的应用，生物样本个体化方法达到了一个新的高度，但尿液标本的成功鉴定取决于样本中DNA的数量和质量。因此，本研究调查了储存条件、样本体积、浓缩方式、提取程序和化学防腐剂对回收DNA数量的影响，以及尿液中DQA1和PM基因座基于PCR的基因分型成功率。将男性和女性志愿者的尿液标本进行分割，并在不同温度下储存长达30天。结果表明，与简单的离心程序相比，使用截留分子量为3000 - 100,000的透析过滤法进行样本纯化并不能提高DNA回收率和分型率。采用有机法和树脂法提取尿液DNA得到了可比的分型结果。较大的样本体积可产生更高量的DNA，但对于1至5毫升的样本体积，分型率不受影响。发现女性样本（14 - 200纳克/毫升）中可定量的DNA量大于男性样本（4 - 60纳克/毫升），并且在室温（RT）和冷冻储存条件下，DNA量均随时间推移而减少。对男性样本的分型还表明，室温储存样本的成功率显著高于冷冻样本，而使用女性样本在测试条件下DNA分型率之间只有微小差异。所有新鲜尿液样本，无论性别、起始样本体积或浓缩方法如何，均成功完成了DQA1 + PM基因型的鉴定。在4℃下，用0.25%叠氮化钠保存样本30天是可以接受的。对于更长的储存时间，在 - 70℃冷冻可能更合适。因此，DQA1 + PM分型在尿液样本个体化中的适用性得到了明确证明。
