Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: Application for transrenal Mycobacterium tuberculosis DNA detection
Design and use of mouse control DNA for DNA biomarker extraction and PCR detection from urine: Application for transrenal Mycobacterium tuberculosis DNA detection
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DOI：
10.1016/j.mimet.2017.02.010
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发表时间：
2017-05-01
影响因子：
2.2
通讯作者：
Pettit, April C.
中科院分区：
文献类型：
--
作者：
Bordelon, Hali;Ricks, Keersten M.;Pettit, April C.
尿液样本越来越多地用于诊断感染，包括大肠杆菌，埃博拉病毒和寨卡病毒。然而，从尿液中提取和浓缩核酸生物标志物对于许多分子检测策略如聚合酶链反应（PCR）是必要的。由于尿液样品通常具有大体积，具有稀释的生物标志物浓度，使得它们易于出现假阴性，因此基于尿液的诊断的另一个障碍是建立适当的控制，特别是排除假阴性。在这项研究中，将小鼠甘油醛3-磷酸脱氢酶（GAPDH）DNA靶标添加到回顾性收集的结核病（TB）感染和TB未感染患者的尿液样本中，以指示完整DNA的提取和PCR抑制剂从尿液样本中的去除。我们在替代尿样上测试了该设计，回顾性的来自秘鲁利马患者的1毫升（mL）尿样和回顾性的来自南非开普敦患者的5 mL尿样。在97%的临床样本中可检测到提取/PCR对照DNA，组间无统计学显著差异。尽管纳入了该对照，但在TB感染组和TB未感染组之间检测到的TB IS 6110 Tr-DNA的量没有差异，除了来自已知HIV感染患者的样品。我们发现与TB未感染/HIV感染患者相比，TB/HIV共感染患者之间的TB IS 6110 Tr-DNA增加（N = 18，p = 0.037）。包含提取/PCR对照DNA以指示成功的DNA提取和PCR抑制剂的去除，应易于适用于作为其他非细胞样品类型的样品制备对照。(C)2017 Elsevier B. V.版权所有。
Urine samples are increasingly used for diagnosing infections including Escherichia coli, Ebola virus, and Zika virus. However, extraction and concentration of nucleic acid biomarkers from urine is necessary for many molecular detection strategies such as polymerase chain reaction (PCR). Since urine samples typically have large volumes with dilute biomarker concentrations making them prone to false negatives, another impediment for urine-based diagnostics is the establishment of appropriate controls particularly to rule out false negatives. In this study, a mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA target was added to retrospectively collected urine samples from tuberculosis (TB)-infected and TB-uninfected patients to indicate extraction of intact DNA and removal of PCR inhibitors from urine samples. We tested this design on surrogate urine samples, retrospective 1 milliliter (mL) urine samples from patients in Lima, Peru and retrospective 5 mL urine samples from patients in Cape Town, South Africa. Extraction/PCR control DNA was detectable in 97% of clinical samples with no statistically significant differences among groups. Despite the inclusion of this control, there was no difference in the amount of TB IS6110 Tr-DNA detected between TB-infected and TB-uninfected groups except for samples from known HIV-infected patients. We found an increase in TB IS6110 Tr-DNA between TB/HIV co-infected patients compared to TB-uninfected/HIV-infected patients (N = 18, p = 0.037). The inclusion of an extraction/PCR control DNA to indicate successful DNA extraction and removal of PCR inhibitors should be easily adaptable as a sample preparation control for other acellular sample types. (C) 2017 Elsevier B.V. All rights reserved.