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患有败血症、创伤和肿瘤的犬血浆游离DNA浓度的测量。

Measurement of plasma cell-free DNA concentrations in dogs with sepsis, trauma, and neoplasia.

作者信息

Letendre Jo-Annie, Goggs Robert

机构信息

Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853.

出版信息

J Vet Emerg Crit Care (San Antonio). 2017 May;27(3):307-314. doi: 10.1111/vec.12592. Epub 2017 Mar 10.

Abstract

OBJECTIVES

To determine if cell-free DNA (cfDNA) was identifiable in canine plasma, to evaluate 3 techniques for the measurement of plasma cfDNA concentrations in dogs presented to an emergency service, and to compare the plasma cfDNA concentrations of healthy dogs to those with sepsis, trauma, and neoplasia.

DESIGN

Retrospective study of banked canine plasma samples collected between May 2014 and December 2014.

SETTING

Dogs presented to the emergency service of a university veterinary teaching hospital.

ANIMALS

Plasma cfDNA was measured on residual plasma samples obtained from 15 dogs with sepsis, 15 dogs with moderate-severe trauma, 15 dogs diagnosed with a sarcoma. Plasma cfDNA was also measured in 15 healthy dogs.

INTERVENTIONS

None.

MEASUREMENTS AND MAIN RESULTS

Assay linearity, repeatability, and reproducibility were evaluated. Quantification of cfDNA was performed in duplicate on diluted citrated plasma and following DNA purification using 2 fluorescence assays (SYBR-Gold; Quant-iT) and by ultraviolet absorbance spectroscopy. Fluorescence intensities (FIs) were converted to cfDNA concentrations using standard curves. Median FI values and cfDNA concentrations were compared to healthy controls using the Kruskal-Wallis test, with adjustment for multiple comparisons. Alpha was set at 0.05. Both assays had excellent linearity, and acceptable repeatability and reproducibility. Compared to controls, plasma cfDNA concentrations were significantly increased in dogs with sepsis or moderate-severe trauma with both assays (P ≤ 0.003). Dogs with neoplasia had significantly increased cfDNA concentrations with the Quant-iT assay only (P = 0.003). When measurements were performed on purified DNA, only dogs with moderate-severe trauma had significantly increased cfDNA concentrations (P < 0.001; SYBR-Gold assay).

CONCLUSIONS

cfDNA can be readily identified in canine plasma using 2 fluorescence assays. DNA extraction offers no advantage over direct measurement. Compared to healthy controls, dogs with sepsis or moderate-severe trauma have significantly increased plasma cfDNA concentrations.

摘要

目的

确定犬血浆中是否可检测到游离DNA(cfDNA),评估三种测量急诊犬血浆cfDNA浓度的技术,并比较健康犬与患有败血症、创伤和肿瘤的犬的血浆cfDNA浓度。

设计

对2014年5月至2014年12月收集的犬血浆样本进行回顾性研究。

地点

一所大学兽医教学医院的急诊服务部门收治的犬。

动物

对从15只患有败血症的犬、15只患有中重度创伤的犬、15只被诊断为肉瘤的犬获取的残余血浆样本测量血浆cfDNA。还对15只健康犬测量血浆cfDNA。

干预措施

无。

测量和主要结果

评估检测的线性、重复性和再现性。使用两种荧光检测法(SYBR-金;Quant-iT)并通过紫外吸收光谱法,对稀释的枸橼酸血浆以及DNA纯化后的样本进行一式两份的cfDNA定量。使用标准曲线将荧光强度(FI)转换为cfDNA浓度。使用Kruskal-Wallis检验将FI中位数和cfDNA浓度与健康对照进行比较,并对多重比较进行校正。α设定为0.05。两种检测法均具有出色的线性以及可接受的重复性和再现性。与对照组相比,两种检测法均显示患有败血症或中重度创伤的犬血浆cfDNA浓度显著升高(P≤0.003)。仅使用Quant-iT检测法时,患有肿瘤的犬cfDNA浓度显著升高(P = 0.003)。当对纯化的DNA进行测量时,仅患有中重度创伤的犬cfDNA浓度显著升高(P < 0.001;SYBR-金检测法)。

结论

使用两种荧光检测法可在犬血浆中轻松检测到cfDNA。DNA提取相对于直接测量无优势。与健康对照相比,患有败血症或中重度创伤的犬血浆cfDNA浓度显著升高。

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