Quantification of plasmid DNA reference materials for Shiga toxin-producing Escherichia coli based on UV, HR-ICP-MS and digital PCR

大肠杆菌 数字聚合酶链反应 质粒 志贺毒素 化学 DNA 色谱法 聚合酶链反应 分子生物学 分析化学(期刊) 生物 基因 生物化学
作者
Wen‐Tzong Liang,Li Xu,Zhiwei Sui,Yan Li,Lanying Li,Yanli Wen,Chunhua Li,Shuzhen Ren,Gang Liu
出处
期刊:Chemistry Central Journal [BioMed Central]
卷期号:10 (1) 被引量:20
标识
DOI:10.1186/s13065-016-0201-0
摘要

The accuracy and metrology traceability of DNA quantification is becoming a critical theme in many fields, including diagnosis, forensic analysis, microorganism detection etc. Thus the research of DNA reference materials (RMs) and consistency of DNA quantification methods has attracted considerable research interest. In this work, we developed 3 plasmid candidate RMs, containing 3 target genes of Escherichia coli O157:H7 (E. coli O157:H7) and other Shiga toxin-producing Escherichia coli (STEC): stx1, stx2, and fliC (h7) respectively. Comprehensive investigation of the plasmid RMs was performed for their sequence, purity, homogeneity and stability, and then the concentration was quantified by three different methods: ultraviolet spectrophotometer (UV), high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) and digital PCR. As a routinely applied method for DNA analysis, UV was utilized for the quantification (OD260) and purity analysis for the plasmids. HR-ICP-MS quantified the plasmid DNA through analysing the phosphorus in DNA molecules. Digital PCR distributed the DNA samples onto a microarray chip containing thousands of reaction chambers, and quantified the DNA copy numbers by analysing the number of positive signals without any calibration curves needed. Based on the high purification of the DNA reference materials and the optimization of dPCR analysis, we successfully achieved good consistency between UV, HR-ICP-MS and dPCR, with relative deviations lower than 10 %. We then performed the co-quantification of 3 DNA RMs with three different methods together, and the uncertainties of their concentration were evaluated. Finally, the certified values and expanded uncertainties for 3 DNA RMs (pFliC, pStx1 and pStx2) were (1.60 ± 0.10) × 1010 copies/μL, (1.53 ± 0.10) × 1010 copies/μL and (1.70 ± 0.11) × 1010 copies/μL respectively.
最长约 10秒,即可获得该文献文件

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
PDF的下载单位、IP信息已删除 (2025-6-4)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
研友_VZG7GZ应助庸人自扰采纳,获得10
刚刚
zxcvvbnm完成签到 ,获得积分10
刚刚
坦率大米完成签到,获得积分10
1秒前
yangying完成签到,获得积分10
2秒前
默默若枫完成签到,获得积分10
2秒前
4秒前
Lawliet完成签到,获得积分10
5秒前
滑蛋肉片发布了新的文献求助10
5秒前
英俊的铭应助蓝色斑马采纳,获得10
6秒前
稳重的胡萝卜完成签到,获得积分10
6秒前
7秒前
坦率鬼卞完成签到,获得积分10
8秒前
年轻的路人完成签到,获得积分10
9秒前
9秒前
fornas完成签到 ,获得积分10
11秒前
12秒前
凩飒给凩飒的求助进行了留言
13秒前
在水一方应助xd采纳,获得10
13秒前
Chushi完成签到,获得积分10
14秒前
领导范儿应助LLX123采纳,获得10
15秒前
17秒前
Alioth发布了新的文献求助10
17秒前
Aierlan611发布了新的文献求助10
18秒前
万能图书馆应助哭泣乌采纳,获得10
19秒前
萝卜发布了新的文献求助10
21秒前
23秒前
23秒前
Lucas应助123采纳,获得10
24秒前
24秒前
胖玻璃球完成签到 ,获得积分10
26秒前
深情安青应助贝壳采纳,获得10
27秒前
庸人自扰发布了新的文献求助10
27秒前
28秒前
28秒前
刘gg发布了新的文献求助10
28秒前
哭泣乌发布了新的文献求助10
31秒前
赘婿应助qqqq采纳,获得10
31秒前
31秒前
CipherSage应助滑蛋肉片采纳,获得10
32秒前
Kurt发布了新的文献求助10
32秒前
高分求助中
Ophthalmic Equipment Market by Devices(surgical: vitreorentinal,IOLs,OVDs,contact lens,RGP lens,backflush,diagnostic&monitoring:OCT,actorefractor,keratometer,tonometer,ophthalmoscpe,OVD), End User,Buying Criteria-Global Forecast to2029 2000
A new approach to the extrapolation of accelerated life test data 1000
Cognitive Neuroscience: The Biology of the Mind 1000
ACSM’s Guidelines for Exercise Testing and Prescription, 12th edition 588
不知道标题是什么 500
A Preliminary Study on Correlation Between Independent Components of Facial Thermal Images and Subjective Assessment of Chronic Stress 500
Technical Brochure TB 814: LPIT applications in HV gas insulated switchgear 500
热门求助领域 (近24小时)
化学 材料科学 医学 生物 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 遗传学 基因 物理化学 催化作用 冶金 细胞生物学 免疫学
热门帖子
关注 科研通微信公众号,转发送积分 3962550
求助须知:如何正确求助?哪些是违规求助? 3508565
关于积分的说明 11141672
捐赠科研通 3241287
什么是DOI,文献DOI怎么找? 1791495
邀请新用户注册赠送积分活动 872888
科研通“疑难数据库(出版商)”最低求助积分说明 803474