大肠杆菌O157∶H7微滴数字PCR定量方法的建立
以大肠杆菌O157∶H7（E.coli O157∶H7）rfb E基因为靶基因,建立了可对其准确定量的微滴数字PCR（dd PCR）方法。对dd PCR反应中的探针浓度进行了优化,考察了方法的线性范围、精密度、定量限和检出限。最终确定dd PCR反应中的最佳探针浓度为300 nmol/L。E.coli O157∶H7基因组DNA浓度范围为4-1.25×105拷贝/20μL dd PCR反应液时,dd PCR方法线性相关系数（R2）为0.999。当DNA浓度为760-88400拷贝/20μL时,方法的精密度最好（RSD〈5%）。本方法的定量限为4拷贝/20μL,检出限为3拷贝/20μL。特异性验...
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| Published in | 分析化学 Vol. 43; no. 3; pp. 319 - 324 |
|---|---|
| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
中国计量科学研究院,北京,100029%中国科学院北京基因组研究所基因组科学与信息重点实验室,北京,100029%北京市计量检测科学研究院 北京100029
2015
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| Subjects | |
| Online Access | Get full text |
| ISSN | 0253-3820 |
| DOI | 10.11895/j.issn.0253-3820.140569 |
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| Abstract | 以大肠杆菌O157∶H7（E.coli O157∶H7）rfb E基因为靶基因,建立了可对其准确定量的微滴数字PCR（dd PCR）方法。对dd PCR反应中的探针浓度进行了优化,考察了方法的线性范围、精密度、定量限和检出限。最终确定dd PCR反应中的最佳探针浓度为300 nmol/L。E.coli O157∶H7基因组DNA浓度范围为4-1.25×105拷贝/20μL dd PCR反应液时,dd PCR方法线性相关系数（R2）为0.999。当DNA浓度为760-88400拷贝/20μL时,方法的精密度最好（RSD〈5%）。本方法的定量限为4拷贝/20μL,检出限为3拷贝/20μL。特异性验证结果表明,建立的dd PCR方法特异性良好,对13份猪肉、牛肉和鸡肉样品的检测结果与定量PCR方法检出结果一致。 |
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| AbstractList | 以大肠杆菌O157∶H7（E.coli O157∶H7）rfb E基因为靶基因,建立了可对其准确定量的微滴数字PCR（dd PCR）方法。对dd PCR反应中的探针浓度进行了优化,考察了方法的线性范围、精密度、定量限和检出限。最终确定dd PCR反应中的最佳探针浓度为300 nmol/L。E.coli O157∶H7基因组DNA浓度范围为4-1.25×105拷贝/20μL dd PCR反应液时,dd PCR方法线性相关系数（R2）为0.999。当DNA浓度为760-88400拷贝/20μL时,方法的精密度最好（RSD〈5%）。本方法的定量限为4拷贝/20μL,检出限为3拷贝/20μL。特异性验证结果表明,建立的dd PCR方法特异性良好,对13份猪肉、牛肉和鸡肉样品的检测结果与定量PCR方法检出结果一致。 以大肠杆菌O157：H7(E. coli O157：H7)rfbE基因为靶基因,建立了可对其准确定量的微滴数字PCR( ddPCR)方法。对ddPCR反应中的探针浓度进行了优化,考察了方法的线性范围、精密度、定量限和检出限。最终确定ddPCR 反应中的最佳探针浓度为300 nmol/L。 E. coli O157：H7基因组 DNA 浓度范围为4~1.25×105拷贝/20μL ddPCR反应液时,ddPCR方法线性相关系数( R2)为0.999。当DNA浓度为760~88400拷贝/20μL 时,方法的精密度最好( RSD<5%)。本方法的定量限为4拷贝/20μL,检出限为3拷贝/20μL。特异性验证结果表明,建立的ddPCR方法特异性良好,对13份猪肉、牛肉和鸡肉样品的检测结果与定量PCR方法检出结果一致。 |
| Abstract_FL | A droplet digital polymerase chain reaction ( ddPCR) method for quantifying E. coli O157:H7 by targeting rfbE gene was developed. The probe concentration in ddPCR was optimized and the linearity range, precision, limit of detection ( LOD) and limit of quantification ( LOQ) were also evaluated. The optimized probe concentration was 300 nmol/L. The ddPCR response was linear over the E. coli O157:H7 genome DNA concentration range from 4 to 1. 25×105 copies in 20 μL ddPCR system and the linear correlation coefficient (R2) was 0. 999. The ddPCR precision (RSD) was less than 5% over the DNA concentration range from 760 to 88400 copies/20 μL. The LOD and LOQ was 3 copies in 20 μL and 4 copies in 20 μL, respectively. Specificity test showed that the ddPCR was specific for detecting E. coli O157:H7. Both ddPCR and standard real time quantitative PCR showed the same results for 16 real samples of chicken meat, pork and beef, which indicated that ddPCR method was suitable for detection of E. coli O157:H7 in food. |
| Author | 董莲华 张玲 姜君 王江南 王晶 陈唯军 |
| AuthorAffiliation | 中国计量科学研究院,北京100029 中国科学院北京基因组研究所基因组科学与信息重点实验室,北京100029 北京市计量检测科学研究院北京100029 |
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| Author_FL | ZHANG Ling WANG Jiang-Nan JING Jun DONG Lian-Hua WANG Jing CHEN Wei-Jun |
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| Keywords | 大肠杆菌O157:H7 Copy number Droplet digital polymerase chain reaction 拷贝数 Escherichia coli O157:H7 微滴数字聚合酶链式反应 |
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| Notes | DONG Lian-Hua , ZHANG Ling , JING Jun , WANG Jiang-Nan , WANG Jing , CHEN Wei-Jun （ National Institute of Metrology, Beijing 100013, China;Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciertees, Beijing 100029 , China; Beijing Institute of Metrology and Test, Beijing 100029, China） 22-1125/O6 Droplet digital polymerase chain reaction; Escherichia coli O157∶ H7; Copy number A droplet digital polymerase chain reaction（ dd PCR） method for quantifying E. coli O157∶ H7 by targeting rfb E gene was developed. The probe concentration in dd PCR was optimized and the linearity range,precision,limit of detection（ LOD） and limit of quantification（ LOQ） were also evaluated. The optimized probe concentration was 300 nmol / L. The dd PCR response was linear over the E. coli O157∶ H7 genome DNA concentration range from 4 to 1. 25 ×10^5copies in 20 μL dd PCR system and the linear correlation coefficient（ R2） was 0. 999. The dd PCR precision（ RSD） was less than 5% over the D |
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| PublicationTitle | 分析化学 |
| PublicationTitleAlternate | Chinese Journal of Analytical Chemistry |
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| PublicationYear | 2015 |
| Publisher | 中国计量科学研究院,北京,100029%中国科学院北京基因组研究所基因组科学与信息重点实验室,北京,100029%北京市计量检测科学研究院 北京100029 |
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| Snippet | 以大肠杆菌O157∶H7（E.coli O157∶H7）rfb E基因为靶基因,建立了可对其准确定量的微滴数字PCR（dd PCR）方法。对dd PCR反应中的探针浓度进行了优化,考察了方法的线性范围、精密度、定量限和检出限。最终确定dd PCR反应中的最佳探针浓度为300 nmol/L。E.coli... 以大肠杆菌O157：H7(E. coli O157：H7)rfbE基因为靶基因,建立了可对其准确定量的微滴数字PCR( ddPCR)方法。对ddPCR反应中的探针浓度进行了优化,考察了方法的线性范围、精密度、定量限和检出限。最终确定ddPCR 反应中的最佳探针浓度为300 nmol/L。 E. coli... |
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| SubjectTerms | 大肠杆菌O157∶H7 微滴数字聚合酶链式反应 拷贝数 |
| Title | 大肠杆菌O157∶H7微滴数字PCR定量方法的建立 |
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