结直肠癌患者粪便细菌实时荧光定量PCR标准品制备方法的比较研究  

Comparison of standard sample preparation methods with real-time fluorescence quantitative PCR from colorectal cancer patient fecal bacteria

作　　者：郭世奎[1,2] 包维民[1,2] 龚昆梅[1,2] 邵剑春[3] 陈弟[3] 王昆华[1,2] 

机构地区：[1]昆明医学院附属昆华医院 [2]云南省第一人民医院普外一科,云南昆明650032 [3]昆明市第一人民医院检验科,云南昆明650011

出　　处：《中国现代普通外科进展》2009年第11期939-942,共4页Chinese Journal of Current Advances in General Surgery

基　　金：云南省应用基础研究基金资助(2005C0074M;2008ZC085M)

摘　　要：目的:比较结直肠癌患者粪便细菌实时荧光定量PCR标准品制备方法的优缺点及获得DNA的质与量。方法:以粪肠球菌作为试验菌株,分别采用标准菌株法、PCR扩增产物纯化法和PCR扩增目的片段割胶纯化法制备标准品,比较三者用于细菌实时荧光定量PCR标准曲线制作的好坏及用核酸蛋白检测仪Biophotmeter测定获得DNA的质与量。并将PCR扩增产物纯化法得到的产物进行测序。结果:PCR扩增目的片段割胶纯化法获得DNA的量及纯度均较低,为29.9±3.2和2.8±0.2,标准菌株法和PCR扩增产物纯化法的量及纯度均较高,分别为62.9±2.7及1.7±0.1,59.1±2.7及1.8±0.1;PCR扩增目的片段割胶纯化法与标准菌株法和PCR扩增产物纯化法相比,差异有统计学意义(P<0.05),标准菌株法和PC R扩增产物纯化法相比,差异无统计学意义(P>0.05),但以标准菌株法为佳。采用标准菌株法及PCR扩增产物纯化法,能获得很好的标准曲线(r=-1.00)。并且纯化产物序列与目的片段序列完全相同。结论:标准菌株法及PCR扩增产物纯化法制备标准品作荧光定量PCR检测比PCR扩增目的片段割胶纯化法更适合肠道相关分子微生态的研究。Objective： To explore the differences among three standard preparation methods with real-time fluorescence quantitative PCR from colorectal cancer patients fecal bacteria, and the quantification and quality of DNA which obtained on the gut-associated analysis of molecular microecotogy. Methods： The standard bacteria was enterococcus faecalis. Using standard bacteria method, PCR amplified product purification method and PCR amplified fragment tapping purification method were applied to compare their products as a standard for the bacterial real-time fluorescence quantitative PCR standard curve and nucleic acid protein detection Determination Biophotmeter was uesd to analyze quantification and quality of the DNA. And PCR amplification product was purified and sequenced. Results： The amount and Durity of DNA from PCR amplified fragment tapping purification method were lower （29.9± 3.2, 2.8± 0.2）, The standard bacteria and PCR amplified product purification method were higher （62.9 ± 2.7, 1.7 ±0.1 59.1± 2.7, 1.8 ± 0.11. The difference between standard bacteria method and PCR amplified product purification method was not significant （P〉0.05）, but the former one was better. Standard bacteria method and PCR amplified product purification method can get a excellent standard curve （r=-1.00）. And the purified PCR amplification products were sequenced, the test sequence was identical with fragment sequence. Conclusion： Comparing with the PCR amplified fragment tapping purification method, standard bacteria method and PCR amplified product purification method are more appropriate for gut-associated molecular micro-ecological research in real-time fluorescence quantitative PCR for colorectal cancer patients.

关 键 词：结直肠肿瘤 粪便 细菌 实时荧光定量PCR 

分 类 号：R735.35[医药卫生—肿瘤]