中国比较医学杂志2012，Vol.22Issue(2)：22-27,6.DOI:10.3969/j.issn.1671.7856.2012.02.006
Trizol RNA提取法在雪貂组织的流感病毒H3N2定量PCR检测中优于RNeasy mini kit(Qiagen)柱子法
Trizol Method for RNA Isolation from Ferret Tissues is Better Than RNeasy Mini Kit (Qiagen) for Diagnosis of Influenza Virus H3N2 Infection
摘要
Abstract
Objective Comparing the methods for total RNA isolation from ferret tissues with Trizol, Rneasy mini kit, modified Rneasy mini kitl and modified Rneasy mini kit2 in removing SYBR Green real time PCR inhibitors for diagnosis of influenza H3N2 virus infection. Methods Comparing the total RNA quality isolated by methods Trizol, Rneasy mini kit, modified Rneasy mini kitl and modified Rneasy mini kit2, respectively, and also the results of assay of SYBR Green real time PCR for the influenza virus H3N2 in tissue total RNA. Results The quality of RNA isolated by the above four methods were measured using a Nano drop 2. 0 UV spectrophotometer. A260/280 of the RNA isolated by Rneasy mini kit was below 1.8, and the other three were between 1. 8 and 2.1. For A260/230, only the RNA isolated by Trizol was about 2. 0, the other three were below 2. 0. RNA isolated by Trizol ran in the 1. 5% agrose gel electrophoresis showed three bands, 28 s, 18 s, and 5 s, while from the same tissues isolated by Rneasy mini kit showed three bands and genomic DNA, 28 s and 18 s. As for modified Rneasy mini kitl and modified Rneasy mini kit2, there were only two bands 28 s and 18 s. Judging form the melting curve of SYBR Green real time PCR, the templates obtained from both turbinates and lung or intestine by the four methods were normal with the same wave crest position for samples and standard. Though the melting curve of tissues was specific, the products of SYBR Green real time PCR were different: the product by Trizol method was a single specific band on the 1.5% agrose gel electrophoresis. By Rneasy mini kit, modified Rneasy mini kitl and modified Rneasy mini kit2, the products were multi-bands. The products of turbinate samples were all a single specific band by the four different methods. Conclusions For turbinate swab samples, the four methods are all right for virus quantification, but for the ferret tissues including lung and intestine, the classical Trizol method is as creditable for as SYBR Green real time PCR.关键词
雪貂/H3N2/RNA提取/SYBR Green荧光定量PCRKey words
Trizol/ RNA isolation/ Ferret/ Lung/ Intestine/ Influenca virus, H3N2/ Diagnosis分类
医药卫生引用本文复制引用
占玲俊,鲍琳琳,李枫棣,吕琦,许黎黎,秦川..Trizol RNA提取法在雪貂组织的流感病毒H3N2定量PCR检测中优于RNeasy mini kit(Qiagen)柱子法[J].中国比较医学杂志,2012,22(2):22-27,6.基金项目
科技重大专项—艾滋病和病毒性肝炎等重大传染病防治:2009ZX10004-402 （）
卫生公益性行业科研专项项目:200802036. （）
