采用巢式实时 PCR 法对人乳头瘤病毒 16 型 E6 致癌基因序列进行超灵敏定量分析。

Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR.

机构信息

División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, Camino de la Presa San José 2055, 78216 San Luis Potosí S,L,P,, México.

出版信息

Infect Agent Cancer. 2010 May 14;5:9. doi: 10.1186/1750-9378-5-9.

DOI:10.1186/1750-9378-5-9

PMID:20470388

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2888785/

Abstract

BACKGROUND

We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR) in the presence of the DNA intercalating agent EvaGreen.

RESULTS

Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp)-long insert of the human papillomavirus type 16 (HPV16) E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 x 102-2.5 x 106 initial pHV101 copies had threshold cycle (Ct) values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell) was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5 degrees C.

CONCLUSIONS

Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.

摘要

背景

我们开发了一种基于常规 PCR 预扩增的超灵敏方法，然后在实时 PCR（qPCR）中通过 DNA 嵌入剂 EvaGreen 进行嵌套扩增。

结果

使用校准了已知数量 pHV101 拷贝的扩增混合物，该混合物携带人类乳头瘤病毒 16 型（HPV16）E6 癌基因的 645 个碱基对（bp）长插入物，通过常规 PCR 生成 645 bp 的 E6-1 扩增子，然后通过嵌套 qPCR 生成 237 bp 的 E6-2 扩增子。直接和嵌套 qPCR 混合物用于 E6-2 扩增，对应于 2.5 x 102-2.5 x 106 个初始 pHV101 拷贝的 Ct 值范围分别为 18.7-29.0 和 10.0-25.0。用包含 50 ng SiHa 细胞系（源自每个细胞一个 HPV16 基因组的侵袭性宫颈癌）DNA 的预扩增混合物的 1/50 体积制备的 qPCR 混合物的 Ct 值为 19.9。从 pHV101 和 SiHa DNA 生成的 E6-2 扩增子的热荧光消光曲线相同，峰值在 85.5 摄氏度。