Establishment of a rapid paper-based extraction method for human papilloma virus DNA and its preliminary evaluation
- Journal of Third Military Medical University Vol. 43, Issue 19, Pages: 1925-1931(2021)
- 作者机构：
1.510006 广州，广州中医药大学研究生院
2.510010 广州，中国人民解放军南部战区总医院基础医学实验室
- 作者简介：
- 基金信息：the Science and Technology Program of Guangzhou(201904010148);the Military Logistics Support Research Program(CGZ16J002);the National Natural Science Foundation for Young Scholars of China(81401752)
DOI：10.16016/j.1000-5404.202103267
CLC： R372;R373.9;R446.5Received：31 March 2021，
Revised：2021-05-28，
Published：15 October 2021
- 稿件说明：
移动端阅览
Weiqi REN, Yu CHEN, Zhongming LIU, et al. Establishment of a rapid paper-based extraction method for human papilloma virus DNA and its preliminary evaluation[J]. Journal of Third Military Medical University, 2021, 43(19): 1925-1931.
Weiqi REN, Yu CHEN, Zhongming LIU, et al. Establishment of a rapid paper-based extraction method for human papilloma virus DNA and its preliminary evaluation[J]. Journal of Third Military Medical University, 2021, 43(19): 1925-1931. DOI： 10.16016/j.1000-5404.202103267.
摘要
目的
2
建立一种简便快速的人乳头瘤病毒(human papillomavirus
HPV)DNA纸基提取方法并对其进行初步评价。
方法
2
以含HPV 16型基因片段的质粒为对象，结合紫外分光光度法和实时荧光定量PCR检测，对6种不同的纸材料进行选择，最终设计成方便拿取的浸蜡试纸条，对18例临床宫颈脱落细胞样本(阳性样本13例，阴性样本5例)进行HPV DNA提取检测，与常规煮沸法进行比较。
结果
2
1张核酸结合区面积为2 mm×10 mm的浸蜡定性滤纸条所提取的临床宫颈脱落细胞样本中的HPV DNA，经实时荧光定量PCR扩增，18例临床宫颈脱落细胞样本中的13例高危型HPV阳性样本均能成功检出，与常规煮沸法结果一致。每个样本提取时间
<
1 min，检出限为8×10
2
copies/mL，相对标准偏差为1.15%。
结论
2
建立的HPV DNA纸基快速提取方法无需移液、加热和离心等操作，洗脱液可直接作为PCR扩增反应的模板，极大简化了临床宫颈脱落细胞样本中HPV DNA的提取步骤。
Abstract
Objective
2
To establish a simple and rapid paper-based extraction method for human papilloma virus (HPV) DNA and to evaluate the method.
Methods
2
The plasmid containing HPV type 16 gene fragment was employed as the test subjects
and then 6 different paper materials were compared and selected by ultraviolet spectrophotometry and real-time fluorescence quantitative PCR (qPCR). Then
according to the efficiency of nucleic acid extraction and the optimal binding area
a convenient paper strip was designed. Finally
the designed paper strip was applied to extract HPV DNA from 18 clinical cervical exfoliated cell samples (13 positives and 5 negatives)
with its performance being evaluated after comparing with the conventional boiling method.
Results
2
The qualitative filter paper with a nucleic acid binding zone of 2 mm×10 mm could successfully extract HPV DNA from clinical cervical exfoliated cells. When this test strip was used for DNA extraction
all the 13 high-risk HPV positives were successfully detected out of the 18 clinical samples
which was consistent with the results of the conventional boiling method. In addition
the extraction time of the test strip was less than 1 min for each sample
with a detection limit of 8×10
2
copies/mL and relative standard deviation of 1.15%.
Conclusion
2
Our paper-based rapid extraction for HPV DNA does not require pipetting
heating and centrifugation
and the eluent can be directly used as the PCR template for amplification
which greatly simplifies the extraction process of HPV DNA from clinical cervical exfoliated cell samples.
关键词
Keywords
references
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