Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus
10.3760/cma.j.issn.1003-9279.2010.01.020
- VernacularTitle:登革病毒RT-PCR核酸检测竞争性假病毒颗粒型内参照品的建立
- Author:
Xiao-Tong HANG
1
;
Jian-Dong LI
;
Quan-Fu ZHANG
;
Chuan LI
;
Shuo ZHANG
;
Mi-Fang LIANG
;
De-Xin LI
Author Information
1. 中国疾病预防与控制中心
- Keywords:
Dengue virus;
Internal control;
Reverse transcriptase polymerase chain reaction
- From:
Chinese Journal of Experimental and Clinical Virology
2010;24(1):56-58
- CountryChina
- Language:Chinese
-
Abstract:
Objective Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus. Methods The internal controls target gene were obtained by insertion of a 180bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. Results The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. Conclusion The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.
