[Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus].
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 180 bp 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.


CONCLUSIONS
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.
Hang
Xiao-Tong
XT
State Key Laboratory for Molecular Virology and Genetic Engineering, Institute for Viral Diseases Control and Prevention, Chinese CDC, Beijing 100052, China.
Li
Jian-Dong
JD
Zhang
Quan-Fu
QF
Li
Chuan
C
Zhang
Shuo
S
Liang
Mi-Fang
MF
Li
De-Xin
DX
D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D003715:Dengue / Q000175:diagnosis / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*
D004279:DNA, Viral