[Use of the recombinant baculovirus BacVP6C for the construction of an internal positive control of rotavirus C].
OBJECTIVE
Unlike group A, a few studies have interested other groups of the rotavirus, especially in Tunisia. The role of rotavirus C (RVC) infection is underestimated because of its sporadic nature. The aim of our study was to develop rapid diagnostic procedures of RVC by using an internal positive control of reverse transcription PCR (RT-PCR).


METHODS
The internal positive control (386pb) was designed from the recombinant baculovirus BacVP6C containing the full length cDNA of the Cowden strain gene 5 (1353pb). A fragment of 596pb was amplified by PCR using the BacVP6C DNA ds as template. Then, a central part of 210pb was deleted and the remaining fragment (386pb) was cloned into pGEM-3Zf(+) plasmid between SP6 and T7 RNA polymerase promoters.


RESULTS
The obtained recombinant plasmid "pIAM1" was then used for the generation of the internal positive control by in vitro transcription. The sensibility of the RT-PCR was about 3.66×10(5) molecules of RNA/μl.


CONCLUSIONS
The use of a shorter positive control, as compared to the wild type, allows increased specificity of the RT-PCR reaction, and could be used for efficient diagnostic and surveillance of RVC-caused diseases.
Abid-Ayadi
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I
Groupe de recherche de virus entériques, département de microbiologie, université de Barcelone, avenue Diagonal 645, 08028 Barcelone, Espagne. islem_a@hotmail.com
Guix
S
S
Pintó
R M
RM
Bosch
A
A
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D035061:Control Groups; D018076:DNA, Complementary / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D042241:Early Diagnosis; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D018411:Spodoptera; D014158:Transcription, Genetic; D014416:Tunisia / Q000453:epidemiology; D014776:Virus Cultivation
D018076:DNA, Complementary; D004274:DNA, Recombinant; D004279:DNA, Viral