The development and application of a SYBR Green I real-time PCR assay for detection of infectious bursal disease virus.
- Author:
Xin ZHOU
1
;
Xia YANG
;
Jun ZHAO
;
Hong-Tao CHANG
;
Xin-Wei WANG
;
Lu CHEN
;
Chuan-Qing WANG
Author Information
1. College of Animal Science and Veterinary Medicine, Henan Agriculture University, Zhengzhou 450002, China. zhouxin20088002@yahoo.cn
- Publication Type:Journal Article
- MeSH:
Animals;
Calibration;
Cell Line;
Conserved Sequence;
DNA Primers;
genetics;
Infectious bursal disease virus;
genetics;
isolation & purification;
Organic Chemicals;
chemistry;
metabolism;
Real-Time Polymerase Chain Reaction;
methods;
Reproducibility of Results;
Spectrometry, Fluorescence;
Viral Proteins;
genetics;
Virus Replication
- From:
Chinese Journal of Virology
2012;28(4):424-430
- CountryChina
- Language:Chinese
-
Abstract:
To meet the needs of detection of infectious bursal disease virus (IBDV) under high efficient culture, a SYBR Green I real-time RT-PCR (qRT-PCR) was developed using a pair of primers specific to the conserved region of VP4 gene of IBDV and compared with TCID50 method by monitoring the proliferation dynamics of IBDV in DF-1 cell line adherent to micro carrier in tubular reactor. The results showed that the RT-PCRassay was linear in the range of 4. 03 X 10(1)-10(9) copies/microL. The IBDV RNA detection limit was 40 copies/microL, which was 1 000 times more sensitive than conventional PCR. No cross-reactions with other viruses was observed. The intra-assay coefficient of variation was less than 0.05%. There was a parallel correlation of IBDV proliferation dynamics in DF-1 cell under Micro carrier suspension and static adherent culture by the qRT-PCR assay and TCID50 method. The detection results of the IBDV samples from tubular and flask culture showed the differences of the micro carrier and adherent culture by both methods. In conclusion, the qRT-PCR assay is more rapid and sensitive than the TCID50 method, which is more appropriate for the real time detection of IBDV.
