Development of SYBR green I based one-step real-time RT-PCR assay for the detection and differentiation of very virulent and classical strains of infectious bursal disease virus
Introduction
Infectious bursal disease (IBD) or Gumboro disease is an acute, highly contagious and immunosuppressive disease affecting the poultry industry worldwide (Muller et al., 2003). The causative agent, infectious bursal disease virus (IBDV) is a non-enveloped virus with bi-segmented, double-stranded RNA genome belonging to the family Birnaviridae. IBDV can be differentiated into two serotypes. Serotype 1 viruses are pathogenic to chickens and differ markedly in their virulence where the virus can be divided as classical, variant, attenuated and very virulent strains (Jackwood et al., 1982, Muller et al., 2003), whilst serotype 2 strains are non-pathogenic for chickens (McFerran et al., 1980).
In the past, reverse transcriptase polymerase chain (RT-PCR) has been used for the detection of IBDV (Tham et al., 1995, Jackwood and Nielsen, 1997) or to quantitate the amount of IBDV in infected bursa of Fabricius (Wu et al., 1997). Hence, RT-PCR has contributed greatly to the improvement of laboratory diagnostics and research on IBDV. Real-time RT-PCR is becoming rapidly a standard method in many diagnostic and research laboratories (Monis et al., 2005). This method continues to expand and to fulfill this demand but there have been several probe-based systems developed, including TaqMan probes (Heid et al., 1996), molecular beacons (Piatek et al., 1998), FRET probes (Chen and Kwok, 1999) and Scorpions (Solinas et al., 2001). An alternative to this system is the use of double-stranded DNA (dsDNA)-specific intercalating dyes such as ethidium bromide (Higuchi et al., 1993), SYBR Green I (Ririe et al., 1997), BEBO (Bengtsson et al., 2003) and LC Green (Wittwer et al., 2003) have been evaluated for the use in real-time PCR applications.
Numerous real-time PCR assays have been developed for the diagnosis of poultry disease and contributed to the control of the disease. Real-time RT-PCR has been applied for the quantitation of IBDV in blood samples from different inbred lines of chickens (Moody et al., 2000) and to detect and quantify virus-specific RNA in bursal samples (Jackwood et al., 2003, Kong et al., 2004a, Li et al., 2007, Peters et al., 2005, Sreedevi and Jackwood, 2007). Therefore, real-time RT-PCR targeting different regions of the IBDV genome, including the VP1, VP2, and the VP4 genes in conjunction with melting curve analysis indicated that real-time PCR is a rapid and sensitive assay for specific detection and differentiation of classical, very virulent and variant IBDV strains (reviewed by Wu et al., 2007). In addition, Hairul Aini et al. (2008) reported that the real-time RT-PCR was at least 10 times more sensitive than colorimetric and conventional agarose gel-based PCR. In this study, an attempt was made to develop a SYBR Green I based real-time PCR as an alternative method for distinguishing vvIBDV and classical IBDV based on discriminatory feature of threshold cycle and melting temperature values.
Section snippets
IBDV reference strains
Two IBDV strains UPM94/273 and D78 were used as reference very virulent (vv) and classical strains, respectively. The IBDV isolate UPM94/273 is classified as vvIBDV based on sequence analysis of segments A and B (Hoque et al., 2001, Chong et al., 2001, Kong et al., 2004b) and pathogenicity studies (Hoque et al., 2001). The D78 (Intervet, Boxmeer, The Netherlands) is classified as a classical IBDV strain.
IBDV strains
Ten IBDV field strains which have been characterized previously: B00/81/Malaysia/AY520910,
One-step real-time RT-PCR detection assay for rapid detection of IBDV strains
The optimized one-step real-time RT-PCR detection method was carried out using both (IF & IVIR) and (IF & RCLA) primer combinations on the reference IBDV strains. As shown in Fig. 1, amplification from very virulent UPM94/273 and classical D78 strains was detected only with match primer combinations, IF & IVIR and IF & RCLA, with the detection of melting temperature (Tm) of 87.2 °C and 86.0 °C, respectively. No specific amplification was detected from mismatch primer combinations.
Amplification of
Discussion
In this study, a specific and sensitive one-step SYBR Green I based real-time RT-PCR assay for the detection and differentiation of very virulent and classical strains of IBDV based on detection of threshold cycle and melting temperature was developed. The optimized real-time RT-PCR assay gave consistent results in detecting and differentiating samples that were positive for very virulent and/or classical strain of IBDV using subtype-specific primers based on primer–template match and mismatch
Acknowledgements
This research was supported by grant number 01-02-04-009 BTK/ER/38 from Ministry of Science, Technology and Innovation, Government of Malaysia and grant number BF054UPM from Malaysian Technology Development Corporation. We thank Hairul Aini Hamzah from Biologics Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia for her excellent technical assistance.
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