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

https://doi.org/10.1016/j.jviromet.2009.06.023Get rights and content

Abstract

A SYBR Green I based one-step real-time reverse transcriptase polymerase chain reaction was developed for the detection and differentiation of very virulent (vv) and classical strains of infectious bursal disease virus (IBDV). The assay showed high PCR efficiency >93% and high reproducibility with coefficient of variation less than 0.5%. When tested on characterized IBDV strains, the very virulent and classical-specific primers detected accurately only vvIBDV and classical IBDV strains, respectively. The diagnostic efficacy of the assay was also tested on 140 bursal samples from experimental infection and 37 bursal samples from cases suspected of IBD. The assay was able to detect IBDV from bursal samples collected at days 3 and 5 post-infection with the vvIBDV strain UPM94/273 and the classical IBDV strain D78. The assay was also able to detect bursal samples infected dually with D78 and UPM94/273. The melting temperature values of the amplification products from the classical and very virulent viral infection were statistically significant (P < 0.05). The specificity of the assay for detecting IBDV from suspected cases was confirmed by sequence analysis of the VP2 gene. The assay showed high sensitivity since bursal samples which were negative for IBDV were confirmed by virus isolation and PCR amplification. Hence, the new assay offers an attractive method for rapid detection of strains of IBDV.

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.

References (36)

  • C.D. Bayliss et al.

    A comparison of the sequences of segment A of four infectious bursal disease virus strains and identification of a variable region in VP2

    J. Gen. Virol.

    (1990)
  • M. Bengtsson et al.

    A new minor groove binding asymmetric cyanine reporter dye for real-time PCR

    Nucleic Acids Res.

    (2003)
  • L.K. Chong et al.

    Nucleotide sequence and phylogenetic analysis of A segment of a highly virulent strain of infectious bursal disease virus

    Acta Virol.

    (2001)
  • C.A. Heid et al.

    Real time quantitative PCR

    Genome Res.

    (1996)
  • R. Higuchi et al.

    Kinetic PCR analysis: real-time monitoring of DNA amplification reactions

    Biotechniques (NY)

    (1993)
  • M.M. Hoque et al.

    Pathogenicity of Ssp1- positive infectious bursal disease virus and molecular characterization of the VP2 hypervariable region

    Avian Pathol.

    (2001)
  • D.J. Jackwood et al.

    Characteristics and serologic studies of two serotypes of infectious bursal disease virus in turkeys

    Avian Dis.

    (1982)
  • D.J. Jackwood et al.

    Detection of infectious bursal disease viruses in commercially reared chickens using the reverse transcriptase/polymerase chain reaction-restriction endonuclease assay

    Avian Dis.

    (1997)
  • Cited by (25)

    • Full-length genome sequencing of a very virulent infectious bursal disease virus isolated in Tunisia

      2021, Poultry Science
      Citation Excerpt :

      RNA extraction was carried out from 200 μL of organ supernatant, using the QIAamp cador Pathogen Mini Kit QIAGEN extraction kit, automated on the QIAcube system. Infectious bursal disease virus detection was performed in a Swift spectrum 48 Thermal Cycler (ESCO), using KAPA SYBR FAST One-Step qRT-PCR Kit in accordance with the manufacturer's instructions and primers as described by Kong et al. (2009). The melting curve analysis was conducted by raising 0.5°C between 55°C and 95°C.

    • Development of real-time PCR assays for single and simultaneous detection of infectious bursal disease virus and chicken anemia virus

      2019, Molecular and Cellular Probes
      Citation Excerpt :

      Several qPCR assays have been published that provide useful references for IBDV and CAV diagnosis and characterization. Most simplex qPCR assays target variable genomic regions or were initially designed to amplify specific IBDV strains [23–32] or CAV genotypes [33–36]. Relatively few studies have been validated for diagnosis and quantification of IBDV [23,27,37] and CAV [33,34,38].

    • Differential activation of intraepithelial lymphocyte-natural killer cells in chickens infected with very virulent and vaccine strains of infectious bursal disease virus

      2018, Developmental and Comparative Immunology
      Citation Excerpt :

      SYBR green-based real-time PCR assay was employed to quantify the viral load of IBDV in CD3-/28.4+IEL-NK cells at various time points following in vitro and in vivo infection. The assay utilises the primers that target the VP4 gene of IBDV as previously described by Kong et al. (2009). The PCR reaction was performed in a CFX96™ Real Time System (BioRad, USA) as follows: 95 °C for 5 min followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 58.7 °C for 30 s, extension at 95 °C for 5 s and melt curve analysis was carried out at 70 °C to 95 °C with increments of 0.5 °C every 5 s per step.

    • Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus

      2014, Journal of Virological Methods
      Citation Excerpt :

      This aim was easily achieved with TaqMan type specific probes. Also SYBR Green chemistry, as previously described for several pathogens (Chai et al., 2013; Franzo et al., 2014; Kong et al., 2009), allowed genotyping based on Tm. However, genetic variability leading to nucleotidic substitution within amplicon could make this analysis difficult or invalidate its effectiveness.

    • Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus

      2012, Journal of Virological Methods
      Citation Excerpt :

      The VP2 hypervariable region also lacks single and conserved nucleotides that could be consistently used to discriminate vvIBDV and non-vvIBDV strains with fluorescent probes. VP4 sequences have also been used for designing real-time assays (Kong et al., 2009; Peters et al., 2005), but this region is moderately conserved, and there are few available sequences in GenBank to guarantee that the assay can be used in genetically diverse strains. The real-time RT-PCR assay bases its discrimination capacity on the identification of a SNP (A229G) included in the VP5/VP2 overlapping region.

    View all citing articles on Scopus
    View full text