Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCR with nucleocapsid gene-specific primers

doi:10.1016/j.jviromet.2009.05.006

Journal of Virological Methods

Volume 160, Issues 1–2, September 2009, Pages 149-156

https://doi.org/10.1016/j.jviromet.2009.05.006 Get rights and content

Abstract

SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (C_t) 18.19 ± 3.63 and a melting temperature (T_m) 86.0 ± 0.28 °C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the C_t value 14.70 ± 2.32 and T_m 87.4 ± 0.21 °C. The assay had a dynamic detection range which spans over a 5 log₁₀ concentration range, 10⁹–10⁵ copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45 ± 0.31% and 1.30 ± 0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV.

Introduction

Newcastle disease (ND) is a highly contagious viral disease of domestic poultry, cage, aviary and wild birds. ND is included as an Office Internationale de Epizootics (OIE) List A disease with enforced statutory control measures in the event of outbreaks of the disease (Aldous and Alexander, 2001). ND is caused by Newcastle disease virus (NDV) which is classified as a member of the order Mononegavirales, family Paramyxoviridae and genus Avulavirus (Mayo, 2002a, Mayo, 2002b). Beard and Hanson (1984) have classified NDV into three major pathotypes based on the clinical signs induced in infected chickens: velogenic (highly virulent), mesogenic (intermediate virulent) and lentogenic (avirulent).

The advent of real-time PCR methods has improved further the significant benefits of RT-PCR. In comparison to conventional gel-based PCR assays, real-time PCR offers increased sensitivity and specificity in a rapid format. Owing to these features, real-time PCR is now one of the most important techniques for the detection and monitoring of virus infections (Watzinger et al., 2006). There are different formats available for real-time PCR. The intercalating dyes SYBR Green I assay is the most cost effective and easier to establish as compared to other real-time detection formats because target-specific fluorogenic probes are not required. However, the major disadvantage is that the dye molecules bind with non-specific PCR products or primer–dimers, which require melting curve analysis to control the specificity of amplified fragments at the end of PCR amplification.

Aldous et al. (2001) and Wise et al. (2004) described a real-time PCR assay for NDV detection and pathotype differentiation using several different TaqMan probes. Tan et al. (2004) described the first SYBR Green I real-time PCR for the detection of NDV, however this assay failed to differentiate the various pathotypes of NDV, Pham et al. (2005a) described a rapid detection and differentiation of NDV by SYBR Green I real-time PCR with melting curve analysis. The investigators used a set of primers which were designed based on the fusion (F) gene for amplification. This primer pair amplified the expected 198 bp product regardless of the pathotypes of NDV. Therefore, only melting curve analysis can be used for differentiation of pathotypes. To date, the use of SYBR Green I real-time PCR incorporated with nucleocapsid (NP) gene-specific primers for detection and differentiation of NDV pathotypes has not been reported. The nucleotide sequences alignment of NP genes from different NDV pathotypes revealed that the velogenic and lentogenic NDV strains showed distinct nucleotide variations at specific position of NP gene. Therefore, attempt was made to detect and differentiate the velogenic and lentogenic NDV strains using SYBR Green I real-time PCR based on the NP gene-specific primers

Section snippets

Primer design

Three sets of primer combinations based on NP gene of NDV were used in the real-time PCR amplification (Table 1). All the primers were designed with the aid of the Primer Premier 5.0™ software. A set of primers, NDVF2 and NDVR2 were designed from conserved region of NP gene of velogenic and lentogenic strains of NDV and used in the reverse transcription to synthesize a 750 bp of cDNA. A forward primer, NDVIF2 was designed from the conserved region of NP gene of both velogenic and lentogenic

Detection and differentiation of NDV strains

After establishing the optimum conditions of the real-time PCR, the assay was able to detect and differentiate the velogenic NDV strain AF2240 and lentogenic NDV strain F. Using the velogenic-specific primer (NDVIF2 and NPV2N), the NDV AF2240 was amplified with a threshold cycle (C_t) 16.43 and melting temperature (T_m) at 86.0 °C (Fig. 1A). However, no amplification was observed when this velogenic-specific primer was tested against the lentogenic NDV strain F (Table 2). On the other hand, the

Discussion

In the present study, a diagnostic method for detection and differentiation of NDV by using NP gene-specific primers in SYBR Green I real-time PCR was developed. The design and application of these primers in this assay were based on the theory of amplification efficiency where primer–template complexes containing mismatches have lower efficiency compared to perfectly matched complexes. Therefore, the primer with perfect match to the template shows low threshold cycle, C_t value (early

Acknowledgement

This research was supported by Grant number 01-2-04-03-BTK/ER/006 from Ministry of Science, Technology and Innovation, Government of Malaysia.

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Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCR with nucleocapsid gene-specific primers

Abstract

Introduction

Section snippets

Primer design

Detection and differentiation of NDV strains

Discussion

Acknowledgement

Vet. Microbiol.

Clin. Chim. Acta

J. Virol. Meth.

J. Virol. Meth.

J. Virol. Meth.

J. Virol. Meth.

Virology

J. Virol. Meth.

Biochem. Biophysic. Res. Commun.

Mol. Aspects Med.