SYBR Green-based duplex real time PCR method for detection of Infectious bronchitis virus, Mass serotype
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Date
2020-02
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Abstract
Infectious bronchitis (IB) is a highly contagious economically important viral disease of poultry, caused
by Infectious bronchitis virus (IBV) belonging to the family Coronaviridae. The Massachusetts (Mass)
serotype of IBV is being used worldwide for regular vaccination against IB since several years. The continuous
application of the Mass strain of IBV for vaccination and mutating nature of virus has yielded a selection
pressure, resulting in the emergence of field strains of non-Mass serotypes. Currently, rapid detection of IBV
infection in the poultry flocks amidst other upper-respiratory avian pathogens is a major challenge, which
demands sensitive and rapid diagnostic methods. The SYBR Green – based RT PCR assay has been proven
to be one of the most effective and sensitive tools for such a diagnosis. A duplex SYBR Green – basedreal time RT PCR assay has been designed for the simultaneous detection of IBV and identification of the
Mass serotype in a single reaction based on melt curve analysis. IBV-specific UTR primers and IBV-Mass-
S1-specific primers used in the duplex real time RT-PCR yielded curves of amplification with two specific
melting curves (Tm1 = 81oC ± 0.5oC and Tm2 = 84oC ± 0.7oC) for IBV – Mass serotype. Owing to the fact
that IBV serotypes other than Mass do not exist in India, the assay was standardised using a known Mass
serotype (H120 vaccine strain) and a well-characterised field isolate IBV –B17. The assay did not yield
amplification curves when viruses other than IBV were assessed. This duplex realtime PCR can thus be used
as a rapid molecular diagnostic tool for identification of IB outbreaks. Further evaluation of the assay for the
simultaneous detection and differentiation of IBV Mass and non-Mass serotypes would throw more light for
the effective control of IB, especially in vaccinated flocks.
Description
TNV_20thMVC_PP_Feb-2020_OP51
Keywords
Veterinary Science