Reverse Transcription Loop-Mediated Isothermal Amplification for the Rapid Detection of Infectious Bronchitis Virus

Abstract

Infectious bronchitis virus (IBV) is a major cause of disease in domestic fowl and causes an acute, highly contagious disease of the respiratory tracts and sometimes urogential tracts (King and Cavanagh, 1991). Current diagnostic assays for IBV include virus isolation in embryonating eggs, tracheal organ culture, or cell culture immunoassays, and molecular assays that detect the viral RNA (Gelb and Jackwood, 1998). Virus isolation is generally considered the gold standard, however, it is expensive and time consuming because several passages may be required to detect the virus. Immunoassays use IBV-specific monoclonal antibodies to detect the virus in direct or indirect fluorescent antibody and enzyme-linked immunosorbent assay formats. Although more rapid and simpler than virus isolation, immunoassays tend to lack specificity and sensitivity to some extent and can not detect all strains of IBV (Karaca and Naqi, 1993; Karaca et al., 1992; Naqi et al., 1993). Molecular assays for the detection of IBV are used commonly because they provide highly specific and sensitive results and detect viral RNA directly from clinical samples or from virus isolated in a laboratory host system. Although RTPCR and real-time RT-PCR are the highly sensitive and specific methods (Cavanagh et al., 1992; Jackwood et al., 1997; Keeler et al., 1998; Kingham et al., 2000; Kwon et al., 1993; Zwaagstra et al., 1992; Liu et al., 2003; Callison et al., 2006), the dependence on special equipment limits their extensive use. A novel nucleic acid amplification method, loop-mediated isothermal amplification (LAMP), employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a singlestranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA