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Title: MULTIPLEX REAL-TIME RT-PCR FOR THE DETECTION OF THREE VIRUSES ASSOCIATED WITH POULT ENTERITIS COMPLEX: TURKEY ASTROVIRUS, TURKEY CORONAVIRUS, AND TURKEY REOVIRUS

Author
item Spackman, Erica
item Kapczynski, Darrell
item SELLERS, HOLLY - UNIV OF GA - ATHENS, GA

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/7/2004
Publication Date: 3/2/2005
Citation: Spackman, E., Kapczynski, D.R., Sellers, H., 2005. Multiplex real-time rt-pcr for the detection of three viruses associated with poult enteritis complex: turkey astrovirus, turkey coronavirus, and turkey reovirus. Avian Diseases. 49:86-91.

Interpretive Summary: Poult enteritis complex (PEC) is an economically important disease of young turkeys characterized by diarrhea, poor weight gain and in some cases high mortality. Many agents may contribute to the development of PEC-like disease. Three viruses have been commonly identified in commercial turkeys which have PEC-like disease (turkey coronavirus, turkey astrovirus and avian reoviruses). Currently, there is little standardization of diagnostics tests for these agents and tests that are available are not sensitive or specific, which has caused problems in consistency for detection of these agents in sick turkeys. In order to improve diagnostics for this disease, a new state-of-the-art technology, real-time RT-PCR, has been applied for the detection of each of these agents. Real-time RT-PCR is rapid, highly sensitive and very specific. Furthermore, this test has been designed to reduce costs by a process called multiplexing, where two or more agents can be tested for simultaneously. This study reports the development optimization and initial validation of the real-time RT-PCR technology for the detection of three of the viruses most often associated with PEC-like disease.

Technical Abstract: Poult enteritis complex (PEC) is an economically important disease of young turkeys characterized by diarrhea, poor weight gain and in some cases high mortality. Although PEC is considered to be a poly-microbial disease, numerous viruses, including turkey coronavirus (TCoV), turkey astrovirus type 2 (TAstV-2) and avian reoviruses (ARV), have been associated with PEC-like disease. Real-time RT-PCR (RRT-PCR), a highly sensitive and specific detection method for viral RNA, was developed in a multiplex format for the simultaneous detection of TAstV-2 and TCoV, and for the detection of 2 genetic types of ARV. Assay sensitivity was determined using in vitro transcribed RNA and varied by target between 150 gene copies for TAstV-2 alone and 2200 copies for TCoV when multiplexed. Virus detection was evaluated with samples collected from poults inoculated at one day-of-age with each of the viruses. Cloacal swabs and intestinal samples were obtained at 1, 2, 3, 4, 6, 9, 14, 17 and 21 days post inoculation, processed and tested for virus detection by RRT-PCR. Cloacal swabs were shown to have similar sensitivity for virus detection as intestinal samples when compared directly. ARV detection by RRT-PCR was compared with virus isolation and had similar sensitivity.