Validation of One-step Multiplex RT-PCR for Diagnosis of Respiratory Viruses Coinfections in Chickens

摘要

Avian influenza (AI), Infectious Bronchitis (IB), and Newcastle disease (ND) are frequently occurring respiratory infections of the chicken. These are highly fatal diseases caused by three devastating viruses in chicken farms in Egypt. Clinical diagnosis, serological tests, and virus isolation are the classical methods currently used for the characterization of these viruses. However, these methods have several concerns related to sensitivity and specificity that can best be managed with rapid diagnostic molecular techniques. Originating from five governorates (Ismailia, Sharkia, Gharbia, Dakahleia and Matrooh), a total of 138 nasal swabs and 144 tissue pools were collected from ten broiler chicken flocks with a history of severe respiratory diseases and tested by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The RT-qPCR positive samples were subjected to multiplex RT-PCR for the diagnosis of three major avian infections (AI, IB, and ND). The results showed that multiplex RT-PCR is sensitive for both uniplex and mRT-PCR as results showed that The lower limit of detection (LOD) for NDV was found to be 10-3, 10-4 for IBV, and 10-5 for AIV. This LOD was found to be corresponding to 15, 14, and 19 picograms for the RNA extracted from the reference strains. Also the results showed that multiplex RT-PCR is very suitable as a routine laboratory test for rapid and specific detection of co-infections in field samples, as it showed that single virus infection is prominent than double and triple virus infections with a percentage of 75%, while double virus co-infection is more frequent than triple virus co-infection with a percentage of 15% and 10 % respectively.