Establishiment And Application Of A Quadruple Real-time Fluorescent RT-PCR For Detecting Avian Metapneumovirus

Avian metapneumovirus(aMPV)causes an acute highly contagious upper respiratory tract infection primarily of turkeys and chickens.Based on genetic and antigenic properties,aMPV can be classified into 4 subgroups:A,B,C and D.In susceptible turkeys,infection of the respiratory tract can occur at any age although young poults appear to be more severely affected.The severity of disease and mortality rate are largely influenced by secondary bacterial infection,but other husbandry factors can also excerabate the severity of disease.Infection with aMPV can result in serious egg production problems in turkey and duck breeding flocks aMPV is an emerging poultry pathogen that has a significant impact on poultry production.The aMPV is widely distributed throughout the world,except in Australia.In this study,we have established a quadruple real-time fluorescent RT-PCR for detecting aMPV in order to clarify the impact of aMPV on domestic poultry and improve the detection level,shorten the detection time,simplify the experimental operation procedures,and to meet the requirements for grassroots and entry-exit rapid detection.We also collected 1920 bird double swabs from living birds market of 8 provinces/municipality for nucleic acid assay to analyze the distribution of aMPV in poultry.In this study,based on the conserved region of G gene of aMPV-A,aMPV-B and aMPV-D,M gene of aMPV-C,corresponding four pairs of primers and four probes were designed,and a quadruple real-time fluorescent RT-PCR detection method was established.We made an evaluation for sensitive and specificity which have established method for detecting aMPV.We have collected 1920 throat and cloacal double swabs in living birds market from 8 provinces/municipality.Taking advantage of a quadruple real-time fluorescent RT-PCR detection method to detect the viral RNA which were extracted from samples.In order to ensure the accuracy of the detection method,we find out the published conventional RT-PCR detection method of avian metapneumovirus through literature for verificationThe results show that a quadruple real-time fluorescent RT-PCR method for aMPV detection was established,by the gradient dilution of ten times of the templates,the detection limit of aMPV-D was found to be 102 copies/?L plasmid sample,and others were found to be 103 copies/p.L plasmid sample,showing high sensitivity.And various subgroups of AIV and other common poultry virus strains in China were collected and used for the quadruple real-time fluorescent RT-PCR detection,and results showed that the normal fluorescence curves appeared in the RNA templates of all subgroups of aMPV,while all subgroups of AIV and other common poultry viruses and the negative control samples didn't,showing high specificity.The results show that 39 out of 1920 samples were positive,including 32 samples were aMPV-B which all chickens were host infection.5 samples were aMPV-C,2 samples were aMPV-A and aMPV-C mixed infection which the host infection were Muscovy duck and goose.We take advantage of the published aMPV common RT-PCR detection method for verification was searched in the literature to verify that the correct target bands can be expanded.The RT-PCR products were sequenced,and the sequencing results-were compared with BLAST on NCBI.For the analysis,the results obtained are consistent with the results of the established detection method.The results completely conformed to the test result by common RT-PCR assay.We established the quadruple real-time fluorescent RT-PCR detection method in this study has good specificity and highly sensitivity,and can be used for the rapid detection of aMPV to distinguish four subgroups simultaneously,which greatly improves the detection efficiency.The test results of 1920 double swab samples from 8 provinces/municipalities showed that some chickens,ducks,and geese were poisoned in China,and the distribution of avian metapneumovirus in birds in some areas of China was analyzed as a next step.The detection of avian metapneumoviruses in poultry populations in other regions provides directions and provides a correct theoretical basis for the prevention and control of avian metapneumoviruses in China.