Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA

Abstract

Avian influenza virus infections are a major cause of morbidity and rapid identification of the virus has important clinical, economical and epidemiological implications. We have developed a one-tube Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for the rapid diagnosis of avian influenza A. A panel of reference influenza strains from various hosts including avian species, human, swine and horse were evaluated in a one tube RT-PCR using primers designed for the amplification of a 218 bp fragment of the NP gene. The PCR products were detected by PCR-ELISA by use of an internal catching probe confirming the NP influenza A origin. The PCR-ELISA was about 100 times more sensitive than detection of PCR products by agarose gel electrophoresis. RT-PCR and detection by PCR-ELISA is comparable in sensitivity to virus propagation in eggs. We also designed primers for the detection of the influenza. A subtypes H5 and H7 shown to have pathogenic potential in poultry. The H5 primers cover the cleavage site of the HA gene and specifically amplify influenza A subtype H5. The H7 primers also cover the HA cleavage site and detected all H7 reference strains investigated. In addition, the H7 primers also amplified very weak and/or additional bands on an agarose gel from other subtypes. However, the H7 origin and the pathogenic potential defined by the presence or absence of basic amino acids at the cleavage site can be determined by sequencing of the PCR product. As far as we know this is the first demonstration of RT-PCR detection on a panel of H7 strains using only one primer set.

Evaluation of the ESPLINE® INFLUENZA A&B‐N Kit for the Diagnosis of Avian and Swine Influenza

“…RT-PCR assays have been used to detect influenza viruses in throat and nasal specimens collected from humans, pigs, and horses (3,6,7,(15)(16)(17). They potentially have high sensitivity, but require a high level of skill and complex laboratory infrastructure and take several hours to perform.…”

Characterization of H5N2 influenza viruses isolated in South Korea and their influence on the emergence of a novel H9N2 influenza virus

“…The swabs and faecal samples were examined by virus isolation in 9-11-day-old embryonated chicken eggs (ECE). The presence and subtype of AIV in the ECE were determined by a haemagglutination assay and RT-PCR methods, as described previously (Fereidouni et al, 2009;Fouchier et al, 2000;Lee et al, 2001;Munch et al, 2001). A total of 201 LPAI viruses of various subtypes were identified by the National Veterinary Research and Quarantine Service (NVRQS), but no HPAI viruses had been isolated.…”

Nucleic acid-based detection of influenza A virus subtypes H7 and N9 with a special emphasis on the avian H7N9 virus

“…Continuous progress is achieved regarding the signal detection of the PCR products, e. g. by PCR-ELISAs [18,19] and in particular by RT-qPCR technologies. Due to a seemingly constant increase in the number of outbreaks of highly pathogenic avian influenza (HPAI) caused by infections with subtype H5 or H7 viruses in many countries, RT-PCR and RT-qPCRs were especially designed for the broad detection and differentiation of these HA genes [18,[20][21][22] and their pathotypes on the basis of the HA cleavage site motif [23][24][25]. Often these assays are combined with a differentiation of the NA subtypes N1, N2, or N7 [26].…”