Development of Real Time RT-PCR Assays for Detection of Type A Influenza Virus and for Subtyping of Avian H5 and H7 Hemagglutinin Subtypes

Abstract

Rapid detection and subtyping of H5 and H7 subtypes influenza A viruses are important for disease control in poultry and potential transmission to humans. Currently, virus isolation and subsequent HA and NA subtyping constitute the standard for avian influenza viruses detection and subtype identification. These methods are highly accurate and sensitive but are also laborious and time-consuming. Reverse transcription PCR and real time reverse transcription PCR assays, suitable tests for rapid detection, have previously been used for the specific diagnosis of H5 and H7 viruses, however, at present, no primer and probe sets are available for the identification of all H5 and H7 strains. Herein, we have developed specific and sensitive real time reverse transcription PCR assays for the detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes and we have also compared these molecular assays with viral isolation in terms of sensitivity. Our results demonstrate that the real time reverse transcription PCR assays are more sensitive, specific, less expensive compared to viral isolation. In conclusion, molecular assays could represent an useful tool for rapid detection and screening of H5 and H7 isolates during influenza A virus outbreaks alternatively to viral isolation.

Evaluating viral interference between Influenza virus and Newcastle disease virus using real-time reverse transcription–polymerase chain reaction in chicken eggs

“…The R2 index for AIV and NDV genes was 0.998 and 0.997, respectively. As the slope of the standard curves of AIV and NDV were 3.046 and 3.220 respectively, the efficiency of the reaction defined by 10(−1/slope) were 2.13 and 2.04, which were within the acceptable range of 1.7 to 2.2 [19]. According to the standard curve, the linear equation for the real-time RT-PCR for AIV and NDV was y = −3.046x + 37.3352 and y = −3.22x + 35.9949, respectively.…”

A diagnostic one-step real-time reverse transcription polymerase chain reaction method for accurate detection of influenza virus type A

“…Viral isolation by conventional culture methods currently serves as the gold standard for the detection and typing of influenza viruses, but important limitations including the laborious and time-consuming process, as well as low sensitivity for the detection of some subtypes of the virus, still exist [26, 27]. Besides, the risk of emergence and human pandemic potential of new influenza A viruses has prompted the attempts to develop more new rapid, sensitive and specific detection methods based on genetic recombination technology and molecular techniques for the most effective management and prevention of the respective infections [28].…”

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].…”