A Complete Molecular Diagnostic Procedure for Applications in Surveillance and Subtyping of Avian Influenza Virus

Abstract

Introduction. The following complete molecular diagnostic procedure we developed, based on real-time quantitative PCR and traditional PCR, is effective for avian influenza surveillance, virus subtyping, and viral genome sequencing. Method. This study provides a specific and sensitive step-by-step procedure for efficient avian influenza identification of 16 hemagglutinin and 9 neuraminidase avian influenza subtypes. Result and Conclusion. This diagnostic procedure may prove exceedingly useful for virological and ecological advancements in global avian influenza research.

Advanced biosensors for detection of pathogens related to livestock and poultry

“…Alternative molecular methods based on PCR and RT-PCR are more sensitive but need extracted genetic material and require the use of equipment which is available only in diagnostic or scientific laboratories. In consequence, on-site detection of avian influenza viruses is rare until now for both early diagnostics and monitoring [ 32 – 34 ]. Similarly, a variety of different serological tests that detect the response of the infected host are established but they are not adapted to strains which have a pandemic potential.…”

Epidemiologic Survey of Avian Influenza Virus Infection in Shorebirds Captured in Hokkaido, Japan

“…A one-step RT-PCR reaction mixture was prepared using a Takara Prime-Script High Fidelity RT-PCR Kit (Takara Bio, Inc., Shiga, Japan). One-step RT-PCR was conducted with a Gene Amp PCR System 9700 (Applied Biosystems, Waltham, Massachusetts, USA) using the following conditions: one cycle of 42 C for 30 min and 94 C for 2 min; 40 cycles of 98 C for 10 s, 30 C for 30 s, and 72 C for 7 min; one cycle of 72 C for 7 min, and a holding step at 4 C. The reaction mixtures were diluted 1:50 in tris-ethylenediaminetetraacetic acid buffer and the diluted PCR reaction mixtures were prepared using KOD-Plus-Ver.2 (Toyobo Life Science, Tokyo, Japan) and used for PCR amplifications of the HA and NA using the reported primer sets (Tseng et al 2014). We conducted PCR amplifications with a Gene Amp PCR System 9700 (Applied Biosystems) using the following conditions: 94 C for 2 min; 35 cycles of 98 C for 10 s, 50 C for 30 s, and 68 C for 2 min, followed by a hold step at 4 C. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen) and direct sequencing was performed using the BigDyet Terminator v3.1 cycle sequencing kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).…”

Avian influenza virus in migratory wild birds and poultry in the United States

“…This method may underrepresent AIV subtype diversity in MWBs due to the low success rate of VI (Runstadler et al, 2007). Some recent studies have suggested that PCR is a better tool for the surveillance of various AIVs circulating in MWBs (Tseng et al, 2014;.…”

“…Primers for each of the 16 HA genes were adopted from previous reports by others (Hoffmann et al, 2016;Tseng et al, 2014; or designed in-house when needed. Primer information is summarized in Table 2.…”

“…Analytical specificity (i.e., lack of cross-reactivity) of the PCR was determined by testing each primer pair with available heterogeneous HA subtypes (Table 1). Since primers were adopted from published literature (Tseng et al, 2014), which established the lack of cross-reactivity with unrelated viruses that could be present in wild bird OPC swabs, no further evaluation was done. Eighty-eight samples were collected during active AIV surveillance of MWBs, while two samples were from passive surveillance (i.e., morbidity/mortality events).…”