Studies on Multiplex RT-PCR for Detection of Avian Influenza Virus Type A Group and Specific H5 and H7 Subtypes
Journal Title: Journal of Veterinary Science & Medicine - Year 2014, Vol 2, Issue 1
Abstract
A multiplex Reverse Transcription Polymerase Chain Reaction (mRT-PCR) for Avian Influenza Virus (AIV) has been developed to simultaneously Detect Nucleoprotein (NP) genes common to all AIV subtypes or type A influenza viruses by AIV NP gene or group primers and hemagglutinin (HA) protein genes specific to H5 and H7 subtypes by H5 and H7 primers, respectively. The AIV group primer bases were selected from conserved regions of AIV NP genes determined by sequence comparison and alignment using DNA STAR primer software (DNA STAR Inc., WI). Primers specific to H5 and H7 subtypes were designed on conserved sequences of HA protein genes of H5 and H7 subtypes, respectively. The mRT-PCR products amplified by AIV group primers gave a 750bp fragment, by H7 primers gave a 518bp fragment and by H5 primers gave a 410bp fragment. Evaluation test of the mRTPCR for AIV indicated that all AIV subtypes H1 through H7 and H9 tested were specifically amplified by AIV group primers, H5 subtypes were amplified by both group primers and H5 primers, and H7 subtypes were amplified by both group and H7 primers. Non-H5 or H7 subtypes were not amplified by the use of H5 or H7 primers. Samples contained both H5 and H7 subtypes were amplified by all 3 primer sets of AIV group, H5 and H7 primers simultaneously in one assay. Other avian viruses had no reaction to any of the AIV primers. The mRT-PCR for AIV detected as low as 2 x 10-3 ng of RNA extracted from H7N2 and H5N2 subtypes or as low as 2.2-2.5 Log10 ELD50 of these two viruses. Sensitivity (Se) and Specificity (Sp) of the mRT-PCR for AIV were measured as 92% and 100%, respectively, in comparison with virus isolation in Embryonated Chicken Eggs (ECE).
Authors and Affiliations
Huaguang Lu
Application of a Nitric Oxide-Releasing Pro-drug for Halting Growth of Human Breastand Canine Mammary Carcinoma Cells
Breast carcinomas remain one of the most common forms of cancer among women. Likewise, mammary cancers are one of the leading causes of death in dogs. Among the principal challenges in tumor therapies, resistance to cyto...
Tick Paralysis in a Snowshoe Hare by Ixodes pacificus Ticks in British Columbia, Canada
We provide the first reported case of tick paralysis in a wildlife animal caused by the western blacklegged tick, Ixodes pacificus Cooley & Kohls. Six I. pacificus females and one male were collected from a feral Snowsho...
An Experimental Reproduction of Necrotic Enteritis in Broiler Chickens
Clostridium perfringens is the main causative agent of avian necrotic enteritis (NE), an enteric infectious disease considered among the most important diseases in the poultry industry. Currently, there are numerous repo...
Gross and HistopathologicalStudy of the Genitalia in Goats:1. Ovaries
Among the 340 samples collected from the pregnant and nonpregnant animals, 126(37.058%) were pregnant, 127(37.35%) were found without gross pathological abnormalities, and the remaining 43(21.64%) samples were found with...
Polypeptide Profiles of Diminazene Aceturate Resistant Trypanosoma evansi Organisms Isolated from a Buffalo
Trypanosomosis was recorded in a buffalo during the month of September 2014 in Proddatur of Andhra Pradesh, India. Disease was confirmed by the presence of Trypanosoma evansi organisms in peripheral blood smears. Buffalo...