APPLICATION OF BIOLOGICAL AND MOLECULAR TECHNIQUES TO THE DIAGNOSIS AND CONTROL OF AVIAN INFLUENZA AND OTHER EMERGING POULTRY PATHOGENS
Location: Exotic and Emerging Avian Viral Diseases Research Unit
Title: Rapid detection of Eurasian and American H7 subtype influenza A viruses using a single TaqManMGB real-time RT-PCR
| Van Borm, Steven - VET RES INST, BELGIUM |
| Boschmans, Marc - VET RES INST, BELGIUM |
| Ozhelvaci, Orkun - VET RES INST, BELGIUM |
| Van Den Berg, Thierry - VET RES INST, BELGIUM |
Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 11, 2009
Publication Date: March 1, 2010
Citation: Van Borm, S., Suarez, D.L., Boschmans, M., Ozhelvaci, O., Van Den Berg, T.P. 2010. Rapid detection of Eurasian and American H7 subtype influenza A viruses using a single TaqManMGB real-time RT-PCR. Avian Diseases. 54:632-638.
Interpretive Summary: Avian influenza virus is a potentially severe and highly contagious disease in poultry. The virus is not normally found in poultry in the U.S. and the severe form of the disease, highly pathogenic avian influenza, is considered a foreign animal disease. We have many policies in place to prevent the introduction of these viruses into the U.S., but the US Department of Agriculture in association with state diagnostic laboratories have also implemented a network of laboratories that can rapidly diagnose outbreaks of disease. With a rapid diagnosis, quarantines and other control measures can be implemented quickly to control the disease. The primary test used for diagnosis is the real-time RT-PCR test that amplifies the viral genetic material to easily detectable levels. The test is done in two steps. First a screening test is used to identify any type A influenza viruses, and if this test is positive then a subtype test to either H5 or H7 influenza is used. The H5 and H7 subtypes are the most important in poultry. Unfortunately the current H7 test used in the U.S. does not identify all H7 viruses. This paper describes an alternative H7 test that appears to detect all H7 influenza viruses with a sensitivity at least as good as the screening test. With further validation this test could potentially replace the current H7 test.
A real time reverse transcription PCR (RRT-PCR) was developed for the detection of all H7 subtype avian influenza viruses. A TaqmanMGB probe targeting a highly conserved HA2 H7 region was developed and either combined with primers targeting Eurasian isolates (primer mix EA), North American isolates (NA), or an equimolar mixture of both (EA+NA). The wide phylogenetic scope (EN+NA) and analytical sensitivity and specificity were validated using a panel of 55 diverse influenza A viruses. The detection limit was determined using serial dilutions of Eurasian isolates A/Ck/BE/06600/2003 and A/Ck/It/1067/v99 and North American isolates A/CK/PA/143586/2001 and A/Quail/PA/20304/1998, to be slightly higher than the detection limit of the generic influenza A matrix RRT-PCR (0.01 to 0.1 EID50/rxn). Using EA+NA or NA, about 1 to 10 EID50/reaction could be detected depending on the virus isolate tested, whereas EA detected as little as 0.1 to 1 EID50/reaction. The diagnostic sensitivity and specificity of this assay (EA primer mix) was determined using 102 clinical swabs from an A/Ck/It/1067/v99 infection experiment. In comparison to Matrix RRT-PCR and virus isolation in embryonating chicken eggs, H7MGB detected more weakly positive swabs. H7MGB diagnostic sensitivity compared to VI was 72.2 % (compared to 33 % for matrix RRT-PCR); and diagnostic specificity was 57.4 % (90.74% for matrix). Overall, this new test is a valuable tool for the detection and identification of any H7 subtype influenza A.