Neuraminidase Subtyping of Avian Influenza Viruses with PrimerHunter-Designed Primers and Quadruplicate Primer Pools
نویسندگان
چکیده
We have previously developed a software package called PrimerHunter to design primers for PCR-based virus subtyping. In this study, 9 pairs of primers were designed with PrimerHunter and successfully used to differentiate the 9 neuraminidase (NA) genes of avian influenza viruses (AIVs) in multiple PCR-based assays. Furthermore, primer pools were designed and successfully used to decrease the number of reactions needed for NA subtyping from 9 to 4. The quadruplicate primer-pool method is cost-saving, and was shown to be suitable for the NA subtyping of both cultured AIVs and uncultured AIV swab samples. The primers selected for this study showed excellent sensitivity and specificity in NA subtyping by RT-PCR, SYBR green-based Real-time PCR and Real-time RT-PCR methods. AIV RNA of 2 to 200 copies (varied by NA subtypes) could be detected by these reactions. No unspecific amplification was displayed when detecting RNAs of other avian infectious viruses such as Infectious bronchitis virus, Infectious bursal disease virus and Newcastle disease virus. In summary, this study introduced several sensitive and specific PCR-based assays for NA subtyping of AIVs and also validated again the effectiveness of the PrimerHunter tool for the design of subtyping primers.
منابع مشابه
PrimerHunter: a primer design tool for PCR-based virus subtype identification
Rapid and reliable virus subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks and global-scale surveillance of highly pathogenic viral subtypes such as avian influenza H5N1. The polymerase chain reaction (PCR) has become the method of choice for virus subtype identification. However, designing subtype-specific PCR primer pairs i...
متن کاملNeuraminidase gene sequence analysis of avian influenza H9N2 viruses isolated from Iran
Influenza A viruses possesses two virion surface glycoproteins including haemagglutinin (HA) and neuraminidase (NA). The NA plays an important role in viral replication and promotes virus release from infected cells and facilitates virus spread throughout the body. To find out any genomic changes that might be occurred on NA gene of avian influenza circulating viruses, we have genetically analy...
متن کاملMultiplex Reverse Transcriptase-PCR Assay for Typing and Subtyping of Influenza A (H5 & H9) Virus in Iran
Avian influenza virus (AIV) infection is a major cause of bird or human mortality and morbidity, therefore the rapid identification of the virus is of important clinical and epidemiological implication. Methods: A multiplex Reverse Transcriptase PCR (RT-PCR) was optimized for the detection of influenza A virus and the H5 and H9 subtypes. The influenza type A specific primers were directed to t...
متن کاملAmplification of four genes of influenza A viruses using a degenerate primer set in a one step RT-PCR method.
We designed a degenerate primer set that yielded full-length amplification of hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural protein (NSP) genes of influenza A viruses in a single reaction mixture. These four genes were amplified from 15 HA (1-15) and 9 NA (1-9) subtypes of influenza A viruses of avian (n=16) origin. In addition, 272 field isolates of avian origin were t...
متن کاملIsolation of H9N2 Subtype of Avian Influenza Viruses during an Outbreak in Chickens in Iran
Avian influenza is an important disease of poultry with the potential to cause major epidemics resulting in significant economic losses. The presence of avian influenza viruses (AIV) in chickens in Iran has not been previously reported. An avian influenza outbreak in broiler, layer and breeder farms occurred during a very hot summer in July 1998. Three AIV isolates designated as 101, 102 and 10...
متن کامل