Polymerase Chain Reaction Based-Assays for Rapid Detection and Subtyping Of Type a Influenza Viruses

Highly pathogenic avian influenza (HPAI) virus causes high morbidity, mortality and still is a big threat in poultry industry. The recently raised awareness of the threat of a new influenza pandemic has stimulated interests in the development of a rapid detection method for influenza A viruses. In this study, four diagnostic methods for the detection of type A influenza viruses were explored. A conventional one-tube nucleoprotein reverse transcriptase polymerase chain reaction (NP RT-PCR) was developed for rapid detection of avian influenza A viruses. This method successfully detected 14 different haemagglutinin (HA) subtypes of different origins. A multiplex RT-PCR that successfully amplified three RNA templates of H5, H7 and H9 in one tube was also developed. The designed primers were specific in amplification of the HA genes of H5, H7 and H9 of type A influenza viruses. No amplification was observed with other avian infectious viruses such as Newcastle disease virus, infectious bronchitis virus and infectious bursa1 disease virus. An enzyme-linked immunosorbent assay (ELISA) detection method was then developed to detect the amplified PCR products. This method was 10 times more sensitive than the detection of PCR product using agarose gel electrophoresis. This method (RT-PCR-ELISA) was as sensitive as virus isolation in specific-pathogen-free (SPF) embryonated eggs. The detection limit of the RT-PCR-ELISA was compared with agarose gel electrophoresis and one-step SYBR Green I real time PCR. The RTPCR- ELISA was 10 times less sensitive than SYBR Green I real time PCR. The whole process for the detection of type A influenza virus and the avian H5, H7 and H9 subtypes, from extraction of RNA to analysis of PCR product by agarose gel electrophoresis or colorimetric assay can be completed within 6 h. It provides a rapid means of identification of the type and subtypes of influenza viruses and would be very useful for their surveillance. The advantage of using an ELSA reader is in removing any element of subjective interpretation as a source of error. The methods developed in this study, were tested on suspected cases. The finding indicated that the methods are rapid, sensitive and specific, and thus would be a method of choice for the surveillance of avian influenza virus. Moreover, the RT-PCR-ELISA method allows handling of a large number of samples and can be used in many diagnostic laboratories. Among the four methods developed, the SYBR Green I real time PCR was the best method in terms of sensitivity and specificity. This is followed by RT-PCR-ELSA, multiplex and conventional RT-PCR assays

Detection of H5, H7 and H9 subtypes of avian influenza viruses by multiplex reverse transcription-polymerase chain reaction.

Subtype-specific multiplex reverse transcription-polymerase chain reaction (RT-PCR) was developed to simultaneously detect three subtypes (H5, H7 and H9) of avian influenza virus (AIV) type A. The sensitivity of the multiplex RT-PCR was evaluated and compared to that of RT-PCR–enzyme-linked immunosorbent assay (ELISA) and conventional RT-PCR. While the sensitivity of the multiplex RT-PCR is as sensitive as the conventional RT-PCR, it is 10 times less sensitive than RT-PCR–ELISA. The multiplex RT-PCR is also as sensitive as the virus isolation method in detecting H9N2 from tracheal samples collected at day 3 and 5 post inoculation. Hence, the developed multiplex RT-PCR assay is a rapid, sensitive and specific assay for detecting of AIV subtypes

Bovine Sex Determining Region Y: Cloning, Optimized Expression, and Purification

<p>Sex determining region Y gene (SRY) is located on Y chromosome and encodes a protein with 229 amino acids. In this study, ORF region of SRY with a length of 690 bp was synthesized using PCR and ligated to pET28a (+), then transformed in E.coli DH5α. E.coli BL21 (DE3) strain was chosen to express recombinant bovine SRY protein. A set of optimization steps was taken including different concentrations of IPTG, glucose, and temperatures at differed incubation times after the induction. Results showed that temperature points and different concentrations of IPTG and glucose had a significant effect (p < 0.01) on total protein and recombinant bovine SRY. After purification, various temperatures and concentrations of IPTG showed meaningful effects (p < 0.01) on the solubility of expressed recombinant SRY. Highest soluble rSRY protein amount was achieved where 0.5 mM IPTG and 0.5% glucose was used at 20°C during induction. In the absence of glucose, the highest amount of soluble recombinant SRY levels were achieved at the concentrations of 0.8 mM of IPTG at 28°C, 20°C, and 1.5 mM IPTG at 37°C during induction for 16, 24, and 8 hours, respectively. Regarding the results obtained in this study, it could be stated that by decreasing temperature and inducer concentration, soluble bovine SRY protein expression increases.</p