ABSTRACT
In order to visually detect H1, N1 and N2 subtype of avian influenza virus (AIV), three reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed. According to the sequences of AIV gene available in GenBank, three degenerate primer sets specific to HA gene of H1 subtype AIV, NA gene of N1 and N2 subtype AIV were designed, and the reaction conditions were optimized. The results showed that all the assays had no cross-reaction with other subtype AIV and other avian respiratory pathogens, and the detection limit was higher than that of conventional RT-PCR. These assays were performed in water bath within 50 minutes. Without opening tube, the amplification result could be directly determined by inspecting the color change of reaction system as long as these assays were fin-ished. Fourteen specimens of H1N1 subtype and eight specimens of H1N2 subtype of AIV were identified from the 120 clinical samples by RT-LAMP assays developed, which was consistent with that of virus isolation. These results suggested that the three newly developed RT-LAMEP assays were simple, specific and sensitive and had potential for visual detection of H1, N1 and N2 subtype of AIV in field.
Subject(s)
Animals , Chickens , DNA Primers , Genetics , Ducks , Influenza A Virus, H1N1 Subtype , Classification , Genetics , Influenza A Virus, H1N2 Subtype , Classification , Genetics , Influenza A virus , Classification , Genetics , Influenza in Birds , Diagnosis , Virology , Nucleic Acid Amplification Techniques , Methods , Poultry Diseases , Diagnosis , Virology , Reverse Transcription , TurkeysABSTRACT
A GeXP based multiplex PCR assay was developed to simultaneously detect six different chicken respiratory viruses including H5, H7, H9 subtypes of avian influenza virus(AIV), new castle disease virus (NDV), infectious bronchitis virus(IBV) and infectious laryngotracheitis virus(ILTV). According to the conserved sequences of genes of each pathogen, seven pairs of specific primers were designed, and the reaction conditions were optimized. The specificity and accuracy of GeXP were examined using samples of single and mixed infections of virus. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of cloned plasmids. To further evaluate the reliability, thirty-four clinical samples were detected by GeXP. The corresponding specific fragments of genes were amplified. The detection limit of GeXP was 10(2) copies/microL when all of 7 pre-mixed plasmids containing target genes of six chicken respiratory viruses were present. In the detection of thirty-four clinical samples, the results of GeXP were accorded with the viral isolation completely. In conclusion, this GeXP assay is a rapid, specific, sensitive and high-throughput method for the detection of chicken respiratory virus infections. It can be applied in rapid differential diagnosis for clinical samples, and also provide an effective tool to prevent and control chicken respiratory diseases with similar clinical symptoms.
Subject(s)
Animals , Chickens , Influenza A virus , Classification , Genetics , Physiology , Influenza in Birds , Diagnosis , Virology , Multiplex Polymerase Chain Reaction , Methods , Poultry Diseases , Diagnosis , Virology , Respiratory Tract Infections , Diagnosis , VirologyABSTRACT
·AIM: To study the effect of an intravitreal injection of angiostatin on vascular leakage in the retina and iris of oxygen-induced retinopathy of prematurity (ROP).·METHODS: Brown Norway rats at postnatal day 7 (P7) were exposed to hyperoxia (750mL/L O2 )for 5 days (P7-12) and then returned to normoxia to induce retinopathy. Angiostatin was reconstituted in sterile Phosphate Buffered Saline(PBS) and diluted to desired different concentrations. Angiostatin solution was injected into the vitreous of the right eye of the ROP rats at P14 and the age-matched normal rats through pars plana using a glass capillary, and the left eye received the same volume of sterile PBS as the control. Vascular permeability was quantified at 1, 2 and 3 days after the injection by measuring albumin leakage from blood vessels into the retina and iris using the Evans blue method and normalized by total protein concentrations. The expression of vascular endothelial growth factor (VEGF) in retina was evaluated using the Western Blot analysis and immuno-histochemistry 24 hours following the injection.·RESULTS: ROP rats showed significant increases of vascular permeability in the retina and iris (P<0.01). Angio-statin reduces vascular permeability in a dose-dependent manner in the retina of ROP rats. The reduction showed a time course trend. [Angiostatin injection reduced retinal vascular permeability by approximately 1.5 and 2-fold at P15 (P<0.05) and P16 (P<0.01), respectively.] Angiostatin injection significantly reduced VEGF levels in the retina of ROP rats but did not affect retinal VEGF levels in normal rats.·CONCLUSION: Angiostatin significantly decreases pa-thological vascular permeability in the retina and iris of ROP rats but not in normal rats. Angiostatin down-regulates VEGF expression in retina of ROP rats. These results suggest that angiostatin may have a therapeutic potential in the treatment of ROP and other diseases with vascular leakage.