Abstract
Reverse transcription-polymerase chain reaction (RT-PCR) has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV). One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP) gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs). The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses.
Highlights
Newcastle disease is a highly contagious disease of birds affecting many domestic and wild avian species
The results of this study demonstrated that the described red blood cells (RBCs) adsorption Reverse transcription-polymerase chain reaction (RT-polymerase chain reaction (PCR)) assay has the potential to be used for the rapid and sensitive detection of Newcastle disease virus (NDV) isolates in a variety of samples, and could be used to detect other viruses with the property of hemagglutination
After NDVs adsorption, we optimized the de-adsorption conditions by suspending the collected cells in saline with different concentrations of EDTA and b-mercaptoethnol, the results showed 5 mM EDTA and 10 mM b-mercaptoethnol could effectively release NDVs from red blood cell at 37°C, while high concentrations of EDTA and b-mercaptoethnol will lead to hemolysis and low virus titer in the supernatant (Table 1)
Summary
Newcastle disease is a highly contagious disease of birds affecting many domestic and wild avian species. Hemagglutionation (HA) and ELISA methods have been applied to detect NDVs in chicken embryos and tissues [2]. The egg passage test is more sensitive than HA and ELISA [3], it will take several days to obtain results, a rapid and sensitive method will be helpful to detect virus in varied samples. RT- PCR has been developed to detect and pathotype Newcastle disease virus (NDV) in clinical samples [4,5,6]. RT-PCR has limited sensitivity in detecting complicated samples, such as feces, tissue samples and contaminated water. An effective and simple virus concentration and purification method will be great helpful to enhance the detection sensitivity [7,8]
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