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United States Department of Agriculture

Agricultural Research Service

Title: Detection of Newcastle Disease Virus Rna by Reverse Transcription Polymerase Chain Reaction Using Formalin-Fixed, Paraffin-Embedded Tissue and Comparison with Immunohistochemistry and in Situ Hybridization

item King, Daniel
item Seal, Bruce

Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: September 7, 2005
Publication Date: November 5, 2005
Citation: Wakamatsu, N., King, D.J., Seal, B.S., Deal, T.M., Brown, C.C. 2005. Detection of newcastle disease virus rna by reverse transcription polymerase chain reaction using formalin-fixed, paraffin-embedded tissue and comparison with immunohistochemistry and in situ hybridization [abstract]. American Association of Veterinary Laboratory Diagnosticians. 48:166.

Technical Abstract: Newcastle disease (ND) is one of the most important avian diseases. Virulent ND has a potential for very serious and rapid spread, and may cause serious economic impact and international trade restrictions for the poultry industry. The clinical signs and gross and microscopic lesions of Newcastle disease virus (NDV) infectious are varied and are dependent on many factors related to the host, virus strain, and environmental stress. There are no pathognomonic lesions associated with any form of the disease. Thus, the use of immunohistochemistry (IHC) and in situ hybridization (ISH) can play an important role in the diagnosis of NDV. In this study, the usefulness of reverse transcription polymerase chain reaction (RT-PCR) from formalin-fixed, paraffin-embedded (FFPE) tissues was examined and compared to the previously utilized IHC and ISH assays. Spleen and lung tissues were collected from 4-week-old chickens experimentally infected with either of two NDV isolates, a low virulence virus (LaSota) and a virulent virus (2002-2003 California). The tissues were harvested immediately postmortem and fixed in 10% neutral buffered formalin for approximately 52 hours prior to processing to paraffin. Just prior to euthanasia, oral and cloacal swabs were collected for viral isolation. Each formalin-fixed tissue embedded in paraffin blocks was trimmed with a razor to obtain 35 mg of tissues. RNA was obtained from the tissues by digestion with proteinase K and subsequent extraction with phenol, chloroform, and isoamyl alcohol. For each RT-PCR, 1 µg nucleic acid was used. By reverse transcription of the RNA and semi-nested PCR with primers for the NDV matrix gene, a 232-bp product was generated and visualized by electrophoresis. The results of PCR were compared to those of IHC for viral nucleoprotein and ISH for matrix gene (850-bp) on 3-µm sections. In addition, results of virus isolation from oral and cloacal swabs were also compared to RT-PCR data. All samples from infected chickens were positive by RT-PCR, including samples that were negative by both IHC and ISH. The RT-PCR positives included tissues from chickens no longer shedding virus detectable by virus isolation from oral or cloacal swabs. The RT-PCR was found to be an effective and sensitive method to detect NDV in FFPE tissues. The development of the NDV RT-PCR assay of FFPE extends the methods that can be conducted without biosecurity concerns beyond the previously used IHC and ISH assays for testing of archived materials.

Last Modified: 4/22/2015
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