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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Virus and Prion Research » Research » Publications at this Location » Publication #79938
Title: DETECTION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV) IN ALVEOLAR MACROPHAGES OF INFECTED PIGS BY SEMI-NESTED SET RT-PCR

Author
item Umthun, Angela
item Mengeling, William

Submitted to: International Virtual Conference on Infectious Diseases of Animals
Publication Type: Proceedings
Publication Acceptance Date: 4/20/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: A semi-nested set PCR was developed to increase the sensitivity of a current RT-PCR method used in RFLP for the differentiation of porcine reproductive and respiratory syndrome virus (PRRSV) strains (Wesley et al., Proc. AASP, 1996). An additional primer was designed outside of one of the original primers which amplify ORF 5. The sensitivity of the semi-nested set PCR was determined using infected MARC-145 cells serially diluted 1:10 into uninfected cells prior to RNA purification. Six infected cells out of 120,000 total cells were sufficient for detecting PRRSV by this method. This is equivalent to 280 infectious units. This is 1000-fold more sensitive than the non-nested PCR method. The semi-nested set RT-PCR method was able to detect PRRSV infected alveolar macrophages isolated from both field animals and experimentally infected pigs. Several of the samples detected by semi-nested PCR were PRRSV negative by the non-nested RT-PCR method. With few exceptions the presence of PRRSV was confirmed by virus isolation from lavage fluid.