Author
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Mengeling, William |
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UMTHUN, ANGELA - FORMER USDA-ARS |
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Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Proceedings Publication Acceptance Date: 10/7/1999 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: The finding that isolates (strains) of porcine reproductive and respiratory syndrome virus (PRRSV) often differ appreciably in base sequence led to the development of a relatively simple and inexpensive test for presumptive strain identification. As first described, the test is performed in 3 basic steps: 1) initial virus amplification by isolation and propagation in cell culture; 2) further amplification of a portion of the viral genome, i.e. open reading frame 5 and short contiguous sequences, by a nonnested polymerase chain reaction (PCR); and 3) restriction fragment length polymorphism analysis of the PCR-amplified product. The test has found wide application for both experimental and field investigations. More recently the test was modified to enhance its timeliness by replacing virus isolation/propagation and nonnested-set PCR with a single nested-set PCR reaction. The modification reduced the time required to obtain test results (from often more than 1 week to less than 2 days) without an appreciable loss of sensitivity. Depending on the sample, as little as 1 median cell culture infectious dose of PRRSV/ml was detected by nested-set PCR. Moreover, nested-set PCR was more sensitive for detecting PRRSV in samples that also contained homologous antibodies or that had been kept under conditions that reduced the likelihood of virus isolation. Additional details of the modified test and its routine application for diagnostic purposes, including strain identification, will be discussed and illustrated. |
