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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Virus and Prion Research » Research » Publications at this Location » Publication #96740
Title: RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF STRAINS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS BY USE OF A NESTED-SET REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION

Author
item UMTHUN, ANGELA - PIONEER
item Mengeling, William

Submitted to: American Journal of Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/24/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: Porcine reproductive and respiratory syndrome (PRRS) made its world debut in North Carolina in 1987. Since that time it has spread throughout North America and much of the rest of the world to become one of the most economically important diseases faced by swine industries worldwide. A relatively simple diagnostic test, previously developed in our laboratory, has been used extensively to identify, and differentiate among, strains of PRRS virus. Such information is particularly important when investigating the source and spread of virus during epidemics of the disease. In this study the test was improved by markedly reducing the time required for test results (from usually more than 1 week to less than 2 days). As a consequence there is the potential for quicker control of the disease with associated financial benefit to the swine producer and the American consumer of pork and pork products.

Technical Abstract: Objective: To increase the timeliness and sensitivity of a procedure using viral nucleic acid amplification followed by restriction fragment length polymorphism (RFLP) analysis for identifying strains of porcine reproductive and respiratory syndrome virus (PRRSV). Sample population: 24 strains of PRRSV. Procedure: A nested-set reverse transcriptase polymerase chain reaction (RT-PCR) was developed and compared in sensitivity to a nonnested-set RT-PCR for amplifying known quantities of infective PRRSV. Once reaction conditions were optimized, nested-set RT-PCR was tested for effectiveness with 24 strains of PRRSV isolated from swine during an interval ranging from 1989 through 1996. Results: The nested-set RT-PCR developed during the study was: 1) 100- to 1000-fold more sensitive than nonnested-set RT-PCR (detecting as little as 1 infective unit of PRRSV/ml of sample); 2) generally as sensitive as the combination of steps, namely virus isolation/propagation and nonnested-set RT-PCR, now used routinely for amplifying PRRSV prior to RFLP analysis; 3) effective in amplifying all of the 24 strains of PRRSV tested; and 4) completed in 1.5 days (including RFLP analysis) compared to the often more than 1 week interval required for the currently used method involving virus isolation and propagation. Conclusion: Nested-set RT-PCR is generally as sensitive as the combination of methods (ie, virus isolation/propagation plus nonnested-set RT-PCR) now used for PRRSV amplification prior to RFLP analysis and it markedly reduces the time required for test results. Clinical relevance: Presumptive identification of PRRSV strains can be provided in a more timely manner.