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

Angela R. Umthun From the Virology Swine Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, PO Box 70, Ames. IA 50010.

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William L. Mengeling From the Virology Swine Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, PO Box 70, Ames. IA 50010.

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 DVM, PhD

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Abstract

Objective

To increase the timeliness and sensitivity of a procedure that uses 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 with a nonnested-set RT-PCR for sensitivity in amplifying known quantities of infective PRRSV. Once reaction conditions were optimized, the nested-set RT-PCR was tested for effectiveness with 24 strains of PRRSV isolated from swine.

Results

The nested-set RT-PCR was 100- to 1,000- fold more sensitive than the nonnested-set RT-PCR, detecting as little as 1 infective unit of PRRSV/ml of sample. It also was generally as sensitive as the combination of steps, namely virus isolation or propagation and nonnested-set RT-PCR, currently used routinely for amplifying PRRSV prior to RFLP analysis, and it was effective for amplifying all of the 24 strains of PRRSV tested. Using this RT-PCR, all tests were completed within 1.5 days (including RFLP analysis), compared with the > 7 days often required for the currently used method involving virus isolation and propagation.

Conclusions

The nested-set RT-PCR was generally as sensitive as the combination of methods now used for PRRSV amplification prior to RFLP analysis, and it can markedly reduce the time required for testing.

Clinical Relevance

Presumptive identification of PRRSV strains can be provided in a more timely manner by use of a nested-set RT-PCR. (Am J Vet Res 1999;60:802–806)

Abstract

Objective

To increase the timeliness and sensitivity of a procedure that uses 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 with a nonnested-set RT-PCR for sensitivity in amplifying known quantities of infective PRRSV. Once reaction conditions were optimized, the nested-set RT-PCR was tested for effectiveness with 24 strains of PRRSV isolated from swine.

Results

The nested-set RT-PCR was 100- to 1,000- fold more sensitive than the nonnested-set RT-PCR, detecting as little as 1 infective unit of PRRSV/ml of sample. It also was generally as sensitive as the combination of steps, namely virus isolation or propagation and nonnested-set RT-PCR, currently used routinely for amplifying PRRSV prior to RFLP analysis, and it was effective for amplifying all of the 24 strains of PRRSV tested. Using this RT-PCR, all tests were completed within 1.5 days (including RFLP analysis), compared with the > 7 days often required for the currently used method involving virus isolation and propagation.

Conclusions

The nested-set RT-PCR was generally as sensitive as the combination of methods now used for PRRSV amplification prior to RFLP analysis, and it can markedly reduce the time required for testing.

Clinical Relevance

Presumptive identification of PRRSV strains can be provided in a more timely manner by use of a nested-set RT-PCR. (Am J Vet Res 1999;60:802–806)

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