Classical swine fever virus: a ring test to evaluate RT-PCR detection methods

• Published in: Veterinary microbiology Vol. 73; no. 2; pp. 159 - 174
• Main Authors: Paton, D.J, McGoldrick, A, Belak, S, Mittelholzer, C, Koenen, F, Vanderhallen, H, Biagetti, M, De Mia, G.-M, Stadejek, T, Hofmann, M.A, Thuer, B
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 13.04.2000; Elsevier Science
• Subjects: Biological and medical sciences; Classical swine fever; Classical swine fever virus; Classical Swine Fever Virus - isolation & purification; DNA, Viral - analysis; Enzyme-Linked Immunosorbent Assay - veterinary; Fundamental and applied biological sciences. Psychology; Laboratories - standards; Microbiology; Molecular diagnosis; pigs; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Polymerase Chain Reaction - veterinary; Ring test; RT-PCR; Sensitivity and Specificity; Swine fever virus; Techniques used in virology; Virology; Virus detection; Ring test; Molecular diagnosis; Virus detection; Classical swine fever; RT-PCR; Pestivirus; Performance evaluation; Reverse transcription; Method; Infection; Virus; Polymerase chain reaction; Viral disease; Swine fever; Diagnosis; Flaviviridae; Veterinary; Detection; Hog cholera virus
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/S0378-1135(00)00142-5

Six laboratories participated in an exercise to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Two sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 34 samples of random primed cDNA. These had been synthesised from viral RNA representative of seven different genetic subtypes of CSFV. The other set comprised 40 clinical samples containing tonsil, spleen, whole blood or serum from a pig that had been experimentally infected with CSFV. Each laboratory tested the samples using one or more PCR/RT-PCR tests that they were accustomed to using. The methods and results of the laboratories were compared with one another. The RT-PCR results obtained from testing the clinical samples were also compared with those obtained by virus isolation and antigen ELISA. The cDNA from three CSFV isolates was detected poorly or not at all by some of the PCR tests. For clinical samples, the order of sensitivity was RT-nested PCR>RT-PCR>virus isolation>ELISA. Both RT-PCR and RT-nested PCR appeared to give some false positive results. Several of the PCR tests appear suitable in terms of specificity and sensitivity. Further trials are necessary to compare results when the same test is performed by different laboratories, and to show that improved control procedures can eliminate problems due to false positive reactions. A limited comparison of extraction and reverse transcription procedures showed similar results in each of three participating laboratories, even though the methods were not standardised.