Development of a reverse-transcription polymerase chain reaction assay with fluorogenic probe

technique for rapid isolation and identification of CSFV, we sought to develop a molecular biologic assay as an accurate, rapid, practical approach to discriminating between vaccine and field strains and thus improve and simplify the diagnosis of CSF. A 5’-Taq nuclease assay has identified alleles that differ by a single base (6). In this assay, probes specific for each virus strain are included in the PCR. The probe that is specific for a vaccine strain and a probe that is specific for the mutation or polymorphism are labeled with a different fluorescent reporter dye at the 5’ end. The 3’ end of each probe carries a dark quencher that suppresses the fluorescence of the reporter dye. When a probe is annealed to a perfectly matched target, the probe is cleaved by the 5’ nuclease activity of Taq polymerase. Cleavage of the reporter dye releases the dye from the constraints of the quencher, which results in increased fluorescence intensity. A mismatch between the probe and the target reduces the efficiency of probe hybridization and promotes displacement of the probe instead of cleavage, which results in decreased fluorescence intensity or an absence of fluorescence. Conventional longer TaqMan probes (16 to 40 base pairs [bp]) cannot reliably distinguish between single mutations. Minor-groovebinder (MGB) probes can be used to analyze mutations that are in
Short Communication
Communication brève
Development of a reverse-transcription polymerase chain reaction assay with fluorogenic probes to discriminate Korean wild-type and vaccine isolates of Classical swine fever virus
(Traduit par Docteur Serge Messier)
Classical swine fever (CSF), a highly contagious and often fatal disease, affects both domestic and wild pigs. The causative agent, Classical swine fever virus (CSFV), is a member of the genus Pestivirus, which belongs to the family Flaviviridae (1). Other important animal pathogens within this genus are Bovine viral diarrhea virus (BVDV) and Border disease virus (BDV), which infects sheep (1). Both BVDV and BDV can naturally infect pigs, and the antibodies generated cross-react with CSFV in serologic asssis difficult (2,3). In Korea, pigs have been vaccinated since the 1970s with the live attenuated strain of CSFV known as K-LOM (4). Although a CSFV eradication program (vaccination, test, and slaughter) is continuous, CSF is still a major viral problem. For this reason, distinguishing between vaccine and wild strains of the virus is important in Korea. The current method for genotyping vaccine-type and wild-type CSFV in Korea includes amplification by means of polymerase chain reaction (PCR), followed by sequencing or digestion with the restriction endonuclease Xho I (5), a process that takes more than 8 h, excluding RNA extraction. Because serologic tests cannot distinguish between antibodies against the vaccine and field strains, and because there is no practical
Ho-Seong Cho, Suk-Jun Park, Nam-Yong Park
Abstract
A 1-step reverse-transcription polymerase chain reaction (RT-PCR) assay using TaqMan minor-groove-binding (MGB) probes was developed to distinguish between vaccine-type and wild-type strains of Classical swine fever virus (CSFV) in Korea. Because attenuated Korean LOM strains have been used in animal vaccination in Korea for some time but CSF remains a serious problem, there was a need for a practical approach to differentiating vaccine and field strains. We examined the fluorescence of 5 vaccine strains, 10 field strains, and 5 mixed samples. Three clusters of the samples could be distinguished: those with only fluorescence of the vaccine-type-specific probe, VIC; those with only fluorescence of the wild-type-specific probe, FAM; and those with both VIC and FAM fluorescence. The RT-PCR assay with fluorogenic probes is sensitive and accurate and is therefore useful for differentiating vaccine and field strains of CSFV in Korea.
Résumé
Une épreuve d’amplification en chaîne par la polymérase avec la transcriptase inverse (RT-PCR) en une étape et utilisant les sondes TaqMan a été développée pour différencier la souche vaccinale et les souches sauvages du virus de la peste porcine classique (CSFV) isolées en Corée. Étant donné que les souches coréennes LOM atténuées sont utilisées en Corée pour la vaccination depuis plusieurs années mais que la CSF demeure un problème sérieux, il y avait une nécessité à développer une approche pratique pour différencier la souche vaccinale et les souches sauvages. La fluorescence de 5 souches vaccinales, 10 isolats cliniques et 5 échantillons mélangés a été examinée. Trois regroupements des échantillons ont été faits : ceux avec de la fluorescence du type de la sonde spécifique au vaccin (VIC); ceux avec de la fluorescence du type de la sonde des isolats sauvages (FAM); et ceux avec une fluorescence double (VIC et FAM). L’épreuve RT-PCR avec des sondes fluorogéniques est sensible et précise et est ainsi utile pour différencier les souches vaccinales et les isolats cliniques de CSFV en Corée.