Closed one-tube reverse transcription nested polymerase chain reaction for the detection of pestiviral RNA with fluorescent probes

McGoldrick, Adrian; Bensaude, Emmanuelle; Ibata, G.; Sharp, Giles; Paton, David

doi:10.1016/s0166-0934(99)00010-5

Cited by 89 publications

(45 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Although many RT-PCR assays have been developed for the detection of pestiviruses, only a few of them specifically detected BDV [9,20]. The main difficulty in designing such assays has been the lack of nucleotide sequence data available for BDV, either in the literature or in international databases.…”

Section: Discussionmentioning

confidence: 99%

“…In a nested RT-PCR employing the 324 × 326 outer and PBD1 × PBD2 inner primers, we observed cross reactivity with other pestiviruses, especially with some CSFV strains (data not shown). This could be eliminated, by incorporating a TaqMan BDV-specific fluorogenic probe, according to the closed one-tube nested RT-PCR method developed by McGoldrick et al [9]. Conflicting results were obtained with the two ovine pestiviruses, 175375 and 59386, and this was shown to be due to contamination of the original BDV stocks with BVDV2.…”

Section: Discussionmentioning

confidence: 99%

“…This method employed the 324 and 326 primers and a second set of panpestivirus reactive inner primers (A11 and A14), as described by McGoldrick et al [8,9]. Alternatively, the inner primers were replaced by primers specific for BDV (PBD1 and PBD2) or BVDV2 (RB1 and RB2, [17]).…”

Section: Closed One-tube Nested Rt-pcrmentioning

confidence: 99%

“…Alternatively, the inner primers were replaced by primers specific for BDV (PBD1 and PBD2) or BVDV2 (RB1 and RB2, [17]). In some RT-PCR experiments the amplification product was hybridised to TaqMan fluorogenic probes specific for BDV or BVDV2 [9].…”

Section: Closed One-tube Nested Rt-pcrmentioning

confidence: 99%

“…The first was a semi-nested RT-PCR assay using primers directed at the E rns gene which was validated on a limited number of BDV strains [20]. Another approach used nested RT-PCR targeted at the conserved 5'noncoding region (5'NCR) of the genome to detect all pestiviruses, followed by BDV discrimination using a specific TaqMan fluorogenic probe [9].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

A RT-PCR assay for the rapid recognition of border disease virus

Vilček

Paton²

2000

Vet. Res.

View full text Add to dashboard Cite

-A reverse transcription -polymerase chain reaction (RT-PCR) method was developed for the specific detection of border disease virus (BDV), using the primers PBD1 and PBD2 flanking a 225 bp DNA fragment, selected from the 5´noncoding region of the pestivirus genome. In tests on 70 pestiviruses it was shown to be BDV-specific. A closed, one-tube nested RT-PCR method employing general pestivirus outer primers (324 and 326), and the same BDV-specific inner primers (PBD1 and PBD2) in conjunction with a BDV-specific fluorogenic TaqMan probe also detected only BDV and was more sensitive. BDV-specific RT-PCR was used in combination with a PCR specific for bovine viral diarrhoea virus type 2 (BVDV2) to ascertain whether virus stocks contained mixtures of BDV and BVDV2. It was shown that the ovine pestivirus strains 175375 and 59386 were originally BDV, but after subculture had become contaminated with BVDV2. This explains a previously reported discrepancy in the genetic typing of 59386. Although the BDV-specific RT-PCR can also detect BDV in clinical samples, the assay is likely to be most useful for the rapid typing of laboratory pestivirus strains. border disease virus / BDV / RT-PCR / typing / pestivirus contaminationRésumé -Identification rapide du border disease virus par RT-PCR. Une méthode d'amplification en chaîne par polymérase après transcription inverse (RT-PCR) a été développée pour la détection spé-cifique du border disease virus (BDV), utilisant les amorces PBD1 et PBD2 flanquant un fragment d'ADN de 225 pb, choisi dans la région 5' non codante du génome des pestivirus. Des tests sur 70 souches de pestivirus ont démontré la spécificité de la méthode. Une méthode de RT-PCR nichée dans un seul tube fermé a également permis de détecter spécifiquement le BDV, tout en améliorant la sensibilité. Cette dernière méthode utilise des amorces externes générales pour les pestivirus (324 et 326), les amorces internes PBD1 et PBD2 ainsi qu'une sonde fluorogénique TaqMan reconnaissant spécifiquement le BDV. Les stocks de virus ont été testés à la fois par RT-PCR spécifique du BDV et RT-PCR spécifique du virus de la diarrhée bovine type 2 (BVDV2), afin de déterminer s'ils contenaient un mélange des deux virus. Il a ainsi été montré que les souches de pestivirus ovins 175375 Vet. Res. 31 (2000)

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%