Use of an internal standard in a TaqMan® nested reverse transcription-polymerase chain reaction for the detection of bovine viral diarrhoea virus

Heath, G. S.; King, Donald P.; Turner, Jane; Wakeley, Philip R.; Banks, M.

doi:10.1016/j.vetmic.2003.09.006

Cited by 14 publications

(6 citation statements)

References 15 publications

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“…Milk is a non-invasive sample type collected from farms on a daily basis and has the advantage that both FMDV and FMDV-specific antibodies can be detected ( Burrows et al, 1971 ; Armstrong, 1997 ), and has been utilised for surveillance of a number of other diseases ( Beaudeau et al, 2001 ; Heath et al, 2003 ). Previous experiments have shown that the mammary gland is an organ that is highly susceptible to FMDV replication, and that FMDV can be detected in milk before the appearance of clinical signs ( Burrows et al, 1971 ; Blackwell and McKercher, 1982 ; Reid et al, 2006 ).…”

Section: Introductionmentioning

confidence: 99%

Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance

Armson

Mioulet

Doel

et al. 2018

Veterinary Microbiology

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Section: Introductionmentioning

confidence: 99%

Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance

Armson

Mioulet

Doel

et al. 2018

Veterinary Microbiology

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“…Similar diagnostic approaches have been used to monitor infection, and to identify herds with infected individuals for other cattle viruses [10,12]. Previous studies with FMD have also demonstrated that the virolactia can precede the presentation of clinical signs in experimentally infected animals [3,5,6].…”

Section: Introductionmentioning

confidence: 99%

Utility of automated real-time RT-PCR for the detection of foot-and-mouth disease virus excreted in milk

et al. 2006

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-Foot-and-mouth disease virus (FMDV) can be excreted in milk and thereby spread infection to susceptible animals in other holdings. The feasibility of using real-time reverse transcription polymerase chain reaction (rRT-PCR) as a diagnostic tool for detection of FMDV in milk was assessed by studying the excretion of virus from experimentally-infected cattle. Fore-and machine milk samples were collected over a 4-week period from two dairy cows infected with FMDV and from two in-contact cows held in the same pen. The whole, skim, cream and cellular debris components of the milks were tested by automated rRT-PCR and results compared to virus isolation (VI) in cell culture. The onset of clinical signs of FMD in all four cows correlated with viraemia, and the presence of FMDV in other clinical samples. rRT-PCR results matched closely with VI in detecting FMDV in all milk components and generally coincided with, but did not consistently precede, the onset of clinical signs. rRT-PCR detected FMDV in milk up to 23 days post inoculation which was longer than VI. Furthermore, the detection limit of FMDV in milk was greater by rRT-PCR than VI and, in contrast to VI, rRT-PCR detected virus genome following heat treatment that simulated pasteurisation. rRT-PCR was also able to detect FMDV in preservativetreated milk. In conclusion, this study showed that automated rRT-PCR is quicker and more sensitive than VI and can be used to detect FMDV in whole milk as well as milk fractions from infected animals. milk / milk components / foot-and-mouth disease virus / real-time RT-PCR / pasteurisation

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“…Young et al (2006) detected a product of 156 bp from 5'UTR using qRT-PCR assay with SYBR Green I from the samples of whole blood collected from cattle with acute BVDV infection. A reliable detection and BVDV classification into genotype I and II has repeatedly been observed only in the studies using qRT-PCR method and specific probes (Heath et al, 2003;Letellier and Kerkhofs, 2003;Baxi et al, 2006;Hoffmann et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

“…Quantitative real-time RT-PCR (qRT-PCR) methods, which have also been developed for application in clinical diagnosis, are predominantly based on differently labeled specific probes using hydrolysis (TaqMan). The released fluorescent light signal is used for quantification and genotyping of BVDV templates (Bhudevi and Weinstock, 2001;Mahlum et al, 2002;Heath et al, 2003;Letellier and Kerkhofs, 2003); in contrast SYBR Green I dye binds non-specifically to doublestranded DNA by intercalation or minor groove binding (Lekanne Deprez et al, 2002;Zipper et al, 2004). Specific identification may be achieved by melting curve analysis that can be used for detecting target nucleic acids characterized by sequence variability.…”

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Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry

Kosinova¹,

Pšikal²,

Robešová³

et al. 2007

Vet. Med.

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Quantitative real-time RT-PCR (qRT-PCR) assay was developed for the detection and quantification of bovine viral diarrhea virus (BVDV) in clinical samples from persistently infected cattle. qRT-PCR was optimized to quantify the number of BVD virus copies using Light Cycler<sup>®</sup> detection system and intercalation fluorogenic dye SYBR Green I. A universal set of primers was selected from a highly conserved 5′ untranslated region (5′UTR) to detect BVDV type I and II simultaneously. Quantification of BVDV cDNA was accomplished using a calibration curve generated from 10-fold serial dilutions of standard plasmid DNA in the range 1−10<sup>8</sup> copies/μl. Analysis of 290 bp amplicons enabled monitoring of the viral RNA/BVDV level in a total of five BVDV strains (BVD-NADL, A03/3004, DB03/2943, KA04/3124, KV05/3412) and sixteen bulk milk samples, and in bovine sera of persistent carriers originating from Czech farms, as well as in a batch of calf serum for cell culture. Melting temperatures of amplicons (Tm) of BVDV strains of the same genotype group I as the NADL reference strain showed variability of the thermal points, however significant differences were observed in Tm values between the representatives of genotype group I and II. Low concentrations of BVD virus in bulk milk samples were also qualitatively identified by conventional RT-PCR. Highly reproducible data were obtained as the coefficients of variation of threshold cycles values in intra-assay and inter-assay were less than 0.85% and 2.76%, respectively. The results give enough evidence of suitability of qRT-PCR assay for quantitative analysis of BVDV in clinical samples.

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Use of an internal standard in a TaqMan® nested reverse transcription-polymerase chain reaction for the detection of bovine viral diarrhoea virus

Cited by 14 publications

References 15 publications

Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance

Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance

Utility of automated real-time RT-PCR for the detection of foot-and-mouth disease virus excreted in milk

Real-time PCR for quantitation of bovine viral diarrhea virus RNA using SYBR Green I fluorimetry

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