Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference

Young, N.J.; Thomas, Carole; Collins, Margaret E.; Brownlie, Joe

doi:10.1016/j.jviromet.2006.08.008

Cited by 39 publications

(43 citation statements)

References 22 publications

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“…The rapid and reliable diagnosis of both persistently and acutely infected cattle is imperative. The use of real-time RT-PCR methods to establish the presence or absence of BVDV RNA in cattle is contributing meaningfully to diagnostic screening and control strategies [33]. …”

Section: Discussionmentioning

confidence: 99%

Genetic characterization of bovine viral diarrhoea (BVD) viruses: confirmation of the presence of BVD genotype 2 in Africa

et al. 2012

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Bovine viral diarrhoea virus (BVDV) has emerged as one of the economically important pathogens in cattle populations with a worldwide distribution causing a complex of disease syndromes. Two genotypes BVDV 1 and 2 exist and are discriminated on the basis of the sequence of the 5′ non-coding region (5 NCR) using real-time PCR. The use of the real-time PCR is more sensitive, specific, and less time consuming than a conventional PCR, and has reduced the risk of cross-contamination of samples. Limited information exists on BVDV genetic subtypes in South Africa. The aim of this study was to determine the genotypes of

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Section: Discussionmentioning

confidence: 99%

Genetic characterization of bovine viral diarrhoea (BVD) viruses: confirmation of the presence of BVD genotype 2 in Africa

et al. 2012

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“…Progress has been made in developing PCR-based assays for detection of BVDV in various clinical 3,8,11,13,16,27,31,32 To validate real-time RT-PCR assay in the authors' laboratory, multiple primers and probes that target the 59-UTR of BVDV were compared, and a previously published combination demonstrated the highest sensitivity and consistency. 13 By following extensive optimization, the real-time RT-PCR assay was able to detect low numbers of viruses in viral stocks or in virus-spiked ear-notch samples.…”

Section: Discussionmentioning

confidence: 99%

Combination of Reverse Transcription Real-Time Polymerase Chain Reaction and Antigen Capture Enzyme-Linked Immunosorbent Assay for the Detection of Animals Persistently Infected with Bovine Viral Diarrhea Virus

et al. 2011

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Abstract. Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle. A successful control program requires early detection and removal of persistently infected (PI) animals. The objective of the current study was to develop, validate, and apply a cost-effective testing scheme for the detection of BVDV PI animals in exposed herds. Pooled samples were screened by using a real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and individual positives were identified with an antigen capture enzyme-linked immunosorbent assay (ACE). The detection limits of the optimized realtime RT-PCR were 10 and 100 RNA copies per reaction for BVDV-1 and BVDV-2, respectively. The semiquantitative results of real-time RT-PCR and ACE or real-time RT-PCR and immunohistochemistry were moderately correlated. The threshold cycle of real-time RT-PCR performed on pooled samples was significantly correlated with the pool size (R 2 5 0.993). The least-cost pool sizes were 50 at a prevalence of 0.25-0.5% and 25 at a prevalence of 0.75-2.0%. By using the combined real-time RT-PCR and ACE procedure, 111 of 27,932 samples (0.4%) tested positive for BVDV. At this prevalence, cost reduction associated with the application of real-time RT-PCR and ACE ranged from 61% to 94%, compared with testing individual samples by ACE, immunohistochemistry, or real-time RT-PCR. Real-time RT-PCR screening also indicated that 92.94% of PI animals were infected with BVDV-1, 3.53% with BVDV-2, and 3.53% with both BVDV-1 and BVDV-2. Analysis of the 59-untranslated region of 22 isolates revealed the predominance of BVDV-1b followed by BVDV-2a.

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“…Several investigators have developed and described qRT-PCR assays using external reference spikes that involve encapsidated RNAs of animal viruses (9,39) or naked RNAs (7,23). The use of intact virions rather than purified naked RNA as an internal control is preferable since these virions can also be used to monitor the efficiency of the viral lysis step itself.…”

Section: Discussionmentioning

confidence: 99%

Detection and Quantification of Infectious Avian Influenza A (H5N1) Virus in Environmental Water by Using Real-Time Reverse Transcription-PCR

Dovas

Papanastassopoulou

Georgiadis

et al. 2010

Appl Environ Microbiol

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Routes of avian influenza virus (AIV) dispersal among aquatic birds involve direct (bird-to-bird) and indirect (waterborne) transmission. The environmental persistence of H5N1 virus in natural water reservoirs can be assessed by isolation of virus in embryonated chicken eggs. Here we describe development and evaluation of a real-time quantitative reverse transcription (RT)-PCR (qRT-PCR) method for detection of H5N1 AIV in environmental water. This method is based on adsorption of virus particles to formalin-fixed erythrocytes, followed by qRT-PCR detection. The numbers of hemagglutinin RNA copies from H5N1 highly pathogenic AIV particles adsorbed to erythrocytes detected correlated highly with the infectious doses of the virus that were determined for three different types of artificially inoculated environmental water over a 17-day incubation period. The advantages of this method include detection and quantification of infectious H5N1 AIVs with a high level of sensitivity, a wide dynamic range, and reproducibility, as well as increased biosecurity. The lowest concentration of H5N1 virus that could be reproducibly detected was 0.91 50% egg infective dose per ml. In addition, a virus with high virion stability (Tobacco mosaic virus) was used as an internal control to accurately monitor the efficiency of RNA purification, cDNA synthesis, and PCR amplification for each individual sample. This detection system could be useful for rapid high-throughput monitoring for the presence of H5N1 AIVs in environmental water and in studies designed to explore the viability and epidemiology of these viruses in different waterfowl ecosystems. The proposed method may also be adapted for detection of other AIVs and for assessment of their prevalence and distribution in environmental reservoirs.

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Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference

Cited by 39 publications

References 22 publications

Genetic characterization of bovine viral diarrhoea (BVD) viruses: confirmation of the presence of BVD genotype 2 in Africa

Genetic characterization of bovine viral diarrhoea (BVD) viruses: confirmation of the presence of BVD genotype 2 in Africa

Combination of Reverse Transcription Real-Time Polymerase Chain Reaction and Antigen Capture Enzyme-Linked Immunosorbent Assay for the Detection of Animals Persistently Infected with Bovine Viral Diarrhea Virus

Detection and Quantification of Infectious Avian Influenza A (H5N1) Virus in Environmental Water by Using Real-Time Reverse Transcription-PCR

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