Interlaboratory Comparison of Six Real-Time PCR Assays for Detection of Bovine Leukemia Virus Proviral DNA

ABSTRACTQuantitative real-time PCR (qPCR) is increasingly being used for the detection of bovine leukemia virus (BLV) proviral DNA. Nevertheless, quality control for the validation and standardization of such tests is currently lacking. Therefore, the present study was initiated by three Office International des Epizooties (OIE) reference laboratories and three collaborating laboratories to measure the interlaboratory variability of six already developed and available BLV qPCR assays. For that purpose, an international panel of 58 DNA samples reflecting the dynamic range of the majority of the assays was distributed to six testing centers. Based on qualitative results, the overall agreement among all six laboratories was moderate. However, significant variability in the measurement of the BLV proviral DNA copy number was observed among different laboratories. Quantitative PCR assays, even when performed by experienced staff, can yield large variability in BLV proviral DNA copy numbers without harmonization. Further standardization of different factors (i.e., utilization of unified protocols and unique calibrators) should increase interlaboratory agreement.

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Development of real time PCR for the detection of Bovine leukemia virus proviral DNA

Veterinary Medicine Journal ◽

10.30896/0042-4846.2020.23.2.20-27 ◽

2020 ◽

Vol 23 (2) ◽

pp. 20-27

Author(s):

I.E. Gafarova ◽

◽

E.S. Lisitsina ◽

Yu.A. Savochkina ◽

N.V. Kirillova ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Proviral Dna

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Strip-dried blood sampling: applicability for bovine leukemia virus detection with ELISA and real-time PCR

Journal of Virological Methods ◽

10.1016/j.jviromet.2018.11.004 ◽

2019 ◽

Vol 263 ◽

pp. 101-104 ◽

Cited By ~ 2

Author(s):

Nikolay Yu. Saushkin ◽

Jeanne V. Samsonova ◽

Alexander P. Osipov ◽

Sergey E. Kondakov

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Virus Detection ◽

Blood Sampling

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Absolute Quantitation of Feline Leukemia Virus Proviral DNA and Viral RNA Loads by TaqMan® Real-time PCR and RT-PCR

Methods in Molecular Biology - Molecular Beacons: Signalling Nucleic Acid Probes, Methods, and Protocols ◽

10.1007/978-1-60327-040-3_6 ◽

2008 ◽

pp. 73-87 ◽

Cited By ~ 9

Author(s):

Valentino Cattori ◽

Regina Hofmann-Lehmann

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leukemia Virus ◽

Viral Rna ◽

Feline Leukemia Virus ◽

Rt Pcr ◽

Absolute Quantitation ◽

Proviral Dna

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Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

Applied and Environmental Microbiology ◽

10.1128/aem.00779-07 ◽

2007 ◽

Vol 73 (20) ◽

pp. 6557-6565 ◽

Cited By ~ 38

Author(s):

Pascal E. Saikaly ◽

Morton A. Barlaz ◽

Francis L. de los Reyes

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genomic Dna ◽

Dynamic Range ◽

Limit Of Detection ◽

Fate And Transport ◽

Biological Warfare ◽

Quantitative Real Time Pcr ◽

Pcr Assays ◽

Detection And Quantification

ABSTRACT Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R 2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R 2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

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