Differences in amplification efficiency of standard curves in quantitative real-time PCR assays and consequences for gene quantification in environmental samples

Abstract Background The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. Methods Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. Results All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. Conclusion Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

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Identification and Validation of Suitable Housekeeping Genes for Normalizing Quantitative Real-Time PCR Assays in Injured Peripheral Nerves

PLoS ONE ◽

10.1371/journal.pone.0105601 ◽

2014 ◽

Vol 9 (8) ◽

pp. e105601 ◽

Cited By ~ 17

Author(s):

Giovanna Gambarotta ◽

Giulia Ronchi ◽

Olivier Friard ◽

Pantaleo Galletta ◽

Isabelle Perroteau ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Peripheral Nerves ◽

Housekeeping Genes ◽

Quantitative Real Time Pcr ◽

Pcr Assays

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Quantitative real-time PCR assays to detect DNA degradation in soy-based food products

Journal of the Science of Food and Agriculture ◽

10.1002/jsfa.3563 ◽

2009 ◽

Vol 89 (7) ◽

pp. 1137-1144 ◽

Cited By ~ 19

Author(s):

Sarah R Murray ◽

Ruth C Butler ◽

Gail M Timmerman-Vaughan

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Food Products ◽

Dna Degradation ◽

Quantitative Real Time Pcr ◽

Pcr Assays

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Potential pitfalls in the quantitative molecular detection ofEscherichia coliO157:H7 in environmental matrices

Canadian Journal of Microbiology ◽

10.1139/w05-149 ◽

2006 ◽

Vol 52 (5) ◽

pp. 482-488 ◽

Cited By ~ 25

Author(s):

Rebekka R.E Artz ◽

Lisa M Avery ◽

Davey L Jones ◽

Ken Killham

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Detection Sensitivity ◽

Escherichia Coli O157 ◽

Soil System ◽

E Coli ◽

Animal Wastes ◽

Pcr Assays

The detection sensitivity and potential interference factors of a commonly used assay based on real-time polymerase chain reaction (PCR) for Escherichia coli O157:H7 using eae gene-specific primers were assessed. Animal wastes and soil samples were spiked with known replicate quantities of a nontoxigenic strain of E. coli O157:H7 in a viable or dead state and as unprotected DNA. The detection sensitivity and accuracy of real-time PCR for E. coli O157:H7 in animal wastes and soil is low compared to enrichment culturing. Nonviable cells and unprotected DNA were shown to produce positive results in several of the environmental samples tested, leading to potential overestimates of cell numbers due to prolonged detection of nonviable cells. This demonstrates the necessity for the specific calibration of real-time PCR assays in environmental samples. The accuracy of the eae gene–based detection method was further evaluated over time in a soil system against an activity measurement, using the bioluminescent properties of an E. coli O157:H7 Tn5luxCDABE construct. The detection of significant numbers of viable but nonculturable (VBNC) as well as nonviable and possibly physically protected cells as shown over a period of 90 days further complicates the use of real-time PCR assays for quick diagnostics in environmental samples and infers that enrichment culturing is still required for the final verification of samples found positive by real-time PCR methods.Key words: Escherichia coli O157:H7, real-time PCR, animal waste, soil, VBNC.

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Development of quantitative real-time PCR and digital droplet-PCR assays for rapid and early detection of the spoilage yeasts Saccharomycopsis fibuligera and Wickerhamomyces anomalus in bread

Food Microbiology ◽

10.1016/j.fm.2021.103894 ◽

2022 ◽

Vol 101 ◽

pp. 103894

Author(s):

Paola Cremonesi ◽

Cristiana Garofalo ◽

Claudia Picozzi ◽

Bianca Castiglioni ◽

Nicola Mangieri ◽

...

Keyword(s):

Early Detection ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Saccharomycopsis Fibuligera ◽

Wickerhamomyces Anomalus ◽

Digital Droplet Pcr ◽

Spoilage Yeasts ◽

Droplet Pcr ◽

Pcr Assays

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Erratum to ‘Generation and validation of new quantitative real time PCR assays to detect Elephant Endotheliotropic herpesviruses 1A, 1B, and 4’ [J. Virol. Methods 237 (2016) 138–142]

Journal of Virological Methods ◽

10.1016/j.jviromet.2016.12.005 ◽

2017 ◽

Vol 240 ◽

pp. 85-86

Author(s):

Taylor Pursell ◽

Jie Tan ◽

RongSheng Peng ◽

Paul D. Ling

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Pcr Assays

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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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