Development of a High-Throughput Method for Quantification of Plasmopara viticola DNA in Grapevine Leaves by Means of Quantitative Real-Time Polymerase Chain Reaction

Plasmopara viticola is a strictly biotrophic oomycete that causes downy mildew, which is one of the most important grapevine diseases. Control of the disease is most often achieved by fungicide applications, which may have severe environmental consequences. Therefore, alternative control strategies based on biocontrol agents (BCAs) are currently in development. Thousands of potential BCAs have to be screened for their antagonist efficacy against Plasmopara viticola. Evaluation of their effect on the pathogen can be achieved by detecting the amount of P. viticola DNA in leaves treated with potential antagonists and infected with the pathogen. In this study, a rapid high-throughput method was developed for relative quantification of P. viticola DNA directly from Vitis vinifera leaves by means of multiplex real-time quantitative polymerase chain reaction (PCR) with TaqMan chemistry. This method allows simultaneous amplification, but independent detection, of pathogen and host DNA by using species-specific primers and TaqMan probes that are labeled with different fluorescent dyes. Including detection of V. vinifera DNA in the tests is fundamental because it provides an endogenous reference and allows normalization for variations caused by sample-to-sample differences in DNA extraction, PCR efficiencies, and pipetting volumes. The developed method allows highly sensitive and specific detection of P. viticola DNA (minimal detectable quantity of 0.1 pg). Moreover, high precision and reproducibility of TaqMan assays were observed over a linear range of four orders of magnitude, confirming the reliability of the developed PCR assay. Potential applications range from screening for BCA efficiency to evaluation of fungicide efficacy, or assessment of host resistance.

Download Full-text

Simultaneous One-Tube Quantification of Host and Pathogen DNA with Real-Time Polymerase Chain Reaction

Phytopathology ◽

10.1094/phyto.2002.92.1.112 ◽

2002 ◽

Vol 92 (1) ◽

pp. 112-116 ◽

Cited By ~ 83

Author(s):

L. M. Winton ◽

J. K. Stone ◽

L. S. Watrud ◽

E. M. Hansen

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Fluorescent Dyes ◽

Quantitative Polymerase Chain Reaction ◽

Positive Control ◽

Douglas Fir ◽

Chain Reaction ◽

Taqman Probes ◽

Polymerase Chain ◽

Pathogen Dna

Phaeocryptopus gaeumannii is a widespread foliar parasite of Douglas-fir. Although normally innocuous, the fungus also causes the defoliating disease Swiss needle cast in heavily infected needles. The extent of P. gaeumannii colonization in Douglas-fir foliage was estimated with real-time quantitative polymerase chain reaction (PCR) using TaqMan chemistry. In order to derive a normalized expression of colonization, both pathogen and host DNA were simultaneously amplified but individually detected by species-specific primers and TaqMan probes labeled with different fluorescent dyes. Detection of host DNA additionally provided an endogenous reference that served as both an internal positive control and adjusted for variation introduced by sample-to-sample differences in DNA extraction and PCR efficiencies. The genes employed for designing the TaqMan probes and primers were β-tubulin for the pathogen and a LEAFY/FLORICAULA-like gene involved in floral development for the tree host. Both probe/primer sets exhibited high precision and reproducibility over a linear range of 4 orders of magnitude. This eliminated the need to analyze samples in multiple dilutions when comparing lightly with heavily infected needles. Quantification of the fungus within needles was successful as early as 1 month after initial infection. Real-time PCR is the only method currently available to quantify P. gaeumannii colonization early in the first year of the colonization process.

Download Full-text

High-throughput applicable genomic sex typing of chicken by TaqMan real-time quantitative polymerase chain reaction

Poultry Science ◽

10.3382/ps.2010-00638 ◽

2010 ◽

Vol 89 (7) ◽

pp. 1451-1456 ◽

Cited By ~ 8

Author(s):

N.F. Rosenthal ◽

H. Ellis ◽

K. Shioda ◽

C. Mahoney ◽

K.R. Coser ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

High Throughput ◽

Quantitative Polymerase Chain Reaction ◽

Chain Reaction ◽

Sex Typing ◽

Polymerase Chain

Download Full-text

Fluorescence-based real-time quantitative polymerase chain reaction (qPCR) technologies for high throughput screening of pathogens

High Throughput Screening for Food Safety Assessment ◽

10.1016/b978-0-85709-801-6.00009-5 ◽

2015 ◽

pp. 219-248 ◽

Cited By ~ 2

Author(s):

C. Löfström ◽

M.H. Josefsen ◽

T. Hansen ◽

M.S.R. Søndergaard ◽

J. Hoorfar

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

High Throughput ◽

High Throughput Screening ◽

Quantitative Polymerase Chain Reaction ◽

Chain Reaction ◽

Polymerase Chain

Download Full-text

Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

Virology Journal ◽

10.1186/s12985-020-01467-y ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Yang Zhang ◽

Chunyang Dai ◽

Huiyan Wang ◽

Yong Gao ◽

Tuantuan Li ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Clinical Samples ◽

Chain Reaction ◽

Pcr Assay ◽

Qrt Pcr ◽

Highly Sensitive ◽

One Step ◽

Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

Download Full-text