Impact of DNA extraction efficiency on the sensitivity of PCR-based plant disease diagnosis and pathogen quantification

Yang, Yalong; Zhou, Qixing; Zahr, Kher; Harding, Michael W.; Feindel, David; Feng, Jie

doi:10.1007/s10658-020-02189-1

Cited by 18 publications

(5 citation statements)

References 18 publications

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“…DNA extraction e ciency affecting the accuracy of plant disease diagnosis or pathogen quanti cation has been studied previously (Yang et al 2021), and experimentally con rmed by the current study. In the current study, a Qiacube was used for all DNA extraction which would increase the consistency of DNA yield across different samples compared to a human technician manually employing a DNA extraction kit.…”

Section: Discussionsupporting

confidence: 57%

Using a GFP-labeled Stagonospora nodorum strain as a DNA extraction efficiency standard in plant disease diagnosis

Fu,

Yang,

Zahr

et al. 2024

Crop Protection

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Section: Discussionsupporting

confidence: 57%

Using a GFP-labeled Stagonospora nodorum strain as a DNA extraction efficiency standard in plant disease diagnosis

Fu,

Yang,

Zahr

et al. 2024

Crop Protection

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“…Given the fact that the efficiency of DNA extraction from mycelia was not 100%, the low limit of the fungal cells for a positive reaction would be larger than 28. According to Yang et al (2021), the extraction efficiencies of pathogen DNA from plant tissue was approximately 50%. Thus, we expected that the P-VL and LAMP-VL could detect V. longisporum DNA from as less as 56 cells.…”

Section: Discussionmentioning

confidence: 99%

Development of a qPCR assay and a LAMP assay forVerticillium longisporumdetection and a triplex qPCR assay for simultaneous detection ofV. longisporum,Leptosphaeria biglobosaandL. maculansfrom canola samples

Fu,

Yang,

Jiang

et al. 2024

Preprint

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Verticillium wilt, Verticillium stem striping, and Verticillium stripe, are common disease names that all denote infection caused by Verticillium longisporum, on canola, or other Brassica crops. In this study, a quantitative PCR (qPCR) assay and a loop-mediated isothermal amplification (LAMP) assay were developed for the detection of V. longisporum from canola stem samples. Both assays are specific to V. longisporum at the species level and ubiquitous at the strain level. The low limit for positive detection of the two assays is 1 pg fungal DNA in a 20-uL reaction or 1,400 fungal cells in 100-mg plant tissue. The qPCR assay was combined with the duplex qPCR assay for the two blackleg pathogens, Leptosphaeria biglobosa and L. maculans to constitute a triplex qPCR system for simultaneous detection of all three pathogens. The usefulness of this triplex qPCR system was verified on canola samples collected from various locations in Alberta, Canada. Using this triplex qPCR system, V. longisporum was detected from one sample, while the two blackleg pathogens were detected at higher frequencies. Since it is sometimes difficult to differentiate Verticillium stripe and blackleg on Alberta canola samples based on visual symptoms, the triplex qPCR system is an important tool for the detection of V. longisporum, especially when its presence is masked or obscured by symptoms of blackleg.

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“…The limit might be lower than 1000 cells, but likely more than 100 cells, as indicated by our test on DNA from 100-cell samples. These results emphasized that DNA extraction had significant influence on the sensitivity of a qPCR diagnosis system (Yang et al 2021). Thus we highly recommend that DNA extraction efficiency should be considered when developing and advocating new PCR-based methods.…”

Section: Sarkes Et Al Manuscript Page 16mentioning

confidence: 94%

Detection of Xanthomonas translucens pv. undulosa from wheat by quantitative PCR

Sarkes

Yang

Dijanovic

et al. 2021

Preprint

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A probe-based quantitative PCR (qPCR) protocol was developed for detection and evaluation of the wheat bacterial leaf streak pathogen Xanthomonas translucens pv. undulosa (Xtu). The protocol can also detect X. translucens pv. translucens (Xtt), but cannot differentiate the two pathovars. When tested on DNA from plant, non-target bacteria and culture of microorganisms from wheat seeds, the qPCR showed a high specificity. On purified Xtu DNA, the qPCR was more sensitive than a loop-mediated isothermal amplification (LAMP) assay. When DNA samples from a set of serial dilutions of Xtu cells were tested, the qPCR method could repeatedly generate quantification cycle (Cq) values from the dilutions containing 1,000 cells. Since 2 uL of the total of 50 uL DNA was used in one reaction, one qPCR reaction could detect the presence of the bacteria in samples containing as few as 40 bacterial cells. The qPCR could detect the bacteria from both infected seed and leaf tissues. For seed testing, a protocol for template preparation was standardized, which allowed one qPCR reaction to test DNA from the surface of one seed. Thus, the qPCR system could theoretically detect Xtu and/or Xtt in samples where the bacteria had an average concentration at or larger than 40 cells per seed.

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Impact of DNA extraction efficiency on the sensitivity of PCR-based plant disease diagnosis and pathogen quantification

Cited by 18 publications

References 18 publications

Using a GFP-labeled Stagonospora nodorum strain as a DNA extraction efficiency standard in plant disease diagnosis

Using a GFP-labeled Stagonospora nodorum strain as a DNA extraction efficiency standard in plant disease diagnosis

Development of a qPCR assay and a LAMP assay forVerticillium longisporumdetection and a triplex qPCR assay for simultaneous detection ofV. longisporum,Leptosphaeria biglobosaandL. maculansfrom canola samples

Detection of Xanthomonas translucens pv. undulosa from wheat by quantitative PCR

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