Molecular methods (digital <scp>PCR</scp> and real‐time <scp>PCR</scp>) for the quantification of low copy <scp>DNA</scp> of Phytophthora nicotianae in environmental samples

Blaya, Josefa; Lloret, Eva; Santísima-Trinidad, Ana B; Ros, Margarita; Pascual, José Antonio

doi:10.1002/ps.4048

Cited by 31 publications

(27 citation statements)

References 37 publications

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“…One possible cause for the higher number of discrepancies in the clinical specimens for causative agents of STIs may be due to target pathogens with a low concentration, as reported by Van et al in an evaluation of BD MAX assay. 37 Digital PCR has been successfully applied in the field of pathogen detection 46,47 and is particularly suited to the low-level detection of nucleic acid even with a highly complex background. 47,48 In the arbitration stage, ultrasensitive digital PCR was performed to resolve discordant results.…”

Section: Discussionmentioning

confidence: 99%

“…37 Digital PCR has been successfully applied in the field of pathogen detection 46,47 and is particularly suited to the low-level detection of nucleic acid even with a highly complex background. 47,48 In the arbitration stage, ultrasensitive digital PCR was performed to resolve discordant results. Thirty-one (77.5%) specimens were confirmed as positive after arbitration testing.…”

Section: Discussionmentioning

confidence: 99%

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Simultaneous detection of eleven sexually transmitted agents using multiplexed PCR coupled with MALDI-TOF analysis

Xiu¹,

Zhang²,

Li³

et al. 2019

IDR

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PurposeSexually transmitted infections (STIs), representing a major global health problem, are caused by different microbes, including bacteria, viruses, and protozoa. Unfortunately, infections of different sexually transmitted pathogens often present similar clinical symptoms, so it is almost impossible to distinguish them clinically. Therefore, the aim of the current study was to develop a sensitive, multitarget, and high-throughput method that can detect various agents responsible for STIs.MethodsWe developed and tested a 23-plex PCR coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) assay (sexually transmitted infection-mass spectrometry, STI-MS) that simultaneously targets 11 different agents, including 8 most common clinical pathogens related to STIs (HSV-1, HSV-2, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, and Haemophilus ducreyi) and 3 controversial microorganisms as pathogens (Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum).ResultsThe results showed that the STI-MS approach can accurately detect the expected agents, without cross-reaction with other organisms. The limit of detection of each STI-MS assay was ranged from 1.739 to 10.009 copies/reaction, using probit analyses. The verification rate for each target organism of the STI-MS ranged from a minimum of 89.3% to a maximum of 100%, using conventional assays and ultrasensitive digital PCR to confirm the STI-MS-positive results. To further evaluate the clinical performance of this assay, 241 clinical specimens (124 urethral/cervical swabs and 117 urine) were tested in parallel using the STI-MS assay and monoplex real-time PCR for each agent. The overall validation parameters of STI-MS were extremely high including sensitivity (from 85.7% to 100%), specificity (from 92.3% to 100%), PPV (from 50% to 100%), and NPV (from 99.1% to 100%) for each target.ConclusionSTI-MS is a useful high-throughput screening tool for detecting mixed infections of STIs and has great potential for application in large-scale epidemiological programs for specific microorganisms of STI.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Simultaneous detection of eleven sexually transmitted agents using multiplexed PCR coupled with MALDI-TOF analysis

Xiu¹,

Zhang²,

Li³

et al. 2019

IDR

View full text Add to dashboard Cite

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“…Applications of dPCR for environmental samples are arising due to a high sensitivity and accurate quantification of target genes in environmental samples [43] and when there are few copies of the DNA target [36, 42, 44–46]. Some authors have reported that dPCR performed better than qPCR for DNA recovered from soils because dPCR seems to be more tolerant of PCR inhibitors [30, 38, 47]. …”

Section: Discussionmentioning

confidence: 99%

Quantitative PCR and Digital PCR for Detection ofAscaris lumbricoidesEggs in Reclaimed Water

Soto

Santísima-Trinidad

Bornay-Llinares

et al. 2017

BioMed Research International

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The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control. Ascaris lumbricoides is one of the soil-transmitted helminths (STH) with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO) recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR) and digital PCR (dPCR) were able to detect low amounts of A. lumbricoides eggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as one A. lumbricoides egg equivalent, while dPCR can detect DNA concentrations as low as five A. lumbricoides egg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20 A. lumbricoides eggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks.

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“…At the end of the 20th century, Brunetto et al [7] proposed the concept of digital ddPCR, which distributes sample DNA evenly into a large number of reaction units and then independently performs PCR amplification on each reaction unit. The ddPCR can obtain a DNA copy a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 number without reference to standard curve or control gene [8][9][10]. ddPCR offers good sensitivity, high precision, and absolute quantification.…”

Section: Introductionmentioning

confidence: 99%

Identification and quantification of cassava starch adulteration in different food starches by droplet digital PCR

et al. 2020

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We report a rapid and accurate quantitative detection method using droplet digital PCR (ddPCR) technology to identify cassava adulteration in starch products. The ddPCR analysis showed that the weight of cassava (M) and cassava-extracted DNA content had a significant linear relationship-the correlation coefficient was R 2 = 0.995, and the maximum coefficient of variation of replicates was 7.48%. The DNA content and DNA copy number (C) measured by ddPCR also had a linear relationship with R 2 = 0.992; the maximum coefficient of variation of replicates was 8.85%. The range of cassava ddPCR DNA content was 25 ng/μL, and the formula M = (C + 32.409)/350.579 was obtained by converting DNA content into the median signal. The accuracy and application potential of the method were verified using the constructed adulteration model. (2020) Identification and quantification of cassava starch adulteration in different food starches by droplet digital PCR. PLoS ONE 15(2): e0228624. https://doi.org/10.

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Molecular methods (digital PCR and real‐time PCR) for the quantification of low copy DNA of Phytophthora nicotianae in environmental samples

Abstract: These results suggest that dPCR is a promising alternative for quantifying soil-borne pathogens in environmental samples, even in early stages of the disease.

Cited by 31 publications

References 37 publications

<p>Simultaneous detection of eleven sexually transmitted agents using multiplexed PCR coupled with MALDI-TOF analysis</p>

<p>Simultaneous detection of eleven sexually transmitted agents using multiplexed PCR coupled with MALDI-TOF analysis</p>

Quantitative PCR and Digital PCR for Detection ofAscaris lumbricoidesEggs in Reclaimed Water

Identification and quantification of cassava starch adulteration in different food starches by droplet digital PCR

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