scholarly journals Comparison of droplet digital PCR and quantitative PCR for the detection of Salmonella and its application for river sediments

2017 ◽  
Vol 15 (4) ◽  
pp. 505-508 ◽  
Author(s):  
Gulshan Singh ◽  
Ayanda Sithebe ◽  
Abimbola M. Enitan ◽  
Sheena Kumari ◽  
Faizal Bux ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Informal Settlement ◽  
Quantitative Polymerase Chain Reaction ◽  
River Sediments ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Analytical Sensitivity ◽  
Aquatic Sediments ◽  
Pcr Inhibitors ◽  
Copy Numbers

Despite advances in microbial detection that quantitative polymerase chain reaction (qPCR) has led to, complex environmental samples, such as sediments, remain a challenge due to presence of PCR inhibitors. Aquatic sediments accumulate particle-bound microbial contaminants and thereby reflect a cumulative microbial load over time. The relatively new droplet digital PCR (ddPCR) has emerged as a direct quantitative method, highly tolerant to PCR inhibitors and relinquishing the necessity for calibration/standard curves. Information is virtually absent where ddPCR has been applied to detect pathogenic organisms in aquatic sediments. This study compared the efficacy of ddPCR with qPCR, for quantification of Salmonella in sediments from the Palmiet River near an informal settlement in Durban, South Africa. ddPCR significantly improved both analytical sensitivity and detection of low concentrations of Salmonella as compared to qPCR. The expected copy numbers measured from both qPCR and ddPCR showed good R2 values (0.999 and 0.994, respectively). The site mostly affected by the informal settlements exhibited Salmonella in the range of 255 ± 37 and 818 ± 30 Salmonella/g (p ≤ 0.0001) in qPCR and ddPCR, respectively. The improved detection of Salmonella in sediments with ddPCR makes it a promising technical method for the quantification of Salmonella in multifarious environmental samples.

Abstract P613: Absolute Gene Quantification Of Intrarenal Renin-angiotensin System Components Using Droplet Digital PCR Analysis

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Ryousuke Satou ◽  
Akemi Katsurada ◽  
Kayoko Miyata ◽  
Andrei Derbenev ◽  
Andrea Zsombok
Keyword(s):  
Copy Number ◽  
System Analysis ◽  
Renin Angiotensin System ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Angiotensin System ◽  
Renin Angiotensin ◽  
Copy Numbers ◽  
Intrarenal Ras

The intrarenal renin-angiotensin system (RAS) has been shown to play crucial roles in the development of hypertension and RAS associated kidney injury including diabetic nephropathy. Although some circulating RAS components are filtered into kidneys and contribute to the regulation of intrarenal RAS activity, evaluating expression levels of RAS components in the kidney is important to elucidate the mechanisms underlying intrarenal RAS activation. Digital PCR is a new technique that has been established to quantify absolute target gene levels, which allows for comparisons of different gene levels. Thus, this study was performed to establish profiles of absolute gene copy numbers for intrarenal RAS components in wild-type (WT) rats, WT and streptozotocin (STZ)-induced diabetic mice. Male Sprague-Dawley rats (N=5) and male C57BL/6J mice were used in this study. The mice were subjected to either control (N=5) or STZ (200 mg/kg, N=4) injection. Seven days after STZ injection, copy numbers of renal cortical angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1 receptor a (AT1a), and AT2 mRNA were determined by a droplet digital PCR. Since (pro)renin proteins produced by juxtaglomerular cells are secreted to circulating system, analysis of renin mRNA was excluded from this evaluation. In the renal cortex of WT rats, the copy number of AGT was higher than other measured RAS components (AGT: 719.2±46.6, ACE: 116.0±14.9, ACE2: 183.6±21.5, AT1a: 196.0±25.2 copies in 1 ng total RNA). AT2 levels were lower than other components (0.068±0.01 copies). In WT mice, ACE exhibited the highest copy number in the components (AGT: 447.2±29.0, ACE: 1662.4±61.2, ACE2: 676.8±41.5, AT1a: 867.0±16.8, AT2: 0.049±0.01 copies). Although STZ-induced diabetes did not change ACE2 and AT1a, ACE levels were reduced (765.5±98.1 copies) and AT2 levels were augmented (0.10±0.01 copies) as previously demonstrated. Accordingly, the absolute quantification by digital PCR established precise gene profiles of intrarenal RAS components, which will provide rationales for targeting the each component in future studies. Furthermore, the results indicate that the high sensitive assay accurately quantifies rare target genes including intrarenal AT2.

scholarly journals Sensitive detection of porcine circovirus 3 by droplet digital PCR

2019 ◽  
Vol 31 (4) ◽  
pp. 604-607
Author(s):  
Yuqi Liu ◽  
Hecheng Meng ◽  
Lei Shi ◽  
Lili Li
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Porcine Circovirus ◽  
Analytical Sensitivity ◽  
Swine Industry ◽  
Potential Threat ◽  
Emerging Virus ◽  
Clinical Detection ◽  
Porcine Circovirus 3 ◽  
High Degree

Porcine circovirus 3 (PCV-3) is a newly emerging virus that poses a potential threat to the swine industry. We developed a sensitive assay utilizing droplet digital PCR (ddPCR) to detect PCV-3. Specificity of the assay was confirmed by the failure of amplification of DNA of other relevant viruses. The detection limit for ddPCR was 1 copy/μL, 10 times greater sensitivity than TaqMan real-time PCR (rtPCR). Both methods showed a high degree of linearity, although TaqMan rtPCR showed less sensitivity than ddPCR for clinical detection. Our findings indicate that ddPCR might offer faster and improved analytical sensitivity for PCV-3 detection.

scholarly journals Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples

Water ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3507
Author(s):  
Mark A. Ibekwe ◽  
Shelton E. Murinda ◽  
Stanley Park ◽  
Amarachukwu Obayiuwana ◽  
Marcia A. Murry ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Environmental Samples ◽  
Point Of Care ◽  
Foodborne Pathogen ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Recombinase Polymerase Amplification ◽  
High Rate ◽  
E Coli

E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was <10 CFU/mL, while ddPCR showed quantification from 1 to 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.

scholarly journals Acoustic array biochip combined with allele-specific PCR for multiple cancer mutation analysis in tissue and liquid biopsy

2021 ◽  
Author(s):  
Nikoletta Naoumi ◽  
Kleita Michaelidou ◽  
George Papadakis ◽  
Agapi E. Simaiaki ◽  
Roman Fernandez ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Liquid Biopsy ◽  
Point Mutations ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Braf V600e ◽  
Analytical Sensitivity ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Regular screening of cancerous point mutations is of importance to cancer management and treatment selection. Although excellent techniques like next-generation sequencing and droplet digital PCR are available, these are still lacking in speed, simplicity and cost-effectiveness. Here a new approach is presented where allele-specific PCR (AS-PCR) is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) array biosensor for the amplification and detection, respectively, of cancer point mutations. For the proof-of-concept, the method was applied to the screening of the BRAF V600E and KRAS G12D mutations in spiked-in and clinical samples. Regarding the BRAF target, an analytical sensitivity of 0.01%, i.e., detection of 1 mutant copy of genomic DNA in an excess of 104 wild type molecules, was demonstrated; moreover, quantitative results during KRAS detection were obtained when an optimized assay was employed with a sensitivity of 0.05%. The assays were validated using tissue and plasma samples obtained from melanoma, colorectal and lung cancer patients. Results are in full agreement with Sanger sequencing and droplet digital PCR, demonstrating efficient detection of BRAF and KRAS mutations in samples having an allele frequency below 1%. The high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness and compatibility with routine work-flow, hold promise for the implementation of this AS-PCR/acoustic methodology in clinical oncology as a tool for tissue and liquid biopsy.

scholarly journals TKI Suspension in CML, the New Challenge for Hematologist. Would Ddpcr Help Us to Improve Outcomes?

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5512-5512
Author(s):  
Marcio M Andrade ◽  
Vanesa Andreu ◽  
Javier Gervas ◽  
M Angeles Montañes ◽  
Gloria Soro ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Isolation ◽  
Final Analysis ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Molecular Response ◽  
Select Patient ◽  
Major Factors

Abstract Background: For molecular assessment in CML, NCCN and European LeukemiaNet had defined the cut-off values. Until now the gold standard for BCR-ABL quantification is the real time quantitative polymerase chain reaction (RQ-PCR), and this technique has a limited sensibility to MR4.0 or MR4.5 in the majority of laboratories worldwide. There are several discontinuation studies for CP-CML patients under imatinib therapy who achieved and sustained at least MR4.0 during 2 years or more, and the fact of achieve a deeper response and the time during this response had been the major factors for good outcomes in these studies, now situated in around 40% of success. The droplet digital PCR (ddPCR), and other kind of digital PCR as the nanofluidic ddPCR, had showed more sensitivity for detect minimal amounts of BCR-ABL transcripts in patients with undetectable BCR-ABL transcripts by RQ-PCR, leading a window for investigation in this field. Aims: to evaluate the usefulness of the ddPCR in detect the presence of transcripts of BCR-ABL in patients with CP-CML under TKI therapy compared with RQ-PCR. Material & methods: A total of 54 samples from 32 patients with CP-CML under TKI therapy were analyzed. Sampling was performing obtaining one sample in EDTA for RQ-PCR and other in a TempusTM tube for ddPCR. The RNA was obtained from peripheral blood using TempusTM Spin RNA isolation kit according manufacturer recommendations. The analysis by ddPCR for each sample was done using 5 μg of the obtained RNA and processed in Qx100 Droplet Digital PCR, Biorad; using primers and probes described by Gabert et al., 2003 using ABL1 for control; the results was analyzed using QuantaLife Biorad software and expressed in absolute copies of BCR-ABL and in ratio BCR-ABL/ABL1. Only experiments with >32,000 copies of ABL1 were considered as valid for ddPCR to consider at least MR4.5. The use of a dual-determination in the same experiment with the ddPCR; one for BCR-ABL and other for ABL1 permit us to relativize the experiment and create results expressed in ratios easy to compare with the results obtained during the analysis by RQ-PCR for the same samples. Period of Study: August-2013 to Jun 2014. Results: The final analysis shows that 26 of the 54 analyzed samples presents values of BCR-ABL/ABL1 ratio within MR4.0 or more in the RQ-PCR test, of them 15 with MR4.5 values and 11 with MR4.0. However only 13 (50%) shows ratio values according to MR4.5/4.0 in the ddPCR analysis. The other 13 samples were compatible to MR3.0 or inferior. The samples that for RQ-PCR obtained values of MR4.5 or more (15), in only 6 (40%) a MR3.0 or inferior value of ratio were obtained by ddPCR, however in samples with values of MR4.0 by RQ-PCR (11), the proportion of samples with MR3.0 or inferior was higher, 63.6% (7 samples). See Table 1. The rest of the samples (28) the molecular results were within MR3.0 or inferior response values by RQ-PCR and for ddPCR this was confirmed, except in 3 samples in with ddPCR show a MR4.0 or superior response value. This led us to hypothesize that the use of ddPCR for confirms the response of the patients with MR4.0 or superior would improve the follow-up of the patients, and the success ratio for discontinuation. Comments: The ddPCR offers a new way to assess CP-CML patients, in especially in patients with >MR4.0 molecular response, and would offer a more accurately tool to select patient for discontinuation trials. Actually around 60% of patients who discontinues therapy lost MR4.0 or MR4.5 at 6 months, in our cohort, 50% of samples classified as MR4.0 for RQ-PCR had MR3.0 or less by ddPCR and would not be suitable for discontinuation trials; maybe if we use ddPCR as the standard for select patient to discontinuation less patients will lost the response. Table 1. Molecular response correlation between RQ-PCR and ddPCR ddPCR(RM<4.0) ddPCR(RM>4.0) Total RQ-PCR (RM4.0) 7 (63.6%) 4 (36.4%) 11 RQ-PCR (RM4.5) 6 (40.0%) 9 (60.0%) 15 Total 13 13 26 Disclosures No relevant conflicts of interest to declare.

scholarly journals Direct Comparison of SARS-CoV-2 Analytical Limits of Detection across Seven Molecular Assays

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Becky Fung ◽  
Allan Gopez ◽  
Venice Servellita ◽  
Shaun Arevalo ◽  
Coral Ho ◽  
...  
Keyword(s):  
High Throughput ◽  
Extraction Method ◽  
Point Of Care ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Analytical Sensitivity ◽  
Limits Of Detection ◽  
Molecular Assays ◽  
Performance Metric ◽  
Roche Cobas

ABSTRACT Analytical sensitivity for SARS-CoV-2 detection is a key performance metric for the evaluation of viral detection assays. We determined analytical limits of detection for seven SARS-CoV-2 assays using serial dilutions of pooled patient material quantified with droplet digital PCR. Limits of detection ranged from ≤10 to 74 copies/ml for commercial high-throughput laboratory analyzers (Roche Cobas, Abbott m2000, and Hologic Panther Fusion) and 167 to 511 copies/ml for sample-to-answer (DiaSorin Simplexa, GenMark ePlex) and point-of-care instruments (Abbott ID NOW). The CDC assay yielded limits of detection ranging from 85 to 499 copies/ml, depending on the extraction method and thermocycler used. These results can help to inform the assay choice for testing approaches to manage the current COVID-19 outbreak.

Development of droplet digital PCR assays to quantify genes involved in nitrification and denitrification, comparison with quantitative real-time PCR and validation of assays in vineyard soil

2020 ◽  
pp. 1-14
Author(s):  
Tanja M. Voegel ◽  
Melissa M. Larrabee ◽  
Louise M. Nelson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Soil Bacteria ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Quantitative Real Time Pcr ◽  
Soil Dna ◽  
Soil C ◽  
Total Bacteria

Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZI, nosZII) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZII increased with time and the soil C/N ratio and NH4+-N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification.

scholarly journals Analysis of the competitiveness between a non-aflatoxigenic and an aflatoxigenic Aspergillus flavus strain on maize kernels by droplet digital PCR

2021 ◽  
Author(s):  
Alexandra Schamann ◽  
Markus Schmidt-Heydt ◽  
Rolf Geisen
Keyword(s):  
Aspergillus Flavus ◽  
Environmental Protection Agency ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Aflatoxin Biosynthesis ◽  
Copy Numbers ◽  
The Us ◽  
Maize Kernels ◽  
Biocontrol Strain ◽  
Genomic Equivalent

AbstractNon-aflatoxigenic Aspergillus flavus strains are used as a biocontrol system on maize fields to decrease the aflatoxin biosynthesis of aflatoxigenic A. flavus strains. A. flavus strain AF36 was the first commercially available biocontrol strain and is authorized for use on maize fields by the US Environmental Protection Agency, e.g., in Texas and Arizona. A droplet digital PCR (ddPCR) assay was developed to analyze the mechanisms of competition and interaction of aflatoxigenic and non-aflatoxigenic A. flavus strains. This assay enables the parallel identification and quantification of the biocontrol strain A. flavus AF36 and the aflatoxigenic A. flavus strain MRI19. To test the assay, spores of both strains were mixed in varying ratios and were incubated on maize-based agar or maize kernels for up to 20 days. Genomic equivalent ratios (genome copy numbers) of both strains were determined by ddPCR at certain times after incubation and were compared to the spore ratios used for inoculation. The aflatoxin biosynthesis was also measured. In general, A. flavus MRI19 had higher competitiveness in the tested habitats compared to the non-aflatoxigenic strain, as indicated by higher final genomic equivalent ratios of this strain compared to the spore ratios used for inoculation. Nevertheless, A. flavus AF36 effectively controlled aflatoxin biosynthesis of A. flavus MRI19, as a clear aflatoxin inhibition was already seen by the inoculation of 10% spores of the biocontrol strain mixed with 90% spores of the aflatoxigenic strain compared to samples inoculated with only spores of the aflatoxigenic A. flavus MRI19.

scholarly journals Multiplex Droplet Digital PCR Quantification of Recurrent Somatic Mutations in Diffuse Large B-Cell and Follicular Lymphoma

2016 ◽  
Vol 62 (9) ◽  
pp. 1238-1247 ◽  
Author(s):  
Miguel Alcaide ◽  
Stephen Yu ◽  
Kevin Bushell ◽  
Daniel Fornika ◽  
Julie S Nielsen ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Somatic Mutations ◽  
B Cell Lymphoma ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Analytical Sensitivity ◽  
Wild Type ◽  
Single Nucleotide ◽  
Large B Cell

Abstract BACKGROUND A plethora of options to detect mutations in tumor-derived DNA currently exist but each suffers limitations in analytical sensitivity, cost, or scalability. Droplet digital PCR (ddPCR) is an appealing technology for detecting the presence of specific mutations based on a priori knowledge and can be applied to tumor biopsies, including formalin-fixed paraffin embedded (FFPE) tissues. More recently, ddPCR has gained popularity in its utility in quantifying circulating tumor DNA. METHODS We have developed a suite of novel ddPCR assays for detecting recurrent mutations that are prevalent in common B-cell non-Hodgkin lymphomas (NHLs), including diffuse large B-cell lymphoma, follicular lymphoma, and lymphoplasmacytic lymphoma. These assays allowed the differentiation and counting of mutant and wild-type molecules using one single hydrolysis probe. We also implemented multiplexing that allowed the simultaneous detection of distinct mutations and an “inverted” ddPCR assay design, based on employing probes matching wild-type alleles, capable of detecting the presence of multiple single nucleotide polymorphisms. RESULTS The assays successfully detected and quantified somatic mutations commonly affecting enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) (Y641) and signal transducer and activator of transcription 6 (STAT6) (D419) hotspots in fresh tumor, FFPE, and liquid biopsies. The “inverted” ddPCR approach effectively reported any single nucleotide variant affecting either of these 2 hotspots as well. Finally, we could effectively multiplex hydrolysis probes targeting 2 additional lymphoma-related hotspots: myeloid differentiation primary response 88 (MYD88; L265P) and cyclin D3 (CCND3; I290R). CONCLUSIONS Our suite of ddPCR assays provides sufficient analytical sensitivity and specificity for either the invasive or noninvasive detection of multiple recurrent somatic mutations in B-cell NHLs.

scholarly journals Quantification of Cadophora luteo-olivacea From Grapevine Nursery Stock and Vineyard Soil Using Droplet Digital PCR

Plant Disease ◽  
2020 ◽  
Vol 104 (8) ◽  
pp. 2269-2274 ◽  
Author(s):  
María Mercedes Maldonado-González ◽  
María del Pilar Martínez-Diz ◽  
Marcos Andrés-Sodupe ◽  
Rebeca Bujanda ◽  
Emilia Díaz-Losada ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Digital Pcr ◽  
Fungal Species ◽  
Droplet Digital Pcr ◽  
Taqman Probe ◽  
Planting Material ◽  
Low Concentrations ◽  
Nursery Stock ◽  
Species Specific ◽  
Detection And Quantification

Cadophora luteo-olivacea is the most prevalent Cadophora species associated with Petri disease and esca of grapevine. Accurate, early, and specific detection and quantification of C. luteo-olivacea are essential to alert growers and nurseries to the presence of the pathogens in soil and to prevent the spread of this pathogen through grapevine planting material. The aim of this study was to develop molecular tools to detect and quantify C. luteo-olivacea inoculum from environmental samples. Species specific primers based on the β-tubulin gene and a TaqMan probe for droplet digital PCR (ddPCR) and quantitative PCR (qPCR) were first developed to detect and quantify purified DNA of the target fungus. Specificity tests showed that the primers were able to amplify the C. luteo-olivacea DNA (20 isolates) while none of the 29 nontarget fungal species (58 isolates) tested were amplified. The ddPCR was shown to be more sensitive compared with qPCR in the detection and quantification of C. luteo-olivacea at very low concentrations and was further selected to accurately detect and quantify the fungus from environmental samples. Twenty-five of the 94 grafting plants (26.6%) analyzed by ddPCR tested positive to C. luteo-olivacea DNA (>3 copies/µl). C. luteo-olivacea was barely detected from vineyard soils. The procedure employed in this study revealed the presence of the pathogen in symptomless vines, which makes implementation of this technique suitable for certification schemes of C. luteo-olivacea-free grapevine planting material.

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