scholarly journals Comparison of real-time PCR and droplet digital PCR for the detection of Xylella fastidiosa in plants

2019 ◽  
Author(s):  
Enora Dupas ◽  
Bruno Legendre ◽  
Valérie Olivier ◽  
Françoise Poliakoff ◽  
Charles Manceau ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Xylella Fastidiosa ◽  
False Negative ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Detection Sensitivity ◽  
Plant Sample ◽  
The European Union

AbstractXylella fastidiosa (Xf) is a quarantine plant pathogen bacterium originating from the Americas and that has emerged in Europe in 2013. Xf can be detected directly on plant macerate using molecular methods such as real-time PCR, which is a sensitive technique. However, some plants may contain components that can act as PCR reaction inhibitors, which can lead to false negative results or an underestimation of the bacterial concentration present in the analyzed plant sample. Droplet digital PCR (ddPCR) is an innovative tool based on the partitioning of the PCR reagents and the DNA sample into thousands of droplets, allowing the quantification of the absolute number of target DNA molecules present in a reaction mixture, or an increase of the detection sensitivity. In this study, a real-time PCR protocol, already used for Xf detection in the framework of official surveys in the European Union, was transferred and optimized for Xf detection using ddPCR. This new assay was evaluated and compared to the initial real-time PCR on five plant matrices artificially inoculated and on naturally infected plants. In our conditions, this new ddPCR enabled the detection of Xf on all artificially inoculated plant macerates with a similar limit of detection, or a slight benefit for Quercus ilex. Moreover, ddPCR improved diagnostic sensitivity as it enabled detection of Xf in samples of Polygala myrtifolia or Q. ilex that were categorized as negative or close to the limit of detection using the real-time PCR. Here, we report for the first time a ddPCR assay for the detection of the bacterium Xf.

scholarly journals Comparison of real-time PCR and droplet digital PCR for the detection of Xylella fastidiosa in plants

2019 ◽  
Vol 162 ◽  
pp. 86-95 ◽  
Author(s):  
Enora Dupas ◽  
Bruno Legendre ◽  
Valérie Olivier ◽  
Françoise Poliakoff ◽  
Charles Manceau ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Xylella Fastidiosa ◽  
Digital Pcr ◽  
Droplet Digital Pcr

scholarly journals Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

2021 ◽  
Author(s):  
Christian Schulze ◽  
Anne-Catrin Geuthner ◽  
Dietrich Mäde
Keyword(s):  
Durum Wheat ◽  
Real Time ◽  
Bread Wheat ◽  
Common Wheat ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Food Fraud ◽  
Single Genome ◽  
Cereal Species

AbstractFood fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR

Food Control ◽  
2019 ◽  
Vol 98 ◽  
pp. 380-388 ◽  
Author(s):  
Xiaofu Wang ◽  
Ting Tang ◽  
Qingmei Miao ◽  
Shilong Xie ◽  
Xiaoyun Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Transgenic Rice ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Processed Foods ◽  
Conventional Pcr ◽  
Rice Line ◽  
Transgenic Rice Line

42P Method optimization for the detection of chimerism by real-time PCR and droplet digital PCR

2021 ◽  
Vol 32 ◽  
pp. S1358
Author(s):  
I.M. Lambrescu ◽  
V.S. Ionescu ◽  
G. Gaina ◽  
A. Popa ◽  
C. Niculite ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Method Optimization

Reverse transcriptase droplet digital PCR vs reverse transcriptase quantitative real‐time PCR for serum HBV RNA quantification

2020 ◽  
Vol 92 (12) ◽  
pp. 3365-3372 ◽  
Author(s):  
Umaporn Limothai ◽  
Natthaya Chuaypen ◽  
Kittiyod Poovorawan ◽  
Watcharasak Chotiyaputta ◽  
Tawesak Tanwandee ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Quantitative Real Time Pcr ◽  
Rna Quantification

scholarly journals Absolute quantification by droplet digital PCR versus analog real-time PCR

2013 ◽  
Vol 10 (10) ◽  
pp. 1003-1005 ◽  
Author(s):  
Christopher M Hindson ◽  
John R Chevillet ◽  
Hilary A Briggs ◽  
Emily N Gallichotte ◽  
Ingrid K Ruf ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr

scholarly journals In-House Validation and Comparison of Two Wheat (Triticum aestivum) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR

2017 ◽  
Vol 11 (5) ◽  
pp. 1281-1290 ◽  
Author(s):  
Annalisa Paternò ◽  
Daniela Verginelli ◽  
Pamela Bonini ◽  
Marisa Misto ◽  
Cinzia Quarchioni ◽  
...  
Keyword(s):  
Triticum Aestivum ◽  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gmo Quantification

scholarly journals SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge

2020 ◽  
Author(s):  
Renfei Lu ◽  
Jian Wang ◽  
Min Li ◽  
Yaqi Wang ◽  
Jia Dong ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Digital Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pharyngeal Swab ◽  
Clinical Specimens ◽  
Positive Results ◽  
Negative Detection

SummaryBackgroundSARS-CoV-2 nucleic acid detection by RT-PCR is one of the criteria approved by China FDA for diagnosis of COVID-19. However, inaccurate test results (for example, high false negative rate and some false positive rate) were reported in both China and US CDC using RT-PCR method. Inaccurate results are caused by inadequate detection sensitivity of RT-PCR, low viral load in some patients, difficulty to collect samples from COVID-19 patients, insufficient sample loading during RT-PCR tests, and RNA degradation during sample handling process. False negative detection could subject patients to multiple tests before diagnosis can be made, which burdens health care system. Delayed diagnosis could cause infected patients to miss the best treatment time window. False negative detection could also lead to prematurely releasing infected patients who still carry residual SARS-CoV-2 virus. In this case, these patients could infect many others. A high sensitivity RNA detection method to resolve the existing issues of RT-PCR is in need for more accurate COVID-19 diagnosis.MethodsDigital PCR (dPCR) instrument DropX-2000 and assay kits were used to detect SARS-CoV-2 from 108 clinical specimens from 36 patients including pharyngeal swab, stool and blood from different days during hospitalization. Double-blinded experiment data of 108 clinical specimens by dPCR methods were compared with results from officially approved RT-PCR assay. A total of 109 samples including 108 clinical specimens and 1 negative control sample were tested in this study. All of 109 samples, 26 were from 21patients reported as positive by officially approved clinical RT-PCR detection in local CDC and then hospitalized in Nantong Third Hospital. Among the 109 samples, dPCR detected 30 positive samples on ORFA1ab gene, 47 samples with N gene positive, and 30 samples with double positive on ORFA1ab and N genes.ResultsThe lower limit of detection of the optimize dPCR is at least 10-fold lower than that of RT-PCR. The overall accuracy of dPCR for clinical detection is 96.3%. 4 out 4 of (100 %) negative pharyngeal swab samples checked by RT-PCR were positive judged by dPCR based on the follow-up investigation. 2 of 2 samples in the RT-PCR grey area (Ct value > 37) were confirmed by dPCR with positive results. 1 patient being tested positive by RT-PCR was confirmed to be negative by dPCR. The dPCR results show clear viral loading decrease in 12 patients as treatment proceed, which can be a useful tool for monitoring COVID-19 treatment.ConclusionsDigital PCR shows improved lower limit of detection, sensitivity and accuracy, enabling COVID-19 detection with less false negative and false positive results comparing with RT-PCR, especially for the tests with low viral load specimens. We showed evidences that dPCR is powerful in detecting asymptomatic patients and suspected patients. Digital PCR is capable of checking the negative results caused by insufficient sample loading by quantifying internal reference gene from human RNA in the PCR reactions. Multi-channel fluorescence dPCR system (FAM/HEX/CY5/ROX) is able to detect more target genes in a single multiplex assay, providing quantitative count of viral load in specimens, which is a powerful tool for monitoring COVID-19 treatment.

scholarly journals Direct Quantitation of SARS-CoV-2 Using Droplet Digital PCR in Suspected Samples With Very Low Viral Load

2021 ◽  
Author(s):  
Michela Deiana ◽  
Chiara Piubelli ◽  
Antonio Mori ◽  
Gian Paolo Chiecchi ◽  
Giulia La Marca ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
False Negative ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Hospitalized Patients ◽  
Lower Sensitivity ◽  
Rt Pcr ◽  
Viral Genes ◽  
Viral Target

Background: The reference test for SARS-CoV-2 detection is the reverse transcriptase real time PCR (real time RT-PCR). However, evidences reported that real time RT-PCR has a lower sensitivity compared with the droplet digital PCR (ddPCR) leading to possible false negative in low viral load cases. Methods: We used ddPCR for viral genes N1 and N2 on 20 negative (no detection) samples from symptomatic hospitalized COVID-patients presenting fluctuating real time RT-PCR results and 10 suspected samples (Ct value>35) from asymptomatic not hospitalized subjects. Results: ddPCR performed on RNA revealed 65% of positivity for at least one viral target in the hospitalized patients group of samples (35% for N1 and N2, 10% only for N1 and 20% only for N2) and 50% in the suspected cases (30% for N1 and N2, while 20% only for N2). On hospitalized patients’ samples, we applied also a direct ddPCR approach on the swab material, achieving an overall positivity of 83%. Conclusion: ddPCR, in particular the direct quantitation on swabs, shows a sensitivity advantage for the SARS-CoV-2 identification and may be useful to reduce the false negative diagnosis, especially for low viral load suspected samples.

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