Detection of KRAS mutations in colon adenocarcinoma by droplet digital PCR.

e15080 Background: The screening of mutations in the KRAS gene is a traditional marker of the effectiveness of targeted therapy for colorectal cancer. Mutations in the 12 codon of the second exon of the KRAS gene are present in 40% of colorectal tumors, and monitoring of the mutational status together with the level of mutant DNA is of particular clinical interest. However, the sensitivity level of the traditionally used real-time polymerase chain reaction (RT-PCR) method is insufficient in some cases. Recently, Droplet Digital PCR (DD-PCR) has been considered as an alternative method, which is more sensitive. The purpose of this study was to compare the methods of detection of somatic mutations in the second exon of the KRAS gene using DD-PCR versus RT-PCR. Methods: This study included 134 patients with colon adenocarcinoma. The presence or absence of activating mutations in the second exon of the KRAS gene in samples of FFPE blocks was detected by RT-PCR using the “Real-time-PCR-KRAS-7M reagent kit” (Biolink, Russia) and Digital Droplet PCR using the “KRAS Screening Multiplex kit” (Bio-Rad, USA). Results: For all 134 samples, selected for the study, the WT status of the KRAS gene was identified by the RT-PCR method. According to the results of DD-PCR for 131 samples (96.2%), a positive amplification response of mutant DNA with more than 200 events was obtained. The number of amplicons varied from 312 to 117917 per sample, the median was 2940 copies. According to recent trials, a clinically significant level of mutational events must exceed 5% of the total number of amplicons in a sample. In our study, 12.9% of cases (17 patients) met this criterion. Conclusions: The DD-PCR method demonstrated a much higher analytical sensitivity for the detection of SNP mutations in comparison with RT-PCR that may be of critical importance in the therapeutic decision.

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

10.20944/preprints202104.0688.v1 ◽

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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Droplet digital PCR versus multiplex real-time PCR method for the detection and quantification of DNA from the four transgenic soy traits MON87769, MON87708, MON87705 and FG72, and lectin

European Food Research and Technology ◽

10.1007/s00217-015-2481-3 ◽

2015 ◽

Vol 241 (4) ◽

pp. 521-527 ◽

Cited By ~ 25

Author(s):

René Köppel ◽

Thomas Bucher ◽

Anna Frei ◽

Hans-Ulrich Waiblinger

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Pcr Method ◽

Detection And Quantification

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Comparison of Real-Time PCR and Droplet Digital PCR for the Determination of JAK2V617F Mutation in Ph'-Negative Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v124.21.5548.5548 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5548-5548

Author(s):

Giulia Fontanelli ◽

Claudia Baratè ◽

Elena Ciabatti ◽

Francesca Guerrini ◽

Riccardo Morganti ◽

...

Keyword(s):

Real Time ◽

Standard Method ◽

Real Time Pcr ◽

Myeloproliferative Neoplasms ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Jak2v617f Mutation ◽

Rt Pcr ◽

Jak2 Mutation ◽

Allele Burden

Abstract Myeloproliferative neoplasms (MPNs) are a group of clonal diseases associated with JAK2 mutation (V617F) in a percentage varying from 50% (Essential Thrombocytemia ET, and Primary Myelofibrosis PMF) to 95% (Polycitemia Vera, PV). The standard method for determining the V617F mutation is today represented by the Real Time PCR (RT-PCR). However, in recent years some studies have focused attention on a new method, the Droplet Digital PCR (DD-PCR), that allows an absolute quantitation of the mutated allele without any reference curve. The main objective of this study was the comparison of two PCR methods (RT-PCR and DD-PCR) for the analysis of the JAK2V617F mutation in order to assess the sensitivity, specificity and feasibility of the two techniques. In the study 225 patients with MPNs JAK2V617F-positive were enrolled; in 99 cases, DNA was still available for further assays and so DD-PCR tests were performed and compared with the results coming from RT-PCR (MutaQuant kit, Ipsogen, Luminy Biotech, Marseille, France). The results show a good sensitivity for both methods; nevertheless, after appropriate dilution tests, the DD-PCR was able to detect the JAK2V617F mutation in the 5x10-4 dilution versus 5x10-3 for the RT-PCR, in front of a fully comparable specificity. A linear regression analysis showed an absolute correlation between the 2 methods (R2=0.91), and the median mutated allele burden was not different when measured by the DD-PCR or the RT-PCR (p=0.67), with the highest median allele burden observed in secondary myelofibrosis (86% by RT-PCR and 91% by DD-PCR) and the lowest one in ET (23% by RT-PCR and 25% by DD-PCR) (see Figure 1 and Figure 2), as expected. Figure 1 Figure 1. Figure 2 Figure 2. Our study demonstrates that a new molecular technique, the DD-PCR, may represent a new frontier in the world of molecular techniques for the detection of JAK2 mutation with a high sensitivity and specificity. In addition the method allows obtain two types of information, qualitative and quantitative, with one analysis, saving time and at a lower cost than the standard method. Disclosures No relevant conflicts of interest to declare.

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Development of droplet digital PCR for the detection of Tilletia laevis, which causes common bunt of wheat, based on the SCAR marker derived from ISSR and real-time PCR

Scientific Reports ◽

10.1038/s41598-020-72976-7 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Tongshuo Xu ◽

Zhaoqun Yao ◽

Jianjian Liu ◽

Han Zhang ◽

Ghulam Muhae Ud Din ◽

...

Keyword(s):

Detection Limit ◽

Real Time ◽

Real Time Pcr ◽

Scar Marker ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Common Bunt ◽

Tilletia Laevis ◽

Pcr Method ◽

Dna Fragment

Abstract Common bunt of wheat caused by Tilletia laevis and/or T. caries (syn. T. tritici), is a major disease in wheat-growing regions worldwide that could lead to 80% or even total loss of production. Even though T. laevis can be distinguished from T. caries on the bases of morphology of teliospores using microscopy technique. However, molecular methods could serve as an additional method to quantify the pathogen. To develop a rapid diagnostic and quantify method, we employed the ISSR molecular marker for T. laevis in this study. The primer ISSR857 generated a polymorphic pattern displaying a 1385 bp T. laevis-specific DNA fragment. A pair of specific primers (L57F/L57R) was designed to amplify a sequence-characterized amplified region (SCAR) (763 bp) for the PCR detection assay. The primers amplified the DNA fragment in the tested isolates of T. laevis but failed in the related species, including T. caries. The detection limit of the primer set (L57F/L57R) was 5 ng/µl of DNA extracted from T. laevis teliospores. A SYBR Green I real-time PCR method for detecting T. laevis with a 100 fg/µl detection limit and droplet digital PCR with a high sensitivity (30 fg/µl detection limit) were developed; this technique showed the most sensitive detection compared to the SCAR marker and SYBR Green I real-time PCR. Additionally, this is the first study related the detection of T. laevis with the droplet digital PCR method.

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Real-time Quantitative RT-PCR method for estimation of mRNA level of CCAAT/enhancer binding protein in the central nervous system ofLymnaea stagnalis

Acta Biologica Hungarica ◽

10.1556/abiol.55.2004.1-4.19 ◽

2004 ◽

Vol 55 (1-4) ◽

pp. 157-161 ◽

Cited By ~ 7

Author(s):

D. Hatakeyama ◽

Hisayo Sadamoto ◽

E. Ito

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Real Time ◽

Binding Protein ◽

Mrna Level ◽

Rt Pcr ◽

Enhancer Binding Protein ◽

Ccaat Enhancer Binding Protein ◽

The Central Nervous System ◽

Pcr Method

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

European Food Research and Technology ◽

10.1007/s00217-021-03786-y ◽

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.

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