Assessment of genetically engineered events in heat-treated and non-treated samples using droplet digital PCR and real-time quantitative PCR

Abstract Background In mature lymphoid disorders, minimal residual disease (MRD) detection based on real time quantitative PCR (RQ-PCR) of immunoglobulin heavy chain gene rearrangement (IgH) has a well-established role in prognostic assessment, particularly in Mantle cell Lymphoma (MCL) and Multiple Myeloma (MM). RQ-PCR has excellent sensitivity and specificity but has a major limitation in its relative quantification nature, as it requires a reference standard curve usually built with dilutions of diagnostic tumor DNA or on plasmids containing the target rearrangement. Droplet Digital PCR (DD-PCR), applying the principle of limiting dilution of DNA and single molecule detection allows a reliable absolute quantification of target. In this study we compared IgH-based MRD detection by RQ-PCR and DD-PCR, to assess whether DD-PCR could achieve the same performances of RQ-PCR in the absence of the limitation mentioned above. Methods Bone marrow (BM) and peripheral blood (PB) samples were collected from patients affected by MCL and MM in which RQ-PCR based MRD analysis was already performed in the context of prospective clinical trials. In all trials patients gave the informed consent for MRD determination. IgH-based MRD detection by RQ-PCR was carried out as previously described [Ladetto et al. BBMT 2000] and results were interpreted according to the Euro-MRD guidelines [van der Velden et al. Leukemia 2007]. DD-PCR was performed by the QX100 Droplet Digital PCR system (Bio-RAD Inc.) on 500 ng of genomic DNA combined with the same Allele Specific Oligonucleotides (ASO)-primers and TaqMan-probes used in the RQ-PCR. Droplets were generated by QX100 droplet generator. End-point PCR (40 cycles) was performed on a T100 Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX100 droplet reader and analyzed by QuantaSoft 1.2 (Bio-Rad Inc). For data interpretation RQ-PCR and DD-PCR results were expressed as amount of target copies per 1E+05 cells. Comparability of MRD results by DD-PCR and RQ-PCR was assessed by means of bivariate correlations between methods analysis (R2.15.1 package irr). Discordances were classified as follows: a positive/negative discordance was defined as major when the positive result was >1E-04 and minor when ≤1E-04; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. Results Overall, 161 samples belonging to 35 patients (18 MCL and 17 MM), 66 MCL and 95 MM were analyzed. 35 samples were taken at diagnosis and 126 at follow-up. 118 were BM while 43 were PB. A significant correlation was found between DD-PCR and RQ-PCR (R2=0.89, p<0.0001) (fig). DD-PCR and RQ-PCR showed superimposable sensitivity (10-5). Specificity in terms of appearance of non-specific amplifications signals in no-template samples (tested for all patients) and reproducibility on 30 replicates (4 samples) were superimposable. 128 out of 161 samples were fully concordant (Choen's K=0.80). MRD detection was concordantly positive in 106/161 (65.8%) samples and concordantly negative in 22/161 samples (13.7%). Only 5/161 (3.1%) samples showed major qualitative discordance. 28/161 (17.4%) samples showed minor qualitative discordance (which might be related to Poisson's statistics). Quantitative discordances were observed in 5/161 (3.1%) of cases (positive non quantifiable (PNQ) cases were conventionally placed to a value intermediate between sensitivity and quantitative range). Interestingly, 17 samples negative by RQ-PCR were scored positive by DD-PCR (median 6 copies, range 2-74) while 16 samples positive by RQ-PCR (median 5 copies, range 2-44) were negative by DD-PCR. Conclusions Here we report for the first time the use of DD-PCR in the context of IgH-based MRD evaluation in lymphoproliferative disorders. DD-PCR is a feasible tool for IGH-based MRD monitoring in MCL and MM, reaching similar sensitivities compared to standardized RQ-PCR. Moreover DD-PCR allows bypassing the need of building a standard curve thus considerably reducing the complexity of IgH-based RQ-PCR (need of purified diagnostic tissue or Flow Cytometry-based quantification of tumor load or diagnosis, or building of a plasmid-derived standard curve). Finally DD-PCR might potentially overcome the problem of positive non-quantifiable samples. These features make DD-PCR a feasible and attractive alternative method for IgH-based MRD assessment. Disclosures: Kubiczkovà: GAP304/10/1395 : Research Funding; MUNI/11/InGA17/2012: Research Funding.

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Real-time quantitative PCR and droplet digital PCR for plant miRNAs in mammalian blood provide little evidence for general uptake of dietary miRNAs

RNA Biology ◽

10.4161/rna.25246 ◽

2013 ◽

Vol 10 (7) ◽

pp. 1080-1086 ◽

Cited By ~ 124

Author(s):

Kenneth W. Witwer ◽

Melissa A. McAlexander ◽

Suzanne E. Queen ◽

Robert J. Adams

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Plant Mirnas ◽

Real Time Quantitative Pcr ◽

Mammalian Blood

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Genes regulating development and behavior exhibited altered expression in Drosophila melanogaster exposed to bisphenol A: use of real-time quantitative PCR (qRT-PCR) and droplet digital PCR (ddPCR) in genotoxicity study

Environmental Science and Pollution Research ◽

10.1007/s11356-020-10805-0 ◽

2020 ◽

Author(s):

Morium Begum ◽

Pallab Paul ◽

Debasmita Das ◽

Kaustav Chakraborty ◽

Ashima Bhattacharjee ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Bisphenol A ◽

Real Time ◽

Quantitative Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Altered Expression ◽

Real Time Quantitative Pcr ◽

Qrt Pcr ◽

And Behavior

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Tolerance of Droplet-Digital PCR vs Real-Time Quantitative PCR to Inhibitory Substances

Clinical Chemistry ◽

10.1373/clinchem.2013.211045 ◽

2013 ◽

Vol 59 (11) ◽

pp. 1670-1672 ◽

Cited By ~ 154

Author(s):

Tanis C Dingle ◽

Ruth Hall Sedlak ◽

Linda Cook ◽

Keith R Jerome

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Real Time Quantitative Pcr

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PSX-B-15 Real-time quantitative PCR versus droplet digital PCR for measurement of peripheral blood leukocytes interferon-tau stimulated genes in beef cattle

Journal of Animal Science ◽

10.1093/jas/skab235.599 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 326-326

Author(s):

Gabriela Dalmaso de Melo ◽

Gessica A Franco-Johannsen ◽

Brooke McAnnally ◽

Reinaldo F Cooke ◽

Ky G Pohler

Keyword(s):

Quantitative Pcr ◽

Reference Gene ◽

Peripheral Blood ◽

Digital Pcr ◽

Beef Cows ◽

Droplet Digital Pcr ◽

Peripheral Blood Leukocytes ◽

Interferon Tau ◽

Blood Leukocytes ◽

Real Time Quantitative Pcr

Abstract We aimed to compare the abundance of interferon-tau stimulated genes (ISG) transcripts in peripheral blood leukocytes of artificially inseminated beef cows using real-time quantitative PCR (RT-qPCR) versus Droplet Digital PCR (ddPCR). Multiparous Bos taurus beef cows (n = 7) were subjected to timed artificial insemination (TAI) on day 0. Pregnancy was determined by transrectal ultrasonography on days 26 and 30 post-TAI, and cows were classified as: pregnant (n=4; embryo detected on days 26 and 30) or non-pregnant (n = 3; no embryo detected). Coccygeal vein blood samples were collected on days 0, 15, 17, 19, 20 and 24 post-TAI. Leukocyte RNA was extracted from the buffy coat fraction using Trizol associated with the DirectZol-RNA kit and transcribed to cDNA. The abundance of ISG (ISG15 and MX2) was assessed by relative quantification to a reference gene (RPS9) using RT-qPCR and by absolute quantification using the QX100TM Droplet DigitalTM PCR System (Bio-rad Laboratories) according to manufacturer’s recommendations. Data were analyzed using PROC MIXED on SAS 9.4. For the RT-qPCR, pregnant cows had greater (P < 0.05) ISG15 and MX2 abundance compared to non-pregnant cows on days 20 (ISG15:0.11±0.1 vs. 0.01±0.001 and MX2:0.73±0.4 vs. 0.06±0.06) and 24 (ISG15:0.34±0.2 vs. 0.01±0.001 and MX2:0.77±0.2 vs. 0.13±0.04). For ddPCR, a greater ISG15 and MX2 copy numbers in pregnant cows was observed on days 15 (ISG15:129 vs. 44 copies/µl and MX2:33 vs. 10 copies/µl) and 20 (ISG15: 216 vs. 30 copies/µl and MX2: 44 vs. 7 copies/µl), and also on day 24 for ISG15 (32 vs. 7 copies/µl) compared to non-pregnant cows. In conclusion, ddPCR was able to detect an earlier expression of ISG in pregnant cows. Future studies are needed to enroll more animals and establish a suitable cutoff value using ddPCR, which could be less subjective for diagnosis as it does not require the use of a reference gene.

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Digital PCR dynamic range is approaching that of real-time quantitative PCR

Biomolecular Detection and Quantification ◽

10.1016/j.bdq.2016.10.001 ◽

2016 ◽

Vol 10 ◽

pp. 31-33 ◽

Cited By ~ 18

Author(s):

Gerwyn M. Jones ◽

Eloise Busby ◽

Jeremy A. Garson ◽

Paul R. Grant ◽

Eleni Nastouli ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Dynamic Range ◽

Digital Pcr ◽

Real Time Quantitative Pcr

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Superiority of Droplet Digital PCR Over Real-Time Quantitative PCR for JAK2 V617F Allele Mutational Burden Assessment in Myeloproliferative Neoplasms: A Retrospective Study

Diagnostics ◽

10.3390/diagnostics10030143 ◽

2020 ◽

Vol 10 (3) ◽

pp. 143

Author(s):

Francesco La Rocca ◽

Vitina Grieco ◽

Vitalba Ruggieri ◽

Emanuela Zifarone ◽

Oreste Villani ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Digital Pcr ◽

Jak2 V617f Mutation ◽

Real Time Quantitative Pcr ◽

Mutational Status ◽

V617f Mutation ◽

Detection And Quantification

JAK2 V617F mutational status is an essential diagnostic index in myeloproliferative neoplasms (MPNs). Although widely used for detection of JAK2 V617F mutation in peripheral blood (PB), sensitive real-time quantitative PCR (qPCR) presents some methodological limitations. Recently, emerging alternative technologies, like digital droplet PCR (ddPCR), have been reported to overcome some of qPCR’s technical drawbacks. The purpose of this study was to compare the diagnostic utility of ddPCR to qPCR for JAK2 V617F detection and quantification in samples from MPNs patients. Sensitivity and specificity of qPCR and ddPCR in the detection of the mutation were assessed by using a calibrator panel of mutated DNA on 195 JAK2 positive MPN samples. Based on our results, ddPCR proved to be a suitable, precise, and sensitive method for detection and quantification of the JAK2 V617F mutation.

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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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