Klinisch nut van Droplet Digital PCR om BCR-ABL1
Klinisch nut van Droplet Digital PCR om BCR-ABL1-transcripten van patiënten met Philadelphia-chromosoom-positieve acute lymfoblastische leukemie te monitoren Publish-chimere antigeenreceptor 19/22 T-celcocktailtherapie
Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) accounts for 20-30% of grownup sufferers with ALL, characterised by translocation of t (9, 22). Tyrosine kinase inhibitors (TKIs) have considerably improved the result despite the fact that there are nonetheless some issues together with relapse attributable to drug-resistant mutations and suboptimal molecular remission depth. Beforehand, we reported the security and efficacy of sequential infusion of CD19/22 chimeric antigen receptor T-cell (CAR-T) immunotherapy within the remedy of relapsed/refractory (R/R) B-cell neoplasms together with instances with Ph+ ALL. Given doable deeper response, extra sufferers had been anticipated to achieve optimum minimal residual illness (MRD) response. Another technique, duplex droplet digital PCR (ddPCR) with excessive sensitivity was established, which may present absolute quantification of MRD with out the necessity for calibration curves. Right here, we retrospectively collected 95 bone marrow samples from 10 sufferers with R/R Ph+, who acquired 19/22 CAR-T-cell cocktail remedy.
Notably, sequential molecular remission for greater than three months (SMR3), a major indicator based mostly on ddPCR after CAR-T infusion was established, which was outlined as a sequential molecular remission for not <three months with destructive MRD. On this cohort, no recurrence was noticed in six sufferers reaching SMR3, the place 4 of whom accepted allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CAR-T cell routine. Sadly, the opposite 4 sufferers who didn’t attain SMR3 relapsed, and didn’t obtain additional particular remedy besides CAR-T routine. To sum up, ddPCR could also be an alternate, particularly when nucleic acid was inadequate in scientific apply. No achievement of SMR3 could also be an early warning of potential relapse after CAR-T and indicating the initiation of different therapies together with allo-HSCT.
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Description: Molecular Biology|Reverse Transcription & PCR |
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Description: Molecular Biology|Reverse Transcription & PCR |
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Description: Molecular Biology|Reverse Transcription & PCR |
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Description: Molecular Biology|Reverse Transcription & PCR |
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Description: Molecular Biology|Reverse Transcription & PCR |
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Description: Biotechnology |
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Description: Ready-to-use Solution containing Taq DNA Polymerase, optimized reaction buffer, MgCl2 and dNTPs |
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Description: Ready-to-use Solution containing Taq DNA Polymerase, optimized reaction buffer, MgCl2 and dNTPs |
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Description: Ready-to-use Solution containing Taq DNA Polymerase, optimized reaction buffer, MgCl2 and dNTPs |
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Description: Part E |
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Description: Part E |
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Taq 2X MeanGreen Master Mix |
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Taq 2X MeanGreen Master Mix |
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ViPrimePLUS OneStep Taq RT-qPCR Master Mix with Low Rox, 150rxns |
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2 × EpiArt HS Taq Master Mix |
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| EM201-01 | Vazyme | 1ml | EUR 16 |
2 × EpiArt HS Taq Master Mix |
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| EM201-02 | Vazyme | 5 ml (5×1ml) | EUR 70 |
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Apex 2X Taq Master Mix, Clear |
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| 42-133 | Genesee Scientific | 100 Reactions/Unit | EUR 54.78 |
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Description: 1.5mM MgCl2 (Final Conc.) |
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Apex 2X Taq Master Mix, Clear |
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| 42-134 | Genesee Scientific | 500 Reactions/Unit | EUR 220.95 |
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Description: 1.5mM MgCl2 (Final Conc.) |
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Description: 1.5mM MgCl2 (Final Conc.) |
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Roseate PCR Master Mix (2x) (Taq Mix (2x)) |
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| 46503 | Sisco Laboratories | 1 ml | EUR 23.09 |
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Description: Part E |
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Roseate PCR Master Mix (2x) (Taq Mix (2x)) |
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| 46503-1 | Sisco Laboratories | 5 x1 ml | EUR 70.13 |
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Description: Part E |
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Kwantitatieve detectie en monitoring van Colletotrichum siamense in rubberbomen met behulp van real-time PCR
Colletotrichum siamense is among the most vital pathogens of rubber bushes in Asia. The correct detection and quantification of C. siamense populations in rubber bushes are of significance for monitoring the epidemics of the illness. On this research, we developed an ITS-based real-time PCR technique to effectively detect C. siamense infecting rubber tree, which reliably detected as little as 100 fg genomic DNA, 100 copies of goal DNA and 20 conidia. The actual-time PCR protocol acknowledged all C. siamense isolates collected from three provinces in China, whereas no amplification was noticed with rubber tree and its different pathogens. Detection and quantification of C. siamense had been carried out in artificially and naturally contaminated rubber leaves.
We may nonetheless detect C. siamense in plant mixes of which solely 0.0001% of the tissue contaminated. An accumulation of C. siamense DNA was noticed throughout the entire an infection course of in any respect three leaf phenological levels, suggesting the real-time PCR technique can be utilized to observe C. siamense improvement in rubber bushes. Lastly, the strategy allowed the detection of C. siamense in naturally contaminated and symptomless leaves of rubber bushes within the fields. In contrast with earlier detection strategies, the real-time PCR technique is extra particular and extra delicate, and will likely be of nice use for research aiming to realize a greater understanding of the epidemiology of Colletotrichum leaf illness, in addition to the prediction of illness threat and the management proposal.
Integratie van microfluïdische monstervoorbereiding met PCR-detectie om de effecten van gelijktijdige scheiding van DNA-remmers en uitwisseling van DNA-oplossingen te onderzoeken
On this paper, we utilized a curved-channel microfluidic machine to separate DNA from PCR-inhibitor-containing water and concurrently wash them into clear water for detection utilizing a conveyable PCR thermocycler. Environmental DNA (eDNA) sampling has turn into an efficient surveying strategy for detecting uncommon organisms. Nevertheless, low focus eDNA molecules could also be masked by PCR inhibitors throughout amplification and detection, rising the chance of false negatives. Due to this fact, applied sciences for on-site DNA separation and washing are urgently wanted. Our machine consisted of a half-circle microchannel with a DNA-inhibitor pattern inlet, a clear buffer inlet, and a number of retailers.
By utilizing the flow-induced inertial forces, 10 μm DNA-conjugated microparticles had been centered on the inner-wall of the curved microchannel whereas separation from 1 μm inhibitor-conjugated microparticles and DNA washing had been achieved concurrently with the Dean circulate. We achieved singleplex focusing, isolation and washing of 10 μm particles at an effectivity of 94.5 ± 2.0%. In duplex experiments with 1 μm and 10 μm particles, bigger particles had been washed with an effectivity of 92.1 ± 1.6% and a purity of 79 ± 2%.
By surface-functionalizing the microparticles with affinity teams in opposition to Atlantic salmon DNA and humic acid (HA), and processing samples of varied concentrations in our machine, we achieved an efficient purification and detection of DNA molecules utilizing the transportable PCR thermocycler. Our technique considerably decreased PCR quantitation cycles from Cq > 38 to Cq = 30.35 ± 0.5, which confirmed enhancement of PCR amplification. The proposed machine takes a promising step ahead in pattern preparation in the direction of an built-in machine that can be utilized for simultaneous purification and answer alternate of DNA in point-of-need environmental monitoring purposes.
SARS-CoV-2-serologie verhoogt diagnostische nauwkeurigheid bij CT-vermoede, PCR-negatieve COVID-19-patiënten tijdens pandemie
Background: Within the absence of PCR detection of SARS-CoV-2 RNA, correct prognosis of COVID-19 is difficult. Low-dose computed tomography (CT) detects pulmonary infiltrates with excessive sensitivity, however findings could also be non-specific. This research assesses the diagnostic worth of SARS-CoV-2 serology for sufferers with distinct CT options however destructive PCR.
Strategies: IgM/IgG chemiluminescent immunoassay was carried out for 107 sufferers with confirmed (group A: PCR + ; CT ±) and 46 sufferers with suspected (group B: repetitive PCR-; CT +) COVID-19, admitted to a German college hospital through the pandemic’s first wave. A standardized, in-house CT classification of radiological indicators of a viral pneumonia was used to evaluate the likelihood of COVID-19.
Outcomes: Seroconversion charges (SR) decided on day 5, 10, 15, 20 and 25 after symptom onset (SO) had been 8%, 25%, 65%, 76% and 91% for group A, and 0%, 10%, 19%, 37% and 46% for group B, respectively; (p < 0.01). In comparison with hospitalized sufferers with a non-complicated course (non-ICU sufferers), seroconversion tended to happen at decrease frequency and delayed in sufferers on intensive care items. SR of sufferers with CT findings categorised as excessive certainty for COVID-19 had been 8%, 22%, 68%, 79% and 93% in group A, in contrast with 0%, 15%, 28%, 50% and 50% in group B (p < 0.01). SARS-CoV-2 serology established a particular prognosis in 12/46 group B sufferers. In 88% (8/9) of sufferers with destructive serology > 14 days after symptom onset (group B), clinico-radiological consensus reassessment revealed possible diagnoses apart from COVID-19. Sensitivity of SARS-CoV-2 serology was superior to PCR > 17d after symptom onset.
Conclusions: Roughly one-third of sufferers with distinct COVID-19 CT findings are examined destructive for SARS-CoV-2 RNA by PCR rendering appropriate prognosis tough. Implementation of SARS-CoV-2 serology testing alongside present CT/PCR-based diagnostic algorithms improves discrimination between COVID-19-related and non-related pulmonary infiltrates in PCR destructive sufferers. Nevertheless, sensitivity of SARS-CoV-2 serology strongly will depend on the time of testing and turns into superior to PCR after the twond week following symptom onset.
