China
Africa
Explore chapters and articles related to this topic
Non-Invasive Prenatal Testing (NIPT)
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Nuria Balaguer, Emilia Mateu-Brull, Miguel Milán
RMD requires precise quantification of mutant and wild-type (WT) alleles in cfDNA and the application of statistical methods to clarify that measured imbalances reflect fetal genotype signal rather than technical noise. Unfortunately, standard NGS protocols are not sensitive enough for these applications since amplification bias between mutant and WT alleles can lead to inaccurate allelic fractions. Possible solutions to these limitations include the use of nested PCR or digital PCR (dPCR) (65,66). Proof-of-principle studies for NIPD using dPCR were reported for several recessive and X-linked conditions, including β-thalassemia (62,67), sickle-cell disease (68), hemophilia (69,70), and recessive forms of deafness (71). However, dPCR is highly sensitive and has a limited capacity for multiplexing compared to NGS, which confines the number of mutations that can be assayed in one test (18).
Leukemias
Published in Pat Price, Karol Sikora, Treatment of Cancer, 2020
The notion of achieving TFR successfully is clearly a significant milestone, since most CML patients have a life expectancy equivalent to that of the general population following appropriate treatment with TKIs. TFR allows the safe and effective discontinuation of TKI therapy, and its inclusion should enable additional personalization of the treatment algorithm and facilitate an improved focus on daily life and improving QoL. It should reduce treatment-related risks, optimize treatment adherence and financial burdens, and better inform on the precise definition of treatment goals and milestones, treatment choice, and monitoring. It is therefore important to identify the best validated methodology, including droplet digital polymerase chain reaction (PCR) (ddPCR), to determine DMR.83
Distribution
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Meanwhile, the heterogeneous asymmetric recombinase polymerase amplification (haRPA) was applied for the phage MS2 detection in combination with the stepwise phage concentration from 1250 L drinking water into 1 mL (Elsäßer et al. 2018). Since then, digital PCR (dPCR) has become a promising technology for absolute quantification of nucleic acid without need of calibration curves. Following dPCR, various digital isothermal amplification methods were also developed which only required isothermal incubation. Among them, a loop-mediated isothermal amplification, or LAMP, became the most popular one and was adapted to the rapid enumeration of the phage MS2 as a model (Huang X et al. 2018; Lin et al. 2019).
Focus on the frontier issue: progress in noninvasive prenatal screening for fetal trisomy from clinical perspectives
Published in Critical Reviews in Clinical Laboratory Sciences, 2023
Meng Tian, Lei Feng, Jinming Li, Rui Zhang
Digital PCR (dPCR) is an efficient and allele-independent method to quantify nucleic acids by counting amplification at a single molecule level [77]. In theory, the DNA samples are diluted to fit approximately one template per well to easily determine the results. However, the true number of template molecules distributed to each well is not precisely one but rather follows a Poisson distribution. After the dPCR process, the amplified positive results are converted and corrected to the real number of input template molecules according to the binomial approximation. The risk of fetal trisomy can be rapidly determined by analyzing the difference in the number of positive wells between the target (e.g. chromosome 21) and reference chromosome (e.g. chromosome 1) [77]. Recently, a size selection strategy was proposed to increase the proportion of cffDNA in maternal samples [78]. In the study, dPCR was performed in 877 pregnant women with an overall accuracy of 99.66%, demonstrating good clinical validity. Overall, dPCR has three critical advantages over sequencing. First, dPCR achieves similar detection performance to NGS using fewer samples. Second, the short time required allows rapid and real-time detection. Most importantly, the low cost of dPCR testing (less than $100) is affordable even when budgets are restricted [79]. Notably, many countries use traditional serum biochemical tests as a primary screening tool followed by the NGS rescreen to balance the cost and performance of detection [80]. With the incorporation of dPCR, NIPS is expected to provide an economical screening strategy without reducing the DRs.
Technical considerations for circulating tumor DNA detection in oncology
Published in Expert Review of Molecular Diagnostics, 2019
Claire Franczak, Pierre Filhine-Tresarrieu, Pauline Gilson, Jean-Louis Merlin, Lewis Au, Alexandre Harlé
Digital PCR assay was developed in the late 90s by Vogelstein and Kinzler in order to transform the exponential nature of PCR to a linear one, leading to sensitivity improvement [76]. With serial dilutions, a single DNA molecule is isolated in a partition and individual PCR is performed in each partition. Two fluorescent probes are then added. One is specific to the target mutant codon of interest and the other hybridizes with a conserved sequence embedded in the amplicon product. In the final step, fluorescence is analyzed to identify partitions containing wild-type sequence and partitions containing mutant PCR product. Binary results are obtained, thus giving the ‘digital’ term in the name of this approach. Sensitivity of this approach is about 0.1% [76,77] (Table 2). Figure 1 depicts the workflow of a dPCR. KRAS mutations in ctDNA assessment by dPCR yielded 87.2% sensitivity and 99.2% specificity [78]. dPCR has been evaluated in numerous studies. In a large pan-cancer analysis of 640 patients, ctDNA was detected in more than 75% of patients with advanced cancers including CRC, breast cancer and melanoma, but less than 50% of patients with thyroid cancer or glioma [78]. Interestingly, 47% of patients with stage I disease of cancer of all kind had ctDNA detected by dPCR including approximately 75% of patients with localized CRC and almost 50% of patients with breast cancer [78].
Implementation and new insights in molecular diagnostics for HIV infection
Published in Expert Review of Molecular Diagnostics, 2018
Hin-Fung Tsang, Lawrence Wing-Chi Chan, Jennifer Chiu-Hung Tong, Heong-Ting Wong, Christopher Koon-Chi Lai, Thomas Chi-Chuen Au, Amanda Kit-Ching Chan, Lawrence Po-Wah Ng, William Chi-Shing Cho, Sze-Chuen Cesar Wong
Another promising application of dPCR in viral diagnostics is the improved detection of low abundance mutant sequences, such as those contributing to antiviral resistance, from high abundance wild-type sequences [25]. In terms of pharmacogenomics, dPCR plays important role in achieving personalized medicine. Taking abacavir, a small molecule reverse transcription inhibitor against HIV, is one of the commonly used treatment options. However, 5–8% of patients taking abacavir would develop life-threatening hypersensitivity against the drug. Mallal et al. identified the association between HLA-B*5701 and abacavir hypersensitivity reaction [26]. To reduce the incidence of abacavir hypersensitivity reaction, genetic screening is now recommended by many HIV treatment guidelines before initiation of abacavir treatment [27].