scholarly journals Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

2021 ◽  
Author(s):  
Suttipat Srisut ◽  
Kanokon Suwannasin ◽  
Rungirun Sugaram ◽  
Arjen M. Dondorp ◽  
Mallika Imwong
Keyword(s):  
Drug Resistance ◽  
Copy Number ◽  
Gene Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Antimalarial Drug Resistance ◽  
Quantification Limit ◽  
Field Samples ◽  
The Cost

Abstract Background: Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum pfplasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with antimalarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to antimalarial drugs.Methods: A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay.Results: There were no significant differences between the GCN results obtained from uniplex andmultiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated.Conclusions: The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of antimalarial drug resistance.

scholarly journals Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

2020 ◽  
Author(s):  
Suttipat Srisut ◽  
Kanokon Suwannasin ◽  
Rungirun Sugaram ◽  
Arjen M. Dondorp ◽  
Mallika Imwong
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Copy Number ◽  
Gene Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Antimalarial Drug Resistance ◽  
Quantification Limit ◽  
Field Samples

Abstract Background: Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum pfplasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with antimalarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to antimalarial drugs.Methods: The ddPCR assays were developed to detect the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay.Results: Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. The accuracy and %RSD value of the duplex ddPCR assay were 94.88% and 3.71, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). Conclusions: The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of antimalarial drug resistance.

scholarly journals Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Suttipat Srisutham ◽  
Kanokon Suwannasin ◽  
Rungniran Sugaram ◽  
Arjen M. Dondorp ◽  
Mallika Imwong
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Copy Number ◽  
Gene Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Quantification Limit ◽  
Field Samples ◽  
The Cost

Abstract Background Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs. Methods A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay. Results There were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated. Conclusions The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.

scholarly journals Detection of MET Gene Copy Number in Cancer Samples Using the Droplet Digital PCR Method

PLoS ONE ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. e0146784 ◽  
Author(s):  
Yanni Zhang ◽  
En-Tzu Tang ◽  
Zhiqiang Du
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Pcr Method

Abstract P613: Absolute Gene Quantification Of Intrarenal Renin-angiotensin System Components Using Droplet Digital PCR Analysis

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Ryousuke Satou ◽  
Akemi Katsurada ◽  
Kayoko Miyata ◽  
Andrei Derbenev ◽  
Andrea Zsombok
Keyword(s):  
Copy Number ◽  
System Analysis ◽  
Renin Angiotensin System ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Angiotensin System ◽  
Renin Angiotensin ◽  
Copy Numbers ◽  
Intrarenal Ras

The intrarenal renin-angiotensin system (RAS) has been shown to play crucial roles in the development of hypertension and RAS associated kidney injury including diabetic nephropathy. Although some circulating RAS components are filtered into kidneys and contribute to the regulation of intrarenal RAS activity, evaluating expression levels of RAS components in the kidney is important to elucidate the mechanisms underlying intrarenal RAS activation. Digital PCR is a new technique that has been established to quantify absolute target gene levels, which allows for comparisons of different gene levels. Thus, this study was performed to establish profiles of absolute gene copy numbers for intrarenal RAS components in wild-type (WT) rats, WT and streptozotocin (STZ)-induced diabetic mice. Male Sprague-Dawley rats (N=5) and male C57BL/6J mice were used in this study. The mice were subjected to either control (N=5) or STZ (200 mg/kg, N=4) injection. Seven days after STZ injection, copy numbers of renal cortical angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1 receptor a (AT1a), and AT2 mRNA were determined by a droplet digital PCR. Since (pro)renin proteins produced by juxtaglomerular cells are secreted to circulating system, analysis of renin mRNA was excluded from this evaluation. In the renal cortex of WT rats, the copy number of AGT was higher than other measured RAS components (AGT: 719.2±46.6, ACE: 116.0±14.9, ACE2: 183.6±21.5, AT1a: 196.0±25.2 copies in 1 ng total RNA). AT2 levels were lower than other components (0.068±0.01 copies). In WT mice, ACE exhibited the highest copy number in the components (AGT: 447.2±29.0, ACE: 1662.4±61.2, ACE2: 676.8±41.5, AT1a: 867.0±16.8, AT2: 0.049±0.01 copies). Although STZ-induced diabetes did not change ACE2 and AT1a, ACE levels were reduced (765.5±98.1 copies) and AT2 levels were augmented (0.10±0.01 copies) as previously demonstrated. Accordingly, the absolute quantification by digital PCR established precise gene profiles of intrarenal RAS components, which will provide rationales for targeting the each component in future studies. Furthermore, the results indicate that the high sensitive assay accurately quantifies rare target genes including intrarenal AT2.

scholarly journals High levels of copy number variation of ampliconic genes across major human Y haplogroups

2017 ◽  
Author(s):  
Danling Ye ◽  
Arslan Zaidi ◽  
Marta Tomaszkiewicz ◽  
Corey Liebowitz ◽  
Michael DeGiorgio ◽  
...  
Keyword(s):  
Copy Number Variation ◽  
Y Chromosome ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Families ◽  
Digital Pcr ◽  
Gene Copy ◽  
Copy Numbers ◽  
Number Variation ◽  
Gene Copy Numbers

AbstractDue to its highly repetitive nature, the human male-specific Y chromosome remains understudied. It is important to investigate variation on the Y chromosome to understand its evolution and contribution to phenotypic variation, including infertility. Approximately 20% of the human Y chromosome consists of ampliconic regions which include nine multi-copy gene families. These gene families are expressed exclusively in testes and usually implicated in spermatogenesis. Here, to gain a better understanding of the role of the Y chromosome in human evolution and in determining sexually dimorphic traits, we studied ampliconic gene copy number variation in 100 males representing ten major Y haplogroups world-wide. Copy number was estimated with droplet digital PCR. In contrast to low nucleotide diversity observed on the Y in previous studies, here we show that ampliconic gene copy number diversity is very high. A total of 98 copy-number-based haplotypes were observed among 100 individuals, and haplotypes were sometimes shared by males from very different haplogroups, suggesting homoplasies. The resulting haplotypes did not cluster according to major Y haplogroups. Overall, only three gene families (DATZ, RBMY, TSPY) showed significant differences in copy number among major Y haplogroups, and the haplogroup of an individual could not be predicted based on his ampliconic gene copy numbers. Finally, we found a significant correlation between copy number variation and individual’s height (for three gene families), but not between the former and facial masculinity/femininity. Our results suggest rapid evolution of ampliconic gene copy numbers on the human Y, and we discuss its causes.

scholarly journals The Copy Number of the spoVA2mob Operon Determines Pressure Resistance of Bacillus Endospores

2019 ◽  
Vol 85 (19) ◽  
Author(s):  
Zhen Li ◽  
Felix Schottroff ◽  
David J. Simpson ◽  
Michael G. Gänzle
Keyword(s):  
High Pressure ◽  
Genome Sequencing ◽  
Copy Number ◽  
Digital Pcr ◽  
Resistance Mechanisms ◽  
Droplet Digital Pcr ◽  
Food Microbiology ◽  
Gene Copy ◽  
Content Type ◽  
Pressure Resistance

ABSTRACT The spoVA2mob operon confers heat resistance to Bacillus spp., and the resistance correlates to the copy number of the operon. Bacillus endospores also exhibit a strong variation in resistance to pressure, but the underlying mechanisms of endospore resistance to pressure are not fully understood. We determined the effects of multiple spoVA2mob operons on high-pressure resistance in Bacillus endospores. The copy numbers of the spoVA2mob operon in 17 strains of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus velezensis, and Bacillus pumilus were determined via droplet digital PCR (ddPCR) and genome sequencing. These strains contained between 0 and 3 copies of the spoVA2mob operon; the quantification of the gene copy number by ddPCR was as accurate as whole-genome sequencing. We further tested the pressure resistance of 17 Bacillus endospores at 600 MPa and 80°C. Strains with one or no spoVA2mob operon had significantly lower pressure resistance than strains with two or three copies of the operons (P < 0.001), indicating that redundant spoVA2mob operons in Bacillus contributed to higher pressure resistance of endospores. The copy number of the spoVA2mob operon was not related to the dipicolinic acid (DPA) content of endospores. Overall, the copy number of the spoVA2mob operon contributes to pressure resistance of Bacillus endospores. This improves our understanding of the pressure resistance mechanisms in Bacillus spp. and may inform the development of high-pressure sterilization in food processing. IMPORTANCE Bacillus spp. are considered pressure-resistant microorganisms, but the resistance mechanisms remain unknown. The spoVA2mob operon is a mobile genetic element, and it can transfer to pathogenic or spoilage organisms by horizontal gene transfer. Results in this study indicate that multiple copies of the spoVA2mob operon mediate high-pressure resistance of Bacillus endospores, and it might contribute to the identification of the source of pressure-resistant pathogens and spoilage organisms that may contaminate the food supply. The droplet digital PCR (ddPCR) system is well suited for analysis in some human diseases due to its high efficiency and capability to provide high precision; however, no relevant studies in food microbiology have been reported so far. This study demonstrates a novel application of ddPCR in food microbiology.

scholarly journals Digital PCR for determination of cytochrome P450 2D6 and sulfotransferase 1A1 gene copy number variations

2017 ◽  
Vol 11 (6) ◽  
pp. 336-341 ◽  
Author(s):  
Yutaro Motoi ◽  
Kazufumi Watanabe ◽  
Hiroyuki Honma ◽  
Yousuke Tadano ◽  
Hiroshi Hashimoto ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Copy Number ◽  
Gene Copy Number ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Gene Copy ◽  
Cytochrome P450 2D6 ◽  
Sulfotransferase 1A1 ◽  
P450 2D6

scholarly journals Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies

2018 ◽  
Vol 64 (12) ◽  
pp. 1762-1771 ◽  
Author(s):  
Mariana Fitarelli-Kiehl ◽  
Fangyan Yu ◽  
Ravina Ashtaputre ◽  
Ka Wai Leong ◽  
Ioannis Ladas ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Gene Copy Number ◽  
Fold Increase ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Clinical Samples ◽  
Liquid Biopsies ◽  
Patients With Cancer ◽  
Free Circulating Dna

Abstract BACKGROUND Although interest in droplet-digital PCR technology (ddPCR) for cell-free circulating DNA (cfDNA) analysis is burgeoning, the technology is compromised by subsampling errors and the few clinical targets that can be analyzed from limited input DNA. The paucity of starting material acts as a “glass ceiling” in liquid biopsies because, irrespective how analytically sensitive ddPCR techniques are, detection limits cannot be improved past DNA input limitations. METHODS We applied denaturation-enhanced ddPCR (dddPCR) using fragmented genomic DNA (gDNA) with defined mutations. We then tested dddPCR on cfDNA from volunteers and patients with cancer for commonly-used mutations. gDNA and cfDNA were tested with and without end repair before denaturation and digital PCR. RESULTS By applying complete denaturation of double-stranded DNA before ddPCR droplet formation the number of positive droplets increased. dddPCR using gDNA resulted in a 1.9–2.0-fold increase in data-positive droplets, whereas dddPCR applied on highly-fragmented cfDNA resulted in a 1.6–1.7-fold increase. End repair of cfDNA before denaturation enabled cfDNA to display a 1.9–2.0-fold increase in data-positive signals, similar to gDNA. Doubling of data-positive droplets doubled the number of potential ddPCR assays that could be conducted from a given DNA input and improved ddPCR precision for cfDNA mutation detection. CONCLUSIONS dddPCR is a simple and useful modification in ddPCR that enables extraction of more information from low-input clinical samples with minor change in protocols. It should be applicable to all ddPCR platforms for mutation detection and, potentially, for gene copy-number analysis in cancer and prenatal screening.

scholarly journals Development and interlaboratory evaluation of a NIST Reference Material RM 8366 for EGFR and MET gene copy number measurements

2019 ◽  
Vol 57 (8) ◽  
pp. 1142-1152 ◽  
Author(s):  
Hua-Jun He ◽  
Biswajit Das ◽  
Megan H. Cleveland ◽  
Li Chen ◽  
Corinne E. Camalier ◽  
...  
Keyword(s):  
Reference Material ◽  
Copy Number ◽  
Target Genes ◽  
Human Cancer ◽  
Gene Copy Number ◽  
Digital Pcr ◽  
Cancer Diagnostics ◽  
Gene Copy ◽  
Human Cancer Cell Lines ◽  
Copy Numbers

Abstract Background The National Institute of Standards and Technology (NIST) Reference Material RM 8366 was developed to improve the quality of gene copy measurements of EGFR (epidermal growth factor receptor) and MET (proto-oncogene, receptor tyrosine kinase), important targets for cancer diagnostics and treatment. The reference material is composed of genomic DNA prepared from six human cancer cell lines with different levels of amplification of the target genes. Methods The reference values for the ratios of the EGFR and MET gene copy numbers to the copy numbers of reference genes were measured using digital PCR. The digital PCR measurements were confirmed by two additional laboratories. The samples were also characterized using Next Generation Sequencing (NGS) methods including whole genome sequencing (WGS) at three levels of coverage (approximately 1 ×, 5 ×  and greater than 30 ×), whole exome sequencing (WES), and two different pan-cancer gene panels. The WES data were analyzed using three different bioinformatic algorithms. Results The certified values (digital PCR) for EGFR and MET were in good agreement (within 20%) with the values obtained from the different NGS methods and algorithms for five of the six components; one component had lower NGS values. Conclusions This study shows that NIST RM 8366 is a valuable reference material to evaluate the performance of assays that assess EGFR and MET gene copy number measurements.

scholarly journals Digital PCR (dPCR) and qPCR Mediated Determination of Transgene Copy Number in the Forage Legume White Clover (Trifolium Repens).

2021 ◽  
Author(s):  
Rafael Narancio ◽  
Ulrik John ◽  
John Mason ◽  
Paula Giraldo ◽  
German Spangenberg
Keyword(s):  
White Clover ◽  
Reference Genes ◽  
Copy Number ◽  
Gene Copy Number ◽  
Single Copy ◽  
Digital Pcr ◽  
Gene Copy ◽  
Relative Quantification ◽  
Transgene Copy Number ◽  
Copy Number Estimation

Abstract Obtaining data on transgene copy number is an integral step in the generation of transgenic plants. Techniques such as Southern blot, segregation analysis, and quantitative PCR (qPCR) have routinely been used for this task, in a range of species. More recently, use of Digital PCR (dPCR) has become prevalent, with a measurement accuracy higher than qPCR reported. Here, the relative merits of qPCR and dPCR for transgene copy number estimation in white clover were investigated. Furthermore, given that single copy reference genes are desirable for estimating gene copy number by relative quantification, and that no single-copy genes have been reported in this species, a search and evaluation of suitable reference genes in white clover was undertaken. Results demonstrated a higher accuracy of dPCR relative to qPCR for copy number estimation in white clover. Two genes, Pyruvate dehydrogenase (PDH), and an ATP-dependent protease, identified as single-copy genes, were used as references for copy number estimation by relative quantification. Identification of single-copy genes in white clover will enable the application of relative quantification for copy number estimation of other genes or transgenes in the species. The results generated here validate the use of dPCR as a reliable strategy for transgene copy number estimation in white clover, and provide resources for future copy number studies in this species.

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