Accurately Detection of A-Globin Gene Copy Number Variants With Two Reactions Using Droplet Digital PCR

2021 ◽  
Author(s):  
Xiuqin Bao ◽  
Danqing Qin ◽  
Jian Ma ◽  
Xiangcheng Zhou ◽  
Jicheng Wang ◽  
...  
Keyword(s):  
Clinical Manifestation ◽  
Copy Number ◽  
Southern China ◽  
Globin Gene ◽  
Copy Number Variants ◽  
Chorionic Villus ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Globin Genes

Abstract Background: α-thalassemia, with carrier rates of 11.31% and 17.55% respectively in Guangdong and Guangxi province, is a highly prevalent disease in Southern China and tropical and subtropical regions and is mainly caused by deletion in α-globin gene (HBA1 and HBA2). The clinical manifestation of α-thalassemia is highly correlated with the copy number of α-globin gene. The decrease of copy number results in α-thalassemia, while duplication or triplication compounded with β-thalassemia may aggravate the clinical manifestation. However, the usual methods we used to measure the copy number variants can only detect the three common types: --SEA, -α3.7 and − α4.2, which may easily miss the rare deletional type and duplication or triplication cases. Therefore, it is essential to establish a new method which allows detection of different copy number variants in α-globin genes, including deletion, duplication and triplication simultaneously and accurately.Methods: 428 peripheral blood and fetal chorionic villus or amniotic fluid samples were recruited in this study. We used a pair of primers and two probes to perform droplet digital PCR.Results: By performing only two reactions, we accurately detected the copy number variants in α-globin genes, including the common form α-thalassemia, triplications such as αααanti4.2 and trisomy 16. The accuracy rate for detecting the copy number of α-globin genes can up to 100%.Conclusions: In conclusion, droplet digital PCR served as an accurate and rapid method for copy number variation detection in clinical screening for α-thalassemia.

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 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 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 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 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

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 High Olfactory Receptor-Rich 11q11 Copy Number in Girls and African American Children

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1943
Author(s):  
Megan Phillips ◽  
Vaithinathan Selvaraju ◽  
Anna Fouty ◽  
Jeganathan Ramesh Babu ◽  
Maninder Sandey ◽  
...  
Keyword(s):  
African American ◽  
Olfactory Receptor ◽  
Copy Number ◽  
Copy Number Variants ◽  
Digital Pcr ◽  
Normal Weight ◽  
World Health ◽  
Gene Copy ◽  
Complex Disorders ◽  
American Children

Copy number variants (CNVs) provide numerous genetic differences between individuals, and they have been linked with multiple human diseases. Obesity is one of the highly heritable complex disorders, which is associated with copy number variance (CNV). A recent report shows that the 11q11 gene, a novel olfactory receptor, and its copy number variants are involved in the early onset of obesity. In the current study, we analyzed the 11q11 gene copy number variance (CNV) based on gender in White/European American (EA) and African American (AA) normal weight and overweight/obese children. Sixty-nine boys and fifty-eight girls between the ages of 6 and 10 years belonging to either EA or AA ethnicity were involved in this study. As per World Health Organization (WHO) guidelines, each participant’s body weight and height were recorded. DNA was extracted from saliva, and the copy number variants for the 11q11 gene were measured using digital PCR. The descriptive analysis of the 11q11 copy number showed significantly more copies in girls compared to boys; similarly, AA participants had significantly increased CNV compared to EA. The normal weight (NW) and overweight/obese (OW/OB) girls were significantly less likely to belong to the low copy number variant (LCNV) group of 11q11 compared to boys; similarly, NW and OW/OB AA children were significantly less likely to belong to the LCNV group. The AA girls in LCNV had significantly higher BMI z-scores. Our findings suggest that the 11q11 copy number in children is race and gender-specific.

scholarly journals Chromosomal Instability Selects Gene Copy-Number Variants Encoding Core Regulators of Proliferation in ER+ Breast Cancer

2014 ◽  
Vol 74 (17) ◽  
pp. 4853-4863 ◽  
Author(s):  
David Endesfelder ◽  
Rebecca A. Burrell ◽  
Nnennaya Kanu ◽  
Nicholas McGranahan ◽  
Mike Howell ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Copy Number ◽  
Chromosomal Instability ◽  
Copy Number Variants ◽  
Gene Copy Number ◽  
Gene Copy

scholarly journals Accurate measurement of transgene copy number in crop plants using droplet digital PCR

2017 ◽  
Vol 90 (5) ◽  
pp. 1014-1025 ◽  
Author(s):  
Ray Collier ◽  
Kasturi Dasgupta ◽  
Yan-Ping Xing ◽  
Bryan Tarape Hernandez ◽  
Min Shao ◽  
...  
Keyword(s):  
Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Crop Plants ◽  
Transgene Copy Number

scholarly journals DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

2017 ◽  
Author(s):  
Elizabeth Nacheva ◽  
Katya Mokretar ◽  
Aynur Soenmez ◽  
Alan M Pittman ◽  
Colin Grace ◽  
...  
Keyword(s):  
Substantia Nigra ◽  
Human Brain ◽  
Frontal Cortex ◽  
Genome Sequencing ◽  
Copy Number ◽  
Dna Isolation ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Whole Genome ◽  
Salting Out

AbstractPotential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array “waves”, and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

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