Routine DNA testing

USDA-ARS?s Scientific Manuscript database

Routine DNA testing. It’s done once you’ve Marker-Assisted Breeding Pipelined promising Qantitative Trait Loci within your own breeding program and thereby established the performance-predictive power of each DNA test for your germplasm under your conditions. By then you are ready to screen your par...

The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

PubMed

Andoh, Kiyohiko; Hattori, Shiho; Mahmoud, Hassan Y A H; Takasugi, Maaya; Shimoda, Hiroshi; Bannai, Hiroshi; Tsujimura, Koji; Matsumura, Tomio; Kondo, Takashi; Kirisawa, Rikio; Mochizuki, Masami; Maeda, Ken

2015-01-02

Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA testing in neurologic diseases.

PubMed

O'Brien, D P; Leeb, T

2014-01-01

DNA testing is available for a growing number of hereditary diseases in neurology and other specialties. In addition to guiding breeding decisions, DNA tests are important tools in the diagnosis of diseases, particularly in conditions for which clinical signs are relatively nonspecific. DNA testing also can provide valuable insight into the risk of hereditary disease when decisions about treating comorbidities are being made. Advances in technology and bioinformatics will make broad screening for potential disease-causing mutations available soon. As DNA tests come into more common use, it is critical that clinicians understand the proper application and interpretation of these test results. Copyright © 2014 by the American College of Veterinary Internal Medicine.

DNA testing in homicide investigations.

PubMed

Prahlow, Joseph A; Cameron, Thomas; Arendt, Alexander; Cornelis, Kenneth; Bontrager, Anthony; Suth, Michael S; Black, Lisa; Tobey, Rebbecca; Pollock, Sharon; Stur, Shawn; Cotter, Kenneth; Gabrielse, Joel

2017-10-01

Objectives With the widespread use of DNA testing, police, death investigators, and attorneys need to be aware of the capabilities of this technology. This review provides an overview of scenarios where DNA evidence has played a major role in homicide investigations in order to highlight important educational issues for police, death investigators, forensic pathologists, and attorneys. Methods This was a nonrandom, observational, retrospective study. Data were obtained from the collective files of the authors from casework during a 15-year period, from 2000 through 2014. Results A series of nine scenarios, encompassing 11 deaths, is presented from the standpoint of the police and death investigation, the forensic pathology autopsy performance, the subsequent DNA testing of evidence, and, ultimately, the final adjudication of cases. Details of each case are presented, along with a discussion that focuses on important aspects of sample collection for potential DNA testing, especially at the crime scene and the autopsy. The presentation highlights the diversity of case and evidence types in which DNA testing played a valuable role in the successful prosecution of the case. Conclusions By highlighting homicides where DNA testing contributed to the successful adjudication of cases, police, death investigators, forensic pathologists, and attorneys will be better informed regarding the types of evidence and situations where such testing is of potential value.

Clinical value of the Treponema pallidum haemagglutination test.

PubMed

Robertson, D H; McMillan, A

1975-04-01

In 1,129 patients attending the Department for Sexually Transmitted Diseases, the serum was examined by three screening tests (VDRL slide, RPCF, and TPHA) and twelve cases of syphilis (1 per cent. of patients attenting the clinic) were discovered. Six of these patients were considered to have latent syphilis (5 acquired, 1 congenital) and were detected only by the TPHA; all six cases were confirmed by the FTA-ABS, The TPHA failed to detect three of the remaining six cases (2 primary and 1 very early, the latter in a contact of a patient with primary syphilis). All six cases were, however, detected by the VDRL. In seven cases, the TPHA was positive in the absence of other evidence of present or previous syphilis. In these cases the FTA-ABS was also negative. The clinical application of the TPHA test in the detection of syphilis is discussed.

Cell-free DNA testing after combined test: factors affecting the uptake.

PubMed

Maiz, Nerea; Alzola, Irune; Murua, Emerson J; Rodríguez Santos, Javier

2016-11-01

First, to assess what was the uptake of cell free DNA (cfDNA) testing after a combined test and the maternal and fetal factors that influenced this decision, and second, to assess the uptake and factors that influence the choice of invasive testing. This observational retrospective study included 1083 singleton pregnancies who had a combined test for screening for Down syndrome between 11 (+) (0) and 13 (+) (6) weeks. Multivariate logistic regression analysis was used to determine which factors affected the uptake of cfDNA test and invasive testing among risk for trisomies 21, 18, and 13, maternal characteristics and fetal nuchal translucency (NT) thickness. Two-hundred fifty-seven (23.7%) women had a cfDNA test, 89 (8.2%) had an invasive test, and 737 (68.1%) had no further test. The uptake of cfDNA increased with the risk for trisomies (p < 0.001), maternal age (p = 0.013), and was higher in nulliparous women (p = 0.004). The uptake of invasive test increased with the risk for trisomies (p < 0.001) and NT thickness (p < 0.001). This study shows that the uptake of cfDNA testing increases with the risk for trisomies, maternal age, and is higher in nulliparous, whereas the uptake of invasive testing increases with the risk for trisomies and NT thickness.

Strategies for Implementing Cell-Free DNA Testing.

PubMed

Cuckle, Howard

2016-06-01

Maternal plasma cell-free (cf) DNA testing has higher discriminatory power for aneuploidy than any conventional multi-marker screening test. Several strategies have been suggested for introducing it into clinical practice. Secondary cfDNA, restricted only to women with positive conventional screening test, is generally cost saving and minimizes the need for invasive prenatal diagnosis but leads to a small loss in detection. Primary cfDNA, replacing conventional screening or retaining the nuchal translucency scan, is not currently cost-effective for third-party payers. Contingent cfDNA, testing about 20% of women with the highest risks based on a conventional test, is the preferred approach. Copyright © 2016 Elsevier Inc. All rights reserved.

Syphilis screening in the Blood Transfusion Service: a report of four years' experience with the Treponema pallidum haemagglutination assay and the subsequent development of a rapid "spin" method.

PubMed Central

Puckett, A; Pratt, G

1987-01-01

A comparison of cardiolipin and a modified Treponema pallidum haemagglutination assay (TPHA) method over a four year period confirmed the superior sensitivity and specificity of TPHA. In 86,495 new donor sera 19 (0.02%) confirmed positive results were detected by TPHA, 10 of which did not react by the cardiolipin test. In 150,789 antenatal samples 49 confirmed positive results were found by TPHA, 30 of which did not react by cardiolipin. No cardiolipin positive, TPHA negative samples were confirmed as positive by the absorbed fluorescence treponemal antibody test, and overall 78% of cardiolipin reactions gave false biological positive results. Cardiolipin tests were continued only because of their speed. A further modification ("spin") of the TPHA has now been developed which is rapid, sensitive, and inexpensive, and in testing 21,807 sera, gave results equivalent to those of the previous "settle" method. Serious consideration should be given to dispensing with cardiolipin tests. PMID:3320095

Forensic aspects of DNA-based human identity testing.

PubMed

Roper, Stephen M; Tatum, Owatha L

2008-01-01

The forensic applications of DNA-based human identity laboratory testing are often underappreciated. Molecular biology has seen an exponential improvement in the accuracy and statistical power provided by identity testing in the past decade. This technology, dependent upon an individual's unique DNA sequence, has cemented the use of DNA technology in the forensic laboratory. This paper will discuss the state of modern DNA-based identity testing, describe the technology used to perform this testing, and describe its use as it relates to forensic applications. We will also compare individual technologies, including polymerase chain reaction (PCR) and Southern Blotting, that are used to detect the molecular differences that make all individuals unique. An increasing reliance on DNA-based identity testing dictates that healthcare providers develop an understanding of the background, techniques, and guiding principles of this important forensic tool.

Multitarget stool DNA testing for colorectal-cancer screening.

PubMed

Imperiale, Thomas F; Ransohoff, David F; Itzkowitz, Steven H; Levin, Theodore R; Lavin, Philip; Lidgard, Graham P; Ahlquist, David A; Berger, Barry M

2014-04-03

An accurate, noninvasive test could improve the effectiveness of colorectal-cancer screening. We compared a noninvasive, multitarget stool DNA test with a fecal immunochemical test (FIT) in persons at average risk for colorectal cancer. The DNA test includes quantitative molecular assays for KRAS mutations, aberrant NDRG4 and BMP3 methylation, and β-actin, plus a hemoglobin immunoassay. Results were generated with the use of a logistic-regression algorithm, with values of 183 or more considered to be positive. FIT values of more than 100 ng of hemoglobin per milliliter of buffer were considered to be positive. Tests were processed independently of colonoscopic findings. Of the 9989 participants who could be evaluated, 65 (0.7%) had colorectal cancer and 757 (7.6%) had advanced precancerous lesions (advanced adenomas or sessile serrated polyps measuring ≥1 cm in the greatest dimension) on colonoscopy. The sensitivity for detecting colorectal cancer was 92.3% with DNA testing and 73.8% with FIT (P=0.002). The sensitivity for detecting advanced precancerous lesions was 42.4% with DNA testing and 23.8% with FIT (P<0.001). The rate of detection of polyps with high-grade dysplasia was 69.2% with DNA testing and 46.2% with FIT (P=0.004); the rates of detection of serrated sessile polyps measuring 1 cm or more were 42.4% and 5.1%, respectively (P<0.001). Specificities with DNA testing and FIT were 86.6% and 94.9%, respectively, among participants with nonadvanced or negative findings (P<0.001) and 89.8% and 96.4%, respectively, among those with negative results on colonoscopy (P<0.001). The numbers of persons who would need to be screened to detect one cancer were 154 with colonoscopy, 166 with DNA testing, and 208 with FIT. In asymptomatic persons at average risk for colorectal cancer, multitarget stool DNA testing detected significantly more cancers than did FIT but had more false positive results. (Funded by Exact Sciences; ClinicalTrials.gov number, NCT01397747.).

Antenatal noninvasive DNA testing: clinical experience and impact.

PubMed

Ferres, Millie A; Hui, Lisa; Bianchi, Diana W

2014-08-01

Nearly two decades ago, the discovery of circulating cell-free fetal DNA in maternal blood created a paradigm shift in prenatal testing. Recent advances in DNA sequencing technology have facilitated the rapid translation of DNA-based testing into clinical antenatal care. In this review, we summarize the technical approaches and current clinical applications of noninvasive testing using cell-free DNA in maternal plasma. We discuss the impact of these tests on clinical care, outline proposed integration models, and suggest future directions for the field. The use of cell-free DNA in maternal blood for the detection of fetal rhesus D antigen status, fetal sex, and common whole chromosomal aneuploidies is now well established, although testing for aneuploidy is still considered screening and not diagnostic. Further advances in technology and bioinformatics may see future clinical applications extend to the noninvasive detection of fetal subchromosomal aneuploidy, single gene disorders, and the entire fetal genome. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Direct-to-consumer DNA testing and the GP.

PubMed

Trent, Ronald

2014-07-01

From early 2000 a new form of DNA genetic testing became available commercially. It bypasses the medical practitioner and can be ordered directly by the individual. To understand direct-to-consumer (DTC) DNA genetic testing and be able to respond appropriately if asked to be involved by a patient. Presently, all but one or two DTC DNA genetic testing laboratories are located outside Australia. The industry promotes itself as a means to better health through giving individuals complete control over their results. When communicating with patients about DTC DNA genetic testing, general practitioners will need to make a determination about the clinical utility of the test and the laboratory validity, both of which can be difficult in the current environment. Assistance may be available through public hospital clinical genetics services, although waiting times can be long. There is likely to be tighter regulation of these types of testing in the future, which may include involvement of medical practitioners.

A survey of the nutritional and haemagglutination properties of legume seeds generally available in the UK.

PubMed

Grant, G; More, L J; McKenzie, N H; Stewart, J C; Pusztai, A

1983-09-01

Eighty-five samples from fifteen different legume seed lines generally available in the UK were examined by measurements of their net protein utilization by rats and by haemagglutination tests with erythrocytes from a number of different animal species. From these results the seeds were classified into four broad groups. Group a seeds from most varieties of kidney (Phaseolus vulgaris), runner (Phaseolus coccineus) and tepary (Phaseolus acutifolius) beans showed high reactivity with all cell types and were also highly toxic. Group b, which contained seeds from lima or butter beans (Phaseolus lunatus) and winged bean (Psophocarpus tetragonolobus), agglutinated only human and pronase-treated rat erythrocytes. These seeds did not support proper growth of the rats although the animals survived the 10 d experimental period. Group c consisted of seeds from lentils (Lens culinaris), peas (Pisum sativum), chick-peas (Cicer arietinum), blackeyed peas (Vigna sinensis), pigeon peas (Cajanus cajan), mung beans (Phaseolus aureus), field or broad beans (Vicia faba) and aduki beans (Phaseolus angularis). These generally had low reactivity with all cells and were non-toxic. Group d, represented by soya (Glycine max) and pinto (Phaseolus vulgaris) beans, generally had low reactivity with all cells but caused growth depression at certain dietary concentrations. This growth depression was probably mainly due to antinutritional factors other than lectins. Lectins from group a seeds showed many structural and immunological similarities. However the subunit composition of the lectin from the tepary bean samples was different from that of the other bean lectins in this or any other groups.

Paternity testing that involves a DNA mixture.

PubMed

Mortera, Julia; Vecchiotti, Carla; Zoppis, Silvia; Merigioli, Sara

2016-07-01

Here we analyse a complex disputed paternity case, where the DNA of the putative father was extracted from his corpse that had been inhumed for over 20 years. This DNA was contaminated and appears to be a mixture of at least two individuals. Furthermore, the mother's DNA was not available. The DNA mixture was analysed so as to predict the most probable genotypes of each contributor. The major contributor's profile was then used to compute the likelihood ratio for paternity. We also show how to take into account a dropout allele and the possibility of mutation in paternity testing. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

PubMed

Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B M; Cornel, Martina C; Sistermans, Erik A

2016-01-01

Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA. © 2016 Elsevier Inc. All rights reserved.

Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing.

PubMed

Yu, Stephanie C Y; Chan, K C Allen; Zheng, Yama W L; Jiang, Peiyong; Liao, Gary J W; Sun, Hao; Akolekar, Ranjit; Leung, Tak Y; Go, Attie T J I; van Vugt, John M G; Minekawa, Ryoko; Oudejans, Cees B M; Nicolaides, Kypros H; Chiu, Rossa W K; Lo, Y M Dennis

2014-06-10

Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.

How good are indirect tests at detecting recombination in human mtDNA?

PubMed

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-07-08

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D' and r(2), Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ(2)) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7-70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed.

How Good Are Indirect Tests at Detecting Recombination in Human mtDNA?

PubMed Central

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-01-01

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D′ and r2, Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ2) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7−70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed. PMID:23665874

The Stool DNA Test is More Accurate than the Plasma Septin 9 Test in Detecting Colorectal Neoplasia

PubMed Central

Ahlquist, David A.; Taylor, William R.; Mahoney, Douglas W.; Zou, Hongzhi; Domanico, Michael; Thibodeau, Stephen N.; Boardman, Lisa A.; Berger, Barry M.; Lidgard, Graham P.

2014-01-01

Background & Aims Several noninvasive tests have been developed for colorectal cancer (CRC) screening. We compared the sensitivities of a multi-marker test for stool DNA (sDNA) and a plasma test for methylated Septin 9 (SEPT9) in identifying patients with large adenomas or CRC. Methods We analyzed paired stool and plasma samples from 30 patients with CRC and 22 with large adenomas from Mayo Clinic archives. Stool (n=46) and plasma (n=49) samples from age- and sex-matched patients with normal colonoscopy results were used as controls. The sDNA test is an assay for methylated BMP3, NDRG4, vimentin, and TFPI2; mutant KRAS; the β-actin gene, and quantity of hemoglobin (by the porphyrin method). It was performed blindly at Exact Sciences (Madison WI); the test for SEPT9 was performed at ARUP Laboratories (Salt Lake City UT). Results were considered positive based on the manufacturer's specificity cutoff values of 90% and 89%, respectively. Results The sDNA test detected adenomas (median 2 cm, range 1–5 cm) with 82% sensitivity (95% confidence interval [CI], 60%–95%); SEPT9 had 14% sensitivity (95% CI, 3%–35%; P=.0001). The sDNA test identified patients with CRC with 87% sensitivity (95% CI, 69%–96%); SEPT9 had 60% sensitivity (95% CI, 41%–77%; P=.046). The sDNA test identified patients with stage I–III CRC with 91% sensitivity (95% CI, 71%–99%); SEPT9 had 50% sensitivity (95% CI, 28%–72%; P=.013); for stage IV CRC, sensitivity values were 75% (95% CI, 35%–97%) and 88% (95% CI, 47%–100%), respectively (P=.56). False-positive rates were 7% for the sDNA test and 27% for SEPT9. Conclusions Based on analyses of paired samples, the sDNA test detects non-metastatic CRC and large adenomas with significantly greater levels of sensitivity than the SEPT9 test. These findings might be used to modify approaches for CRC prevention and early detection. PMID:22019796

Non-invasive prenatal testing using cell-free fetal DNA in maternal circulation.

PubMed

Liao, Gary J W; Gronowski, Ann M; Zhao, Zhen

2014-01-20

The identification of cell-free fetal DNA (cffDNA) in maternal circulation has made non-invasive prenatal testing (NIPT) possible. Maternal plasma cell free DNA is a mixture of maternal and fetal DNA, of which, fetal DNA represents a minor population in maternal plasma. Therefore, methods with high sensitivity and precision are required to detect and differentiate fetal DNA from the large background of maternal DNA. In recent years, technical advances in the molecular analysis of fetal DNA (e.g., digital PCR and massively parallel sequencing (MPS)) has enabled the successful implementation of noninvasive testing into clinical practice, such as fetal sex assessment, RhD genotyping, and fetal chromosomal aneuploidy detection.With the ability to decipher the entire fetal genome from maternal plasma DNA, we foresee that an increased number of non-invasive prenatal tests will be available for detecting many single-gene disorders in the near future. This review briefly summarizes the technical aspects of the NIPT and application of NIPT in clinical practice.

Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing

PubMed Central

Yu, Stephanie C. Y.; Chan, K. C. Allen; Zheng, Yama W. L.; Jiang, Peiyong; Liao, Gary J. W.; Sun, Hao; Akolekar, Ranjit; Leung, Tak Y.; Go, Attie T. J. I.; van Vugt, John M. G.; Minekawa, Ryoko; Oudejans, Cees B. M.; Nicolaides, Kypros H.; Chiu, Rossa W. K.; Lo, Y. M. Dennis

2014-01-01

Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring. PMID:24843150

Unique DNA-barcoded aerosol test particles for studying aerosol transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harding, Ruth N.; Hara, Christine A.; Hall, Sara B.

Data are presented for the first use of novel DNA-barcoded aerosol test particles that have been developed to track the fate of airborne contaminants in populated environments. Until DNATrax (DNA Tagged Reagents for Aerosol eXperiments) particles were developed, there was no way to rapidly validate air transport models with realistic particles in the respirable range of 1–10 μm in diameter. The DNATrax particles, developed at Lawrence Livermore National Laboratory (LLNL) and tested with the assistance of the Pentagon Force Protection Agency, are the first safe and effective materials for aerosol transport studies that are identified by DNA molecules. The usemore » of unique synthetic DNA barcodes overcomes the challenges of discerning the test material from pre-existing environmental or background contaminants (either naturally occurring or previously released). The DNATrax particle properties are demonstrated to have appropriate size range (approximately 1–4.5 μm in diameter) to accurately simulate bacterial spore transport. As a result, we describe details of the first field test of the DNATrax aerosol test particles in a large indoor facility.« less

Unique DNA-barcoded aerosol test particles for studying aerosol transport

DOE PAGES

Harding, Ruth N.; Hara, Christine A.; Hall, Sara B.; ...

2016-03-22

Data are presented for the first use of novel DNA-barcoded aerosol test particles that have been developed to track the fate of airborne contaminants in populated environments. Until DNATrax (DNA Tagged Reagents for Aerosol eXperiments) particles were developed, there was no way to rapidly validate air transport models with realistic particles in the respirable range of 1–10 μm in diameter. The DNATrax particles, developed at Lawrence Livermore National Laboratory (LLNL) and tested with the assistance of the Pentagon Force Protection Agency, are the first safe and effective materials for aerosol transport studies that are identified by DNA molecules. The usemore » of unique synthetic DNA barcodes overcomes the challenges of discerning the test material from pre-existing environmental or background contaminants (either naturally occurring or previously released). The DNATrax particle properties are demonstrated to have appropriate size range (approximately 1–4.5 μm in diameter) to accurately simulate bacterial spore transport. As a result, we describe details of the first field test of the DNATrax aerosol test particles in a large indoor facility.« less

Relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: development and application of a bayesian random-effects model.

PubMed

Coudeville, Laurent; Bailleux, Fabrice; Riche, Benjamin; Megas, Françoise; Andre, Philippe; Ecochard, René

2010-03-08

Antibodies directed against haemagglutinin, measured by the haemagglutination inhibition (HI) assay are essential to protective immunity against influenza infection. An HI titre of 1:40 is generally accepted to correspond to a 50% reduction in the risk of contracting influenza in a susceptible population, but limited attempts have been made to further quantify the association between HI titre and protective efficacy. We present a model, using a meta-analytical approach, that estimates the level of clinical protection against influenza at any HI titre level. Source data were derived from a systematic literature review that identified 15 studies, representing a total of 5899 adult subjects and 1304 influenza cases with interval-censored information on HI titre. The parameters of the relationship between HI titre and clinical protection were estimated using Bayesian inference with a consideration of random effects and censorship in the available information. A significant and positive relationship between HI titre and clinical protection against influenza was observed in all tested models. This relationship was found to be similar irrespective of the type of viral strain (A or B) and the vaccination status of the individuals. Although limitations in the data used should not be overlooked, the relationship derived in this analysis provides a means to predict the efficacy of inactivated influenza vaccines when only immunogenicity data are available. This relationship can also be useful for comparing the efficacy of different influenza vaccines based on their immunological profile.

Relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: development and application of a bayesian random-effects model

PubMed Central

2010-01-01

Background Antibodies directed against haemagglutinin, measured by the haemagglutination inhibition (HI) assay are essential to protective immunity against influenza infection. An HI titre of 1:40 is generally accepted to correspond to a 50% reduction in the risk of contracting influenza in a susceptible population, but limited attempts have been made to further quantify the association between HI titre and protective efficacy. Methods We present a model, using a meta-analytical approach, that estimates the level of clinical protection against influenza at any HI titre level. Source data were derived from a systematic literature review that identified 15 studies, representing a total of 5899 adult subjects and 1304 influenza cases with interval-censored information on HI titre. The parameters of the relationship between HI titre and clinical protection were estimated using Bayesian inference with a consideration of random effects and censorship in the available information. Results A significant and positive relationship between HI titre and clinical protection against influenza was observed in all tested models. This relationship was found to be similar irrespective of the type of viral strain (A or B) and the vaccination status of the individuals. Conclusion Although limitations in the data used should not be overlooked, the relationship derived in this analysis provides a means to predict the efficacy of inactivated influenza vaccines when only immunogenicity data are available. This relationship can also be useful for comparing the efficacy of different influenza vaccines based on their immunological profile. PMID:20210985

Application of forensic DNA testing in the legal system.

PubMed

Primorac, D; Schanfield, M S

2000-03-01

DNA technology has taken an irreplaceable position in the field of the forensic sciences. Since 1985, when Peter Gill and Alex Jeffreys first applied DNA technology to forensic problems, to the present, more than 50,000 cases worldwide have been solved through the use of DNA based technology. Although the development of DNA typing in forensic science has been extremely rapid, today we are witnessing a new era of DNA technology including automation and miniaturization. In forensic science, DNA analysis has become "the new form of scientific evidence" and has come under public scrutiny and the demand to show competence. More and more courts admit the DNA based evidence. We believe that in the near future this technology will be generally accepted in the legal system. There are two main applications of DNA analysis in forensic medicine: criminal investigation and paternity testing. In this article we present background information on DNA, human genetics, and the application of DNA analysis to legal problems, as well as the commonly applied respective mathematics.

Can indirect tests detect a known recombination event in human mtDNA?

PubMed

White, Daniel James; Gemmell, Neil John

2009-07-01

Whether human mitochondrial DNA (mtDNA) recombines sufficiently to influence its evolution, evolutionary analysis, and disease etiology, remains equivocal. Overall, evidence from indirect studies of population genetic data suggests that recombination is not occurring at detectable levels. This may be explained by no, or low, recombination or, alternatively, current indirect tests may be incapable of detecting recombination in human mtDNA. To investigate the latter, we have tested whether six well-established indirect tests of recombination could detect recombination in a human mtDNA data set, in which its occurrence had been empirically confirmed. Three showed statistical evidence for recombination (r(2) vs. distance, the Homoplasy test, Neighborhood Similarity Score), and three did not (D' vs. distance, Max Chi Squared, Pairwise Homoplasy Index). Possible reasons for detection failure are discussed. Further, evidence from earlier studies suggesting a lack of recombination in mtDNA in humans is reconsidered, taking into account the appropriateness of the tests used, based on our new findings.

The use of acid phosphatase test papers for DNA profiling.

PubMed

Reshef, A; Barash, M; Gallili, N; Michael, A; Brauner, P

2005-01-01

The acid phosphatase (AP) test is a routine assay used to screen casework items for the possible presence of semen. This colour test is carried out on filter paper which is retained after testing. Two-year-old AP test papers were found to contain sufficient DNA for short tandem repeat (STR) profiling. Prior to polymerase chain reaction (PCR) amplification, the DNA was preferentially separated into sperm depleted and sperm enriched cell fractions. The implication of these findings for past and present cases is discussed.

Post-conviction DNA testing: the UK's first ‘exoneration’ case?

PubMed Central

Johnson, Paul; Williams, Robin

2005-01-01

The routine incorporation of forensic DNA profiling into the criminal justice systems of the United Kingdom has been widely promoted as a device for improving the quality of investigative and prosecutorial processes. From its first uses in the 1980s, in cases of serious crime, to the now daily collection, analysis and comparison of genetic samples in the National DNA Database, DNA profiling has become a standard instrument of policing and a powerful evidential resource for prosecutors. However, the use of post-conviction DNA testing has, until recently, been uncommon in the United Kingdom. This paper explores the first case, in England, of the contribution of DNA profiling to a successful appeal against conviction by an imprisoned offender. Analysis of the details of this case is used to emphasise the ways in which novel forms of scientific evidence remain subject to traditional and heterogeneous tests of relevance and credibility. PMID:15112595

Stakeholder attitudes and needs regarding cell-free fetal DNA testing.

PubMed

Hill, Melissa; Lewis, Celine; Chitty, Lyn S

2016-04-01

To explore stakeholder views on cell-free DNA testing and highlight findings important for successful implementation and the provision of best practice in counseling. Noninvasive tests based on the analysis of cell-free fetal DNA are now widely available in clinical practice and applications are expanding rapidly. It is essential that stakeholder views are considered in order to identify and address any ethical and social issues. We provide an overview of stakeholder viewpoints and then focus on the key issues of informed decision making, test uptake, service delivery and information sources. Stakeholders are positive about the introduction of cell-free fetal DNA testing into clinical practice. They describe both practical and psychological benefits arising from tests that are safe and can potentially be performed earlier in pregnancy. Key concerns, which include the potential for these tests to have a negative impact on informed decision making and increased societal pressure to have testing, can be addressed through careful parent-directed counseling. As applications for these tests expand it is increasingly important to develop innovative approaches to facilitate good understanding for parents who are offered noninvasive prenatal testing.

DNA nanotechnology from the test tube to the cell.

PubMed

Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A; Seelig, Georg

2015-09-01

The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology - applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems - lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

Clinical perspective of cell-free DNA testing for fetal aneuploidies.

PubMed

Gratacós, Eduard; Nicolaides, Kypros

2014-01-01

Cell-free DNA testing in maternal blood provides the most effective method of screening for trisomy 21, with a reported detection rate of 99% and a false positive rate of less than 0.1%. After many years of research, this method is now commercially available and is carried out in an increasing number of patients, and there is an expanding number of conditions that can be screened for. However, the application of these methods in clinical practice requires a careful analysis. Current first-trimester screening strategies are based on a complex combination of tests, aiming at detecting fetal defects and predicting the risk of main pregnancy complications. It is therefore necessary to define the optimal way of combining cell-free DNA testing with current first-trimester screening methods. In this concise review we describe the basis of cell-free DNA testing and discuss the potential approaches for its implementation in combination with current tests in the first trimester. © 2014 S. Karger AG, Basel.

Analytical validation of BRAF mutation testing from circulating free DNA using the amplification refractory mutation testing system.

PubMed

Aung, Kyaw L; Donald, Emma; Ellison, Gillian; Bujac, Sarah; Fletcher, Lynn; Cantarini, Mireille; Brady, Ged; Orr, Maria; Clack, Glen; Ranson, Malcolm; Dive, Caroline; Hughes, Andrew

2014-05-01

BRAF mutation testing from circulating free DNA (cfDNA) using the amplification refractory mutation testing system (ARMS) holds potential as a surrogate for tumor mutation testing. Robust assay validation is needed to establish the optimal clinical matrix for measurement and cfDNA-specific mutation calling criteria. Plasma- and serum-derived cfDNA samples from 221 advanced melanoma patients were analyzed for BRAF c.1799T>A (p.V600E) mutation using ARMS in two stages in a blinded fashion. cfDNA-specific mutation calling criteria were defined in stage 1 and validated in stage 2. cfDNA concentrations in serum and plasma, and the sensitivities and specificities of BRAF mutation detection in these two clinical matrices were compared. Sensitivity of BRAF c.1799T>A (p.V600E) mutation detection in cfDNA was increased by using mutation calling criteria optimized for cfDNA (these criteria were adjusted from those used for archival tumor biopsies) without compromising specificity. Sensitivity of BRAF mutation detection in serum was 44% (95% CI, 35% to 53%) and in plasma 52% (95% CI, 43% to 61%). Specificity was 96% (95% CI, 90% to 99%) in both matrices. Serum contains significantly higher total cfDNA than plasma, whereas the proportion of tumor-derived mutant DNA was significantly higher in plasma. Using mutation calling criteria optimized for cfDNA improves sensitivity of BRAF c.1799T>A (p.V600E) mutation detection. The proportion of tumor-derived cfDNA in plasma was significantly higher than in serum. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Early Adoption of a Multitarget Stool DNA Test for Colorectal Cancer Screening.

PubMed

Finney Rutten, Lila J; Jacobson, Robert M; Wilson, Patrick M; Jacobson, Debra J; Fan, Chun; Kisiel, John B; Sweetser, Seth; Tulledge-Scheitel, Sidna M; St Sauver, Jennifer L

2017-05-01

To characterize early adoption of a novel multitarget stool DNA (MT-sDNA) screening test for colorectal cancer (CRC) screening and to test the hypothesis that adoption differs by demographic characteristics and prior CRC screening behavior and proceeds predictably over time. We used the Rochester Epidemiology Project research infrastructure to assess the use of the MT-sDNA screening test in adults aged 50 to 75 years living in Olmsted County, Minnesota, in 2014 and identified 27,147 individuals eligible or due for screening colonoscopy from November 1, 2014, through November 30, 2015. We used electronic Current Procedure Terminology and Health Care Common Procedure codes to evaluate early adoption of the MT-sDNA screening test in this population and to test whether early adoption varies by age, sex, race, and prior CRC screening behavior. Overall, 2193 (8.1%) and 974 (3.6%) individuals were screened by colonoscopy and MT-sDNA, respectively. Age, sex, race, and prior CRC screening behavior were significantly and independently associated with MT-sDNA screening use compared with colonoscopy use after adjustment for all other variables (P<.05 for all). The rates of adoption of MT-sDNA screening increased over time and were highest in those aged 50 to 54 years, women, whites, and those who had a history of screening. The use of the MT-sDNA screening test varied predictably by insurance coverage. The rates of colonoscopy decreased over time, whereas overall CRC screening rates remained steady. The results of the present study are generally consistent with predictions derived from prior research and the diffusion of innovation framework, pointing to increasing use of the new screening test over time and early adoption by younger patients, women, whites, and those with prior CRC screening. Copyright © 2017 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.

Positive cell-free fetal DNA testing for trisomy 13 reveals confined placental mosaicism.

PubMed

Hall, April L; Drendel, Holli M; Verbrugge, Jennifer L; Reese, Angela M; Schumacher, Katherine L; Griffith, Christopher B; Weaver, David D; Abernathy, Mary P; Litton, Christian G; Vance, Gail H

2013-09-01

We report on a case in which cell-free fetal DNA was positive for trisomy 13 most likely due to confined placental mosaicism. Cell-free fetal DNA testing analyzes DNA derived from placental trophoblast cells and can lead to incorrect results that are not representative of the fetus. We sought to confirm commercial cell-free fetal DNA testing results by chorionic villus sampling and amniocentesis. These results were followed up by postnatal chromosome analysis of cord blood and placental tissue. First-trimester cell-free fetal DNA test results were positive for trisomy 13. Cytogenetic analysis of chorionic villus sampling yielded a mosaic karyotype of 47,XY,+13[10]/46,XY[12]. G-banded analysis of amniotic fluid was normal, 46,XY. Postnatal cytogenetic analysis of cord blood was normal. Karyotyping of tissues from four quadrants of the placenta demonstrated mosaicism for trisomy 13 in two of the quadrants and a normal karyotype in the other two. Our case illustrates several important aspects of this new testing methodology: that cell-free fetal DNA may not be representative of the fetal karyotype; that follow-up with diagnostic testing of chorionic villus sampling and/or amniotic fluid for abnormal test results should be performed; and that pretest counseling regarding the full benefits, limitations, and possible testing outcomes of cell-free fetal DNA screening is important.

DNA-testing for BRCA1/2 prior to genetic counselling in patients with breast cancer: design of an intervention study, DNA-direct.

PubMed

Sie, Aisha S; Spruijt, Liesbeth; van Zelst-Stams, Wendy A G; Mensenkamp, Arjen R; Ligtenberg, Marjolijn J; Brunner, Han G; Prins, Judith B; Hoogerbrugge, Nicoline

2012-05-08

Current practice for patients with breast cancer referred for genetic counseling, includes face-to-face consultations with a genetic counselor prior to and following DNA-testing. This is based on guidelines regarding Huntington's disease in anticipation of high psychosocial impact of DNA-testing for mutations in BRCA1/2 genes. The initial consultation covers generic information regarding hereditary breast cancer and the (im)possibilities of DNA-testing, prior to such testing. Patients with breast cancer may see this information as irrelevant or unnecessary because individual genetic advice depends on DNA-test results. Also, verbal information is not always remembered well by patients. A different format for this information prior to DNA-testing is possible: replacing initial face-to-face genetic counseling (DNA-intake procedure) by telephone, written and digital information sent to patients' homes (DNA-direct procedure). In this intervention study, 150 patients with breast cancer referred to the department of Clinical Genetics of the Radboud University Nijmegen Medical Centre are given the choice between two procedures, DNA-direct (intervention group) or DNA-intake (usual care, control group). During a triage telephone call, patients are excluded if they have problems with Dutch text, family communication, or of psychological or psychiatric nature. Primary outcome measures are satisfaction and psychological distress. Secondary outcome measures are determinants for the participant's choice of procedure, waiting and processing times, and family characteristics. Data are collected by self-report questionnaires at baseline and following completion of genetic counseling. A minority of participants will receive an invitation for a 30 min semi-structured telephone interview, e.g. confirmed carriers of a BRCA1/2 mutation, and those who report problems with the procedure. This study compares current practice of an intake consultation (DNA-intake) to a home informational

[Hot topics of circulating tumor DNA testing in breast cancer].

PubMed

Liu, Y H; Zhou, B; Xu, L; Xin, L

2017-02-01

The progress of gene detection technologies represented by next generation sequencing (NGS) and digital PCR laid a foundation for studies of circulating tumor DNA (ctDNA) in breast cancer. In 2014, the NGS workgroup organized by the College of American Pathologists (CAP) published the College of American Pathologists ' Laboratory Standards for Next - Generation Sequencing Clinical Tests, which provides a blueprint for the standardization of gene testing. In 2015, the Guidelines for Diagnostic Next - generation Sequencing published by the European Society of Human Genetics claimed that NGS is unacceptable in clinical practice before studies guided by guidelines are approved. Although existing studies show the benefits of ctDNA testing in disease monitoring and prognosis analyzing, we have a ways to go to normalize the procedure and build strict detection criteria.

Bioinformatics Approaches for Fetal DNA Fraction Estimation in Noninvasive Prenatal Testing

PubMed Central

Peng, Xianlu Laura; Jiang, Peiyong

2017-01-01

The discovery of cell-free fetal DNA molecules in plasma of pregnant women has created a paradigm shift in noninvasive prenatal testing (NIPT). Circulating cell-free DNA in maternal plasma has been increasingly recognized as an important proxy to detect fetal abnormalities in a noninvasive manner. A variety of approaches for NIPT using next-generation sequencing have been developed, which have been rapidly transforming clinical practices nowadays. In such approaches, the fetal DNA fraction is a pivotal parameter governing the overall performance and guaranteeing the proper clinical interpretation of testing results. In this review, we describe the current bioinformatics approaches developed for estimating the fetal DNA fraction and discuss their pros and cons. PMID:28230760

Bioinformatics Approaches for Fetal DNA Fraction Estimation in Noninvasive Prenatal Testing.

PubMed

Peng, Xianlu Laura; Jiang, Peiyong

2017-02-20

The discovery of cell-free fetal DNA molecules in plasma of pregnant women has created a paradigm shift in noninvasive prenatal testing (NIPT). Circulating cell-free DNA in maternal plasma has been increasingly recognized as an important proxy to detect fetal abnormalities in a noninvasive manner. A variety of approaches for NIPT using next-generation sequencing have been developed, which have been rapidly transforming clinical practices nowadays. In such approaches, the fetal DNA fraction is a pivotal parameter governing the overall performance and guaranteeing the proper clinical interpretation of testing results. In this review, we describe the current bioinformatics approaches developed for estimating the fetal DNA fraction and discuss their pros and cons.

Attitudes to predictive DNA testing in familial adenomatous polyposis.

PubMed Central

Whitelaw, S; Northover, J M; Hodgson, S V

1996-01-01

Attitudes to predictive DNA testing for familial adenomatous polyposis were documented in 62 affected adults. Patient views on prenatal testing and termination of pregnancy for this disorder were sought, as were opinions on the most suitable age to offer predictive testing for at risk children and the most appropriate age to begin screening. While 15 (24%) of those questioned stated that they would proceed to termination of pregnancy if a prenatal test indicated that the unborn baby was affected, in clinical practice no one has yet requested this option. Six (10%) people who had refrained from having children for fear of passing on the polyposis gene felt that the arrival of prenatal testing would enable them to consider planning a family. The majority of patients (93%) said they would like their children tested by DNA analysis at birth or in infancy, but felt that 10 to 12 years was the most appropriate time to discuss the diagnosis with the child. PMID:8818937

Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood.

PubMed

Nicolaides, Kypros H; Syngelaki, Argyro; del Mar Gil, Maria; Quezada, Maria Soledad; Zinevich, Yana

2014-01-01

To investigate potential performance of cell-free DNA (cfDNA) testing in maternal blood in detecting fetal triploidy. Plasma and buffy coat samples obtained at 11-13 weeks' gestation from singleton pregnancies with diandric triploidy (n=4), digynic triploidy (n=4), euploid fetuses (n=48) were sent to Natera, Inc. (San Carlos, Calif., USA) for cfDNA testing. Multiplex polymerase chain reaction amplification of cfDNA followed by sequencing of single nucleotide polymorphic loci covering chromosomes 13, 18, 21, X, and Y was performed. Sequencing data were analyzed using the NATUS algorithm which identifies copy number for each of the five chromosomes. cfDNA testing provided a result in 44 (91.7%) of the 48 euploid cases and correctly predicted the fetal sex and the presence of two copies each of chromosome 21, 18 and 13. In diandric triploidy, cfDNA testing identified multiple paternal haplotypes (indicating fetal trisomy 21, trisomy 18 and trisomy 13) suggesting the presence of either triploidy or dizygotic twins. In digynic triploidy the fetal fraction corrected for maternal weight and gestational age was below the 0.5th percentile. cfDNA testing by targeted sequencing and allelic ratio analysis of single nucleotide polymorphisms covering chromosomes 21, 18, 13, X, and Y can detect diandric triploidy and raise the suspicion of digynic triploidy. © 2013 S. Karger AG, Basel.

Detection of Cytomegalovirus (CMV) Infection in Wheezing Infants by Urine DNA and Serum IgG Testing.

PubMed

Zeng, Zhao-Cheng; Chang, Qing; Sun, Zhi-Wei; Song, Ming-Mei; Jin, Xin-Ling; Jiang, Shu-Ya; Yang, Xia

2017-03-11

BACKGROUND The aim of this study was to investigate the involvement of CMV infection in wheezing infants and the association between CMV-DNA and immunoglobulins (Igs). MATERIAL AND METHODS A total of 243 wheezing infants and 3,000 parturients were enrolled in this study. The infants were randomly grouped to receive blood HCMV-DNA tests (n=46) or urine HCMV-DNA tests (n=197). Furthermore, all participants had serum CMV-specific IgM and IgG testing. Afterwards, 10 HCMV-IgG positive infants were randomly selected for simultaneous blood and urine HCMV-DNA tests, and 25 HCMV-IgG positive puerperants were randomly selected for urine HCMV-DNA tests. RESULTS The detection rate of urine HCMV-DNA was significantly higher than that of blood HCMV-DNA (67.5% vs. 13.0%, p<0.001). Fifteen (6.2%) and 190 (80.0%) infants showed positive CMV-specific IgM and IgG results (p<0.001), respectively. Among the 10 HCMV-IgG positive infants tested further, only two infants had positive HCMV-DNA blood tests, while all of the 10 infants had positive HCMV-DNA urine tests. However, HCMV-DNA was not detected in the urine of the 25 randomly selected parturients positive for HCMV-IgG. CONCLUSIONS CMV infection may be one of the causes of wheezing in infants; CMV infection can be detected by urine-HCMV-DNA and serum HCMV-IgG testing. Infants were more susceptible to CMV infection than parturients.

Clinical evaluation of the COBAS Amplicor HBV monitor test for measuring serum HBV DNA and comparison with the Quantiplex branched DNA signal amplification assay in Taiwan.

PubMed

Dai, C-Y; Yu, M-L; Chen, S-C; Lin, Z-Y; Hsieh, M-Y; Wang, L-Y; Tsai, J-F; Chuang, W-L; Chang, W-Y

2004-02-01

To evaluate the performance characteristics and clinical usefulness of the COBAS Amplicor HBV monitor (COBAS-AM) test in Taiwan and to examine its correlation with the Quantiplex branched DNA signal amplification (bDNA) assay for measuring serum hepatitis B virus (HBV) DNA concentrations. HBV DNA was measured by the COBAS-AM test in 149 sera from chronic HBV infected patients that had previously been analysed by the bDNA assay. The COBAS-AM test showed good reproducibility, with acceptable intra-assay and interassay coefficients of variation (1.6% and 0.9%, respectively) and good linearity (r2=0.98). The overall sensitivity of the COBAS-AM test was significantly higher than that of the bDNA assay (95.3% v 83.2%): 69.6% of samples with HBV DNA below the detection limit of the bDNA assay could be measured by the COBAS-AM test. There was a significant correlation between the results of the two assays (r=0.901; p<0.0001). On average, the results derived from the COBAS-AM test were 0.55 log lower than those of the bDNA assay. HBV DNA concentrations were significantly higher among HBV e antigen (HBeAg) positive patients than negative ones, and higher among patients with abnormal alanine aminotransferase (ALT) concentrations than those with normal ALT concentrations (p=0.0003). The COBAS-AM assay, more sensitive in HBeAg negative samples than the bDNA assay, can effectively measure HBV DNA concentrations in Taiwanese patients. HBV DNA values measured by the COBAS-AM test and bDNA assay correlate significantly.

Early Adoption of a Multi-target Stool DNA Test for Colorectal Cancer Screening

PubMed Central

Finney Rutten, Lila J.; Jacobson, Robert M.; Wilson, Patrick M.; Jacobson, Debra J.; Fan, Chun; Kisiel, John B.; Sweetser, Seth R.; Tulledge-Scheitel, Sidna M.; St. Sauver, Jennifer L.

2017-01-01

Objective To characterize early adoption of a novelmulti-target stool deoxyribonucleic acid (MTsDNA) screening test for colorectal cancer (CRC) and test the hypothesis that adoption differs by demographic characteristics, prior CRC screening behavior, and proceeds predictably over time. Patients and Methods We used the Rochester Epidemiology Project infrastructure to assess MTsDNA screening test use among adults aged 50–75 years, and identified 27,147 individuals eligible/due for screening colonoscopy from November 1, 2014 through November 30, 2015, and living in Olmsted County, Minnesota in2014. We used electronic Current Procedure Terminology and Health Care Common Procedure codes to evaluate early adoption of MTsDNA screening test in this population and to test whether early adoption varies by age, sex, race, and prior screening behavior. Results Overall, 2,193 (8.1%) and 974 (3.6%) of individuals were screened by colonoscopy and MT-sDNA, respectively. Age, sex, race, and prior screening were significantly and independently associated with MT-sDNA screening use compared to colonoscopy use after adjustment for all other variables. Rates of adoption of MTsDNA screening increased over time and were highest among those aged 50–54 years, females, whites, and had a prior history of screening. MT-sDNA screening use varied predictably by insurance coverage. Rates of colonoscopy decreased over time, while overall CRC screening rates remained steady. Conclusion Our results are generally consistent with predictions derived from prior research and Diffusion of Innovation framework, pointing to increasing use of the new screening test over time, and early adoption by younger patients, females, whites and those with prior CRC screening. PMID:28473037

Should cell-free DNA testing be used to target antenatal rhesus immune globulin administration?

PubMed

Ma, Kimberly K; Rodriguez, Maria I; Cheng, Yvonne W; Norton, Mary E; Caughey, Aaron B

2016-01-01

To compare the rates of alloimmunization with the use of cell-free DNA (cfDNA) screening to target antenatal rhesus immune globulin (RhIG) prenatally, versus routine administration of RhIG in rhesus D (RhD)-negative pregnant women in a theoretic cohort using a decision-analytic model. A decision-analytic model compared cfDNA testing to routine antenatal RhIG administration. The primary outcome was maternal sensitization to RhD antigen. Sensitivity and specificity of cfDNA testing were assumed to be 99.8% and 95.3%, respectively. Univariate and bivariate sensitivity analyses, Monte Carlo simulation, and threshold analyses were performed. In a cohort of 10,000 RhD-negative women, 22.6 sensitizations would occur with utilization of cfDNA, while 20 sensitizations would occur with routine RhIG. Only when the sensitivity of the cfDNA test reached 100%, the rate of sensitization was equal for both cfDNA and RhIG. Otherwise, routine RhIG minimized the rate of sensitization, especially given RhIG is readily available in the United States. Adoption of cfDNA testing would result in a 13.0% increase in sensitization among RhD-negative women in a theoretical cohort taking into account the ethnic diversity of the United States' population.

Human papillomavirus DNA testing as an adjunct to cytology in cervical screening programs.

PubMed

Lörincz, Attila T; Richart, Ralph M

2003-08-01

Our objective was to review current large studies of human papillomavirus (HPV) DNA testing as an adjunct to the Papanicolaou test for cervical cancer screening programs. We analyzed 10 large screening studies that used the Hybrid Capture 2 test and 3 studies that used the polymerase chain reaction test in a manner that enabled reliable estimates of accuracy for detecting or predicting high-grade cervical intraepithelial neoplasia (CIN). Most studies allowed comparison of HPV DNA and Papanicolaou testing and estimates of the performance of Papanicolaou and HPV DNA as combined tests. The studies were selected on the basis of a sufficient number of cases of high-grade CIN and cancer to provide meaningful statistical values. Investigators had to demonstrate the ability to generate reasonably reliable Hybrid Capture 2 or polymerase chain reaction data that were either minimally biased by nature of study design or that permitted analytical techniques for addressing issues of study bias to be applied. Studies had to provide data for the calculation of test sensitivity, specificity, predictive values, odds ratios, relative risks, confidence intervals, and other relevant measures. Final data were abstracted directly from published articles or estimated from descriptive statistics presented in the articles. In some studies, new analyses were performed from raw data supplied by the principal investigators. We concluded that HPV DNA testing was a more sensitive indicator for prevalent high-grade CIN than either conventional or liquid cytology. A combination of HPV DNA and Papanicolaou testing had almost 100% sensitivity and negative predictive value. The specificity of the combined tests was slightly lower than the specificity of the Papanicolaou test alone, but this decrease could potentially be offset by greater protection from neoplastic progression and cost savings available from extended screening intervals. One "double-negative" HPV DNA and Papanicolaou test indicated

Personal DNA testing in college classrooms: perspectives of students and professors.

PubMed

Daley, Lori-Ann A; Wagner, Jennifer K; Himmel, Tiffany L; McPartland, Kaitlyn A; Katsanis, Sara H; Shriver, Mark D; Royal, Charmaine D

2013-06-01

Discourse on the integration of personal genetics and genomics into classrooms is increasing; however, limited data have been collected on the perspectives of students and professors. We conducted a cross-sectional survey of undergraduate and graduate students as well as professors at two major universities to assess attitudes regarding the use of personal DNA testing and other personalized activities in college classrooms. Students indicated that they were more likely to enroll (60.2%) in a genetics course if it offered personal DNA testing; undergraduate students were more likely than graduate students to enroll if personal DNA testing was offered (p=0.029). Students who majored in the physical sciences were less likely to enroll than students in the biological or social sciences (p=0.019). Students also indicated that when course material is personalized, the course is more interesting (94.6%) and the material is easier to learn (87.3%). Professors agreed that adding a personalized element increases student interest, participation, and learning (86.0%, 82.6%, and 72.6%, respectively). The results of this study indicate that, overall, students and professors had a favorable view of the integration of personalized information, including personal DNA testing, into classroom activities, and students welcomed more opportunities to participate in personalized activities.

The Sperm Chromatin Structure Assay (SCSA(®)) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility.

PubMed

Evenson, Donald P

2016-06-01

Thirty-five years ago the pioneering paper in Science (240:1131) on the relationship between sperm DNA integrity and pregnancy outcome was featured as the cover issue showing a fluorescence photomicrograph of red and green stained sperm. The flow cytometry data showed a very significant difference in sperm DNA integrity between fertile and subfertile bulls and men. This study utilized heat (100°C, 5min) to denature DNA at sites of DNA strand breaks followed by staining with acridine orange (AO) and measurements of 5000 individual sperm of green double strand (ds) DNA and red single strand (ss) DNA fluorescence. Later, the heat protocol was changed to a low pH protocol to denature the DNA at sites of strand breaks; the heat and acid procedures produced the same results. SCSA data are very advantageously dual parameter with 1024 channels (degrees) of both red and green fluorescence. Hundreds of publications on the use of the SCSA test in animals and humans have validated the SCSA as a highly useful test for determining male breeding soundness. The SCSA test is a rapid, non-biased flow cytometer machine measurement providing robust statistical data with exceptional precision and repeatability. Many genotoxic experiments showed excellent dose response data with very low coefficient of variation that further validated the SCSA as being a highly powerful assay for sperm DNA integrity. Twelve years following the introduction of the SCSA test, the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labelling (TUNEL) test (1993) for sperm was introduced as the only other flow cytometric assay for sperm DNA fragmentation. However, the TUNEL test can also be done by light microscopy with much less statistical robustness. The COMET (1998) and Sperm Chromatin Dispersion (SCD; HALO) (2003) tests were introduced as light microscope tests that don't require a flow cytometer. Since these tests measure only 50-200 sperm per sample, they suffer from the lack of

Selection response to DNA testing for canine ceroid lipofuscinosis in Tibetan terriers.

PubMed

Kluth, Susanne; Eckardt, Judith; Distl, Ottmar

2014-09-01

A late onset form of canine ceroid lipofuscinosis (CCL) is prevalent in Tibetan terriers. The disease is inherited as a monogenic recessive trait caused by aberrant exon skipping in ATP13A2. The aim of the present study was to analyse the frequencies of this mutation in Tibetan terriers registered with the German club for Tibetan dog breeds (Internationaler Klub für Tibetische Hunderassen, KTR) from 1987 to 2012 and to determine responses to selection following the introduction of DNA testing in 2010. The study included DNA extracted from blood samples from 1120/1240 (90.3%) Tibetan terriers registered with the KTR, including 405/420 (96.4%) registered breeding dogs. Mutant allele frequencies before the introduction of DNA testing were 0.20-0.28 in the registered and breeding dog populations, respectively, decreasing to 0.09 and 0.14, respectively, following the introduction of DNA testing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Problem-based test: replication of mitochondrial DNA during the cell cycle.

PubMed

Sétáló, György

2013-01-01

Terms to be familiar with before you start to solve the test: cell cycle, generation time, S-phase, cell culture synchronization, isotopic pulse-chase labeling, density labeling, equilibrium density-gradient centrifugation, buoyant density, rate-zonal centrifugation, nucleoside, nucleotide, kinase enzymes, polymerization of nucleic acids, re-replication block, cell fractionation, Svedberg (sedimentation constant = [ S]), nuclear DNA, mitochondrial DNA, heavy and light mitochondrial DNA chains, heteroplasmy, mitochondrial diseases Copyright © 2013 Wiley Periodicals, Inc.

Double Aneuploidy Detected by Cell-Free DNA Testing and Confirmed by Fetal Tissue Analysis.

PubMed

Echague, Charlene G; Petersen, Scott M

2016-06-01

Double aneuploidies account for 0.21-2.8% of spontaneous abortions resulting from chromosomal abnormalities. Rarely, cell-free DNA testing detects multiple aneuploidies; however, to discern among maternal, placental, and fetal origin, further evaluation is required. A 49-year-old woman, gravida 5 para 0, underwent cell-free DNA testing at 11 4/7 weeks of gestation, which revealed a fetus that was high risk for trisomies 18 and 21. On ultrasonography at 14 weeks of gestation, she was diagnosed with a missed abortion and underwent surgical management. Fetal and placental tissues were sent for analysis and were positive for trisomies 18 and 21, confirming the results of cell-free DNA testing. Our case highlights the ability of cell-free DNA testing to recognize a double aneuploidy confirmed by fetal tissue analysis.

Serological reactions in Rhesus monkeys inoculated with the 17D strain of yellow fever virus.

PubMed

GROOT, H

1962-01-01

Haemagglutination-inhibition tests, which depend on the appearance of haemagglutination-inhibiting antibodies in the serum in virus infections, are in common use in the study of arthropod-borne diseases. This paper contains the results of an investigation into the appearance and pattern of haemagglutination-inhibiting antibodies in the serum of rhesus monkeys inoculated intracerebrally with the 17D strain of yellow fever virus during the testing of seed lots of yellow fever vaccine. These antibodies appeared on the tenth day after inoculation, and were still demonstrable four years later. In all of the eight monkeys tested complement-fixing and neutralizing antibodies against yellow fever antigens also developed, and in six out of the eight heterologous antigens developed.

Lay perceptions of predictive testing for diabetes based on DNA test results versus family history assessment: a focus group study.

PubMed

Wijdenes-Pijl, Miranda; Dondorp, Wybo J; Timmermans, Danielle Rm; Cornel, Martina C; Henneman, Lidewij

2011-07-05

This study assessed lay perceptions of issues related to predictive genetic testing for multifactorial diseases. These perceived issues may differ from the "classic" issues, e.g. autonomy, discrimination, and psychological harm that are considered important in predictive testing for monogenic disorders. In this study, type 2 diabetes was used as an example, and perceptions with regard to predictive testing based on DNA test results and family history assessment were compared. Eight focus group interviews were held with 45 individuals aged 35-70 years with (n = 3) and without (n = 1) a family history of diabetes, mixed groups of these two (n = 2), and diabetes patients (n = 2). All interviews were transcribed and analysed using Atlas-ti. Most participants believed in the ability of a predictive test to identify people at risk for diabetes and to motivate preventive behaviour. Different reasons underlying motivation were considered when comparing DNA test results and a family history risk assessment. A perceived drawback of DNA testing was that diabetes was considered not severe enough for this type of risk assessment. In addition, diabetes family history assessment was not considered useful by some participants, since there are also other risk factors involved, not everyone has a diabetes family history or knows their family history, and it might have a negative influence on family relations. Respect for autonomy of individuals was emphasized more with regard to DNA testing than family history assessment. Other issues such as psychological harm, discrimination, and privacy were only briefly mentioned for both tests. The results suggest that most participants believe a predictive genetic test could be used in the prevention of multifactorial disorders, such as diabetes, but indicate points to consider before both these tests are applied. These considerations differ with regard to the method of assessment (DNA test or obtaining family history) and also differ from

Post-mortem diagnosis of chronic Chagas's disease comparative evaluation of three serological tests on pericardial fluid.

PubMed

Lopes, E R; Chapadeiro, E; Batista, S M; Cunha, J G; Rocha, A; Miziara, L; Ribeiro, J U; Patto, R J

1978-01-01

In an attempt to improve the post-mortem diagnosis of Chagas's disease the authors performed haemagglutination tests (HAT), fluorescent Trypanosoma cruzi antibody tests (FAT), and complement fixation tests (CFT) on the pericardial fluid obtained at autopsy of 50 individuals with Chagas's heart disease, and 93 patients in whom this disease was not thought to be present. The results demonstrate that all three tests are efficient for the post-mortem diagnosis of Chagas's disease but suggest that their combined use would detect more cases than would one isolated reaction only.

Investigating CSI: portrayals of DNA testing on a forensic crime show and their potential effects.

PubMed

Ley, Barbara L; Jankowski, Natalie; Brewer, Paul R

2012-01-01

The popularity of forensic crime shows such as CSI has fueled debate about their potential social impact. This study considers CSI's potential effects on public understandings regarding DNA testing in the context of judicial processes, the policy debates surrounding crime laboratory procedures, and the forensic science profession, as well as an effect not discussed in previous accounts: namely, the show's potential impact on public understandings of DNA and genetics more generally. To develop a theoretical foundation for research on the "CSI effect," it draws on cultivation theory, social cognitive theory, and audience reception studies. It then uses content analysis and textual analysis to illuminate how the show depicts DNA testing. The results demonstrate that CSI tends to depict DNA testing as routine, swift, useful, and reliable and that it echoes broader discourses about genetics. At times, however, the show suggests more complex ways of thinking about DNA testing and genetics.

Antigenic properties of the envelope of influenza virus rendered soluble by surfactant-solvent systems

PubMed Central

Larin, N. M.; Gallimore, P. H.

1971-01-01

Dissociating chemical treatments employing surfactant-solvent systems were applied to purified influenza A and B viruses to obtain viral preparations possessing a significantly higher or lower haemagglutinating activity than the intact virus. All preparations, whether with high or low haemagglutinating activity, with the exception of envelope protein solubilized by Triton X-100, were significantly lacking in the ability to excite the formation of haemagglutination-inhibiting and virus-neutralizing antibodies in inoculated ferrets. In contrast to other treatments, Triton X-100 treatment of virus significantly enhanced the antigenicity of viral protein as judged by virus neutralization and haemagglutination inhibition tests. Yet the haemagglutinating activity of the envelope protein solubilized with Triton X-100 was about 1% that of the intact virus. Results suggest that the correlation assumed to exist between the haemagglutinating activity of influenza virus and its ability to excite the formation of humoral antibodies is coincidental. Another important point is that the specific antigenicity of viral protein may be lost or enhanced owing to effects, other than solubilization, by surface-active agents. PMID:5291750

Nanostructured SERS-electrochemical biosensors for testing of anticancer drug interactions with DNA.

PubMed

Ilkhani, Hoda; Hughes, Taylor; Li, Jing; Zhong, Chuan Jian; Hepel, Maria

2016-06-15

Widely used anti-cancer treatments involving chemotherapeutic drugs result in cancer cell damage due to their strong interaction with DNA. In this work, we have developed laboratory biosensors for screening chemotherapeutic drugs and to aid in the assessment of DNA modification/damage caused by these drugs. The sensors utilize surface-enhanced Raman scattering (SERS) spectroscopy and electrochemical methods to monitor sensory film modification and observe the drug-DNA reactivity. The self-assembled monolayer protected gold-disk electrode (AuDE) was coated with a reduced graphene oxide (rGO), decorated with plasmonic gold-coated Fe2Ni@Au magnetic nanoparticles functionalized with double-stranded DNA (dsDNA), a sequence of the breast cancer gene BRCA1. The nanobiosensors AuDE/SAM/rGO/Fe2Ni@Au/dsDNA were then subjected to the action of a model chemotherapeutic drug, doxorubicin (DOX), to assess the DNA modification and its dose dependence. The designed novel nanobiosensors offer SERS/electrochemical transduction, enabling chemically specific and highly sensitive analytical signals generation. The SERS measurements have corroborated the DOX intercalation into the DNA duplex whereas the electrochemical scans have indicated that the DNA modification by DOX proceeds in a concentration dependent manner, with limit of detection LOD=8 µg/mL (S/N=3), with semilog linearity over 3 orders of magnitude. These new biosensors are sensitive to agents that interact with DNA and facilitate the analysis of functional groups for determination of the binding mode. The proposed nanobiosensors can be applied in the first stage of the drug development for testing the interactions of new drugs with DNA before the drug efficacy can be assessed in more expensive testing in vitro and in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

The clinical utility of HPV DNA testing in cervical cancer screening strategies.

PubMed

Bhatla, Neerja; Moda, Nidhi

2009-09-01

Cervical cancer continues to be the commonest cause of death among women in developing countries, largely due to the failure to the inability to sustain effective cytology-based screening programs. While this burden may come down following implementation of the human papillomavirus (HPV) vaccine, screening will still be required. HPV DNA testing is a promising new technology for cervical cancer prevention and is the most reproducible of all cervical cancer screening tests. Presently, the two assays most widely used for the detection of genital types are the polymerase chain reaction (PCR) and Hybrid Capture 2 assays (hc2). Rapid, affordable tests are expected to be available soon. HPV DNA testing can be used in a variety of clinical scenarios that include primary screening in women older than 30 yr; as an adjunctive test to cytology; in the triage of women with an equivocal cytologic report, e.g., ASC-US; or for follow-up post-treatment for cervical intraepithelial neoplasia (CIN). HPV DNA testing can also be performed on self-collected samples, which allows screening in remote areas and also in women who refuse gynecologic examination.

The utilization of circulating cell-free fetal DNA testing and decrease in invasive diagnostic procedures: an institutional experience.

PubMed

Pettit, K E; Hull, A D; Korty, L; Jones, M C; Pretorius, D H

2014-10-01

To characterize the patient population utilizing circulating cell-free fetal DNA (ccffDNA) testing at a large academic center and evaluate trends in the performance of invasive diagnostic procedures. A retrospective cohort study of all patients who underwent cell-free DNA testing from May to December 2012 was performed. During the study period, 206 patients had cell-free DNA testing. Of those, 75% (155/206) were of ages ⩾ 35 years. Of those undergoing ccffDNA testing, 41% had positive aneuploidy screening and 38% had abnormal ultrasound findings. Only 7% of the patients with negative ccffDNA testing opted for an invasive diagnostic procedure compared with 60% with positive testing (P<0.01). The rate of invasive procedures decreased from 5.9% of all visits to the center during a similar 8-month period in 2010 to 4.1% of all visits during the study period (P<0.01). Our data suggest that ccffDNA testing leads to reduced uptake of invasive procedures.

Stretching chimeric DNA: A test for the putative S-form

NASA Astrophysics Data System (ADS)

Whitelam, Stephen; Pronk, Sander; Geissler, Phillip L.

2008-11-01

Double-stranded DNA "overstretches" at a pulling force of about 65 pN, increasing in length by a factor of 1.7. The nature of the overstretched state is unknown, despite its considerable importance for DNA's biological function and technological application. Overstretching is thought by some to be a force-induced denaturation and by others to consist of a transition to an elongated, hybridized state called S-DNA. Within a statistical mechanical model, we consider the effect upon overstretching of extreme sequence heterogeneity. "Chimeric" sequences possessing halves of markedly different AT composition elongate under fixed external conditions via distinct, spatially segregated transitions. The corresponding force-extension data vary with pulling rate in a manner that depends qualitatively and strikingly upon whether the hybridized S-form is accessible. This observation implies a test for S-DNA that could be performed in experiment.

UK NHS pilot study on cell-free DNA testing in screening for fetal trisomies: factors affecting uptake.

PubMed

Gil, M M; Giunta, G; Macalli, E A; Poon, L C; Nicolaides, K H

2015-01-01

This study reports on the clinical implementation of cell-free DNA (cfDNA) testing, contingent on the results of the combined test, in screening for fetal trisomies 21, 18 and 13 in two UK National Health Service hospitals. Women with a combined-test risk of ≥ 1:100 (high risk) were offered the options of chorionic villus sampling (CVS), cfDNA testing or no further testing and those with a risk of 1:101 to 1:2500 (intermediate risk) were offered cfDNA or no further testing. The objective of the study was to examine the factors affecting patient decisions concerning their options. Combined screening was performed in 6651 singleton pregnancies in which the risk for trisomies was high in 260 (3.9%), intermediate in 2017 (30.3%) and low in 4374 (65.8%). Logistic regression analysis was used to determine which factors among maternal characteristics, fetal nuchal translucency thickness (NT) and risk for trisomies were significant predictors of opting for CVS in the high-risk group and opting for cfDNA testing in the intermediate-risk group. In the high-risk group, 104 (40.0%) women opted for CVS; predictors for CVS were increasing fetal NT and increasing risk for trisomies, while the predictor against CVS was being of Afro-Caribbean racial origin (r = 0.366). In the intermediate-risk group, 1850 (91.7%) women opted for cfDNA testing; predictors for cfDNA testing were increasing maternal age, increasing risk for trisomies and university education, while predictors against cfDNA testing were being of Afro-Caribbean racial origin, smoking and being parous (r = 0.105). This study has identified factors that can influence the decision of women undergoing combined screening in favor of or against CVS and in favor of or against cfDNA testing. Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.

Is HPV DNA testing specificity comparable to that of cytological testing in primary cervical cancer screening? Results of a meta-analysis of randomized controlled trials.

PubMed

Pileggi, Claudia; Flotta, Domenico; Bianco, Aida; Nobile, Carmelo G A; Pavia, Maria

2014-07-01

Human-papillomavirus (HPV) DNA testing has been proposed as an alternative to primary cervical cancer screening using cytological testing. Review of the evidence shows that available data are conflicting for some aspects. The overall goal of the study is to update the performance of HPV DNA as stand-alone testing in primary cervical cancer screening, focusing particularly on the aspects related to the specificity profile of the HPV DNA testing in respect to cytology. We performed a meta-analysis of randomized controlled clinical trials. Eight articles were included in the meta-analysis. Three outcomes have been investigated: relative detection, relative specificity, and relative positive predictive value (PPV) of HPV DNA testing versus cytology. Overall evaluation of relative detection showed a significantly higher detection of CIN2+ and CIN3+ for HPV DNA testing versus cytology. Meta-analyses that considered all age groups showed a relative specificity that favored the cytology in detecting both CIN2+ and CIN3+ lesions whereas, in the ≥30 years' group, specificity of HPV DNA and cytology tests was similar in detecting both CIN2+ and CIN3+ lesions. Results of the pooled analysis on relative PPV showed a not significantly lower PPV of HPV DNA test over cytology. A main key finding of the study is that in women aged ≥30, has been found an almost overlapping specificity between the two screening tests in detecting CIN2 and above-grade lesions. Therefore, primary screening of cervical cancer by HPV DNA testing appears to offer the right balance between maximum detection of CIN2+ and adequate specificity, if performed in the age group ≥30 years. © 2013 UICC.

Cost-Effectiveness of Cervical Cancer Screening With Human Papillomavirus DNA Testing and HPV-16,18 Vaccination

PubMed Central

Goldhaber-Fiebert, Jeremy D.; Stout, Natasha K.; Salomon, Joshua A.; Kuntz, Karen M.; Goldie, Sue J.

2011-01-01

Background The availability of human papillomavirus (HPV) DNA testing and vaccination against HPV types 16 and 18 (HPV-16,18) motivates questions about the cost-effectiveness of cervical cancer prevention in the United States for unvaccinated older women and for girls eligible for vaccination. Methods An empirically calibrated model was used to assess the quality-adjusted life years (QALYs), lifetime costs, and incremental cost-effectiveness ratios (2004 US dollars per QALY) of screening, vaccination of preadolescent girls, and vaccination combined with screening. Screening varied by initiation age (18, 21, or 25 years), interval (every 1, 2, 3, or 5 years), and test (HPV DNA testing of cervical specimens or cytologic evaluation of cervical cells with a Pap test). Testing strategies included: 1) cytology followed by HPV DNA testing for equivocal cytologic results (cytology with HPV test triage); 2) HPV DNA testing followed by cytology for positive HPV DNA results (HPV test with cytology triage); and 3) combined HPV DNA testing and cytology. Strategies were permitted to switch once at age 25, 30, or 35 years. Results For unvaccinated women, triennial cytology with HPV test triage, beginning by age 21 years and switching to HPV testing with cytology triage at age 30 years, cost $78 000 per QALY compared with the next best strategy. For girls vaccinated before age 12 years, this same strategy, beginning at age 25 years and switching at age 35 years, cost $41 000 per QALY with screening every 5 years and $188 000 per QALY screening triennially, each compared with the next best strategy. These strategies were more effective and cost-effective than screening women of all ages with cytology alone or cytology with HPV triage annually or biennially. Conclusions For both vaccinated and unvaccinated women, age-based screening by use of HPV DNA testing as a triage test for equivocal results in younger women and as a primary screening test in older women is expected to be more

Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2013-01-01

The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

Kastle-Meyer blood test reagents are deleterious to DNA.

PubMed

Sloots, James; Lalonde, Wendy; Reid, Barbara; Millman, Jonathan

2017-12-01

The Kastle-Meyer (KM) test is a quick and easy chemical test for blood used in forensic analyses. Two practical variations of this test are the KM-rub (indirect) test and the more sensitive KM-direct test, the latter of which is performed by applying reagents directly to a suspected blood stain. This study found that sodium hydroxide present in the KM reagents eliminated the potential to generate a DNA profile when applied directly to small quantities of blood. A modified approach to the KM-rub test that increases its sensitivity is presented as a method to replace destructive KM-direct testing. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Clinical Utility of Epstein-Barr Virus DNA Testing in the Treatment of Nasopharyngeal Carcinoma Patients.

PubMed

Kim, Kelly Y; Le, Quynh-Thu; Yom, Sue S; Ng, Raymond H W; Chan, K C Allen; Bratman, Scott V; Welch, John J; Divi, Rao L; Petryshyn, Raymond A; Conley, Barbara A

2017-08-01

Epstein-Barr virus (EBV) DNA analysis has been shown to be useful for early detection, prognostication, and monitoring of treatment response of nasopharyngeal carcinoma (NPC), and the recent literature provides growing evidence of the clinical utility of EBV DNA testing, particularly to inform treatment decisions for NPC patients. Despite the fact that NPC is a rare disease, the NRG Oncology cooperative group has successfully activated a phase 2/3 randomized clinical trial for NPC with international partners and in that process has discovered that the development of a harmonized EBV DNA test is absolutely critical for integration into clinical trials and for future deployment in clinical and central laboratories. In November 2015, the National Cancer Institute (NCI) convened a workshop of international experts in the treatment of NPC and EBV testing to provide a forum for discussing the state of EBV DNA testing and its clinical utility, and to stimulate consideration of future studies and clinical practice guidelines for EBV DNA. This review provides a summary of that discussion. Published by Elsevier Inc.

High-Risk Palliative Care Patients' Knowledge and Attitudes about Hereditary Cancer Testing and DNA Banking.

PubMed

Quillin, John M; Emidio, Oluwabunmi; Ma, Brittany; Bailey, Lauryn; Smith, Thomas J; Kang, In Guk; Yu, Brandon J; Owodunni, Oluwafemi Patrick; Abusamaan, Mohammed; Razzak, Rab; Bodurtha, Joann N

2017-12-04

Even at the end of life, testing cancer patients for inherited susceptibility may provide life-saving information to their relatives. Prior research suggests palliative care inpatients have suboptimal understanding of genetic importance, and testing may be underutilized in this clinical setting. These conclusions are based on limited research. This study aimed to estimate genetic testing prevalence among high-risk palliative care patients in a National Cancer Institute-designated comprehensive cancer center. We also aimed to understand these patients' understanding of, and attitudes toward, hereditary cancer testing and DNA banking. Palliative care in-patients with cancer completed structured interviews, and their medical records were reviewed. Among patients at high risk for hereditary cancer, we assessed history of genetic testing/DNA banking; and related knowledge and attitudes. Among 24 high-risk patients, 14 (58.3%) said they/their relatives had genetic testing or they had been referred for a genetics consultation. Of the remaining 10 patients, seven (70%) said they would "probably" or "definitely" get tested. Patients who had not had testing were least concerned about the impact of future testing on their family relationships; two (20%) said they were "extremely concerned" about privacy related to genetic testing. Of patients without prior testing, five (50%) said they had heard or read "a fair amount" about genetic testing. No high-risk patients had banked DNA. Overall, 23 (95.8%) said they had heard or read "almost nothing" or "relatively little" about DNA banking. Written materials and clinician discussion were most preferred ways to learn about genetic testing and DNA banking. Overall, this study demonstrates underutilization of genetics services at the end of life continues to be problematic, despite high patient interest.

Swabbing Students: Should Universities Be Allowed to Facilitate Educational DNA Testing?

PubMed Central

Callier, Shawneequa L.

2012-01-01

Recognizing the profound need for greater patient and provider familiarity with personalized genomic medicine, many university instructors are including personalized genotyping as part of their curricula. During seminars and lectures students run polymerase chain reactions on their own DNA or evaluate their experiences using direct-to-consumer genetic testing services subsidized by the university. By testing for genes that may influence behavioral or health-related traits, however, such as alcohol tolerance and cancer susceptibility, certain universities have stirred debate on the ethical concerns raised by educational genotyping. Considering the potential for psychosocial harm and medically relevant outcomes, how far should university-facilitated DNA testing be permitted to go? The analysis here distinguishes among these learning initiatives and critiques their approaches to the ethical concerns raised by educational genotyping. PMID:22452475

Ethical, Legal and Social Issues in Japan on the Determination of Blood Relationship via DNA Testing.

PubMed

Toya, Waki

2017-01-01

DNA paternity testing has recently become more widely available in Japan. The aim of this paper is to examine the issues surrounding (1) the implementing agency, whether the testing is conducted in a commercial direct-to-consumer (DTC) setting or a judicial non-DTC setting, and (2) the implementation conditions and more specifically the legal capacity of the proband (test subject). Literature research in Japanese and English was conducted. Some countries prohibit commercial DNA testing without the consent of the proband or her or his legally authorized representative. But as in some cases, the results of DTC paternity testing have proven to be unreliable. I propose a complete prohibition of DTC DNA paternity testing in Japan. In many cases of paternity testing, the proband is a minor. This has led to debate about whether proxy consent is sufficient for paternity testing or whether additional safeguards (such as a court order) are required. In cases where commercial DNA testing has been conducted and the test results are produced in court as evidence, the court must judge whether or not to admit these results as evidence. Another important issue is whether or not paternity testing should be legally mandated in certain cases. If we come to the conclusion that DNA test results are the only way to conclusively establish a parent-child relationship, then our society may prioritize even more genetic relatedness over other conceptions of a parent-child relationship. This prioritization could adversely affect families created through assisted reproductive technology (ART), especially in situations where children are not aware of their biological parentage. This paper argues for a complete prohibition of DTC DNA paternity testing in Japan, and highlights that broader ethical and legal deliberation on such genetic services is required.

Is it time to sound an alarm about false-positive cell-free DNA testing for fetal aneuploidy?

PubMed

Mennuti, Michael T; Cherry, Athena M; Morrissette, Jennifer J D; Dugoff, Lorraine

2013-11-01

Testing cell-free DNA (cfDNA) in maternal blood samples has been shown to have very high sensitivity for the detection of fetal aneuploidy with very low false-positive results in high-risk patients who undergo invasive prenatal diagnosis. Recent observation in clinical practice of several cases of positive cfDNA tests for trisomy 18 and trisomy 13, which were not confirmed by cytogenetic testing of the pregnancy, may reflect a limitation of the positive predictive value of this quantitative testing, particularly when it is used to detect rare aneuploidies. Analysis of a larger number of false-positive cases is needed to evaluate whether these observations reflect the positive predictive value that should be expected. Infrequently, mechanisms (such as low percentage mosaicism or confined placental mosaicism) might also lead to positive cfDNA testing that is not concordant with standard prenatal cytogenetic diagnosis. The need to explore these and other possible causes of false-positive cfDNA testing is exemplified by 2 of these cases. Additional evaluation of cfDNA testing in clinical practice and a mechanism for the systematic reporting of false-positive and false-negative cases will be important before this test is offered widely to the general population of low-risk obstetric patients. In the meantime, incorporating information about the positive predictive value in pretest counseling and in clinical laboratory reports is recommended. These experiences reinforce the importance of offering invasive testing to confirm cfDNA results before parental decision-making. Copyright © 2013 Mosby, Inc. All rights reserved.

Cell-free fetal DNA testing: A pilot study of obstetric healthcare provider attitudes towards clinical implementation

PubMed Central

Sayres, Lauren; Allyse, Megan; Norton, Mary; Cho, Mildred

2011-01-01

Objective To provide a preliminary assessment of obstetric healthcare provider opinions surrounding implementation of cell-free fetal DNA testing. Methods A 37-question pilot survey was used to address questions around the translation and use of non-invasive prenatal testing using cell-free fetal DNA. The survey was distributed and collected at a Continuing Medical Education course on obstetrics and gynecology. Results Of 62 survey respondents, 73% are female and 87% hold MD/DO degrees. Respondents generally agree that patients want prenatal diagnostic information to help make decisions about a pregnancy and that cell-free fetal DNA testing will encourage the testing of more patients for more conditions. However, there is an overall lack of knowledge or conviction about using this technology. Genetic counseling and professional society approval are deemed important to implementation whereas the possibility of direct-to-consumer testing and government regulation produce mixed responses. Respondents indicate that they are more likely to offer cell-free fetal DNA testing for chromosomal abnormalities and single-gene disorders, but are cautious with respect to determination of sex and behavioral or late-onset conditions. Conclusion Preliminary assessment indicates uncertainty among obstetric providers about the details of implementing cell-free fetal DNA testing and suggests expanded research on perspectives of this stakeholder group. PMID:21793012

Clinical and serological tests for arboviruses in free-living domestic pigeons (Columba livia).

PubMed

Ramos, Bruna Alves; Chiang, Jannifer Oliveira; Martins, Lívia Carício; Chagas, Liliane Leal das; Silva, Franko de Arruda E; Ferreira, Milene Silveira; Freitas, Maria Nazaré Oliveira; Alcantara, Bianca Nascimento de; Silva, Sandro Patroca da; Miranda, Stefânia Araújo; Sepulvreda, Barbara Alves; Corrêa, Layna Thayssa Guimarães; Negrão, Andréa Maria Góes; Vasconcelos, Pedro Fernando da Costa; Casseb, Alexandre do Rosário

2017-08-01

In this study, we evaluated the role of free-living domestic pigeons (Columba livia) as a reservoir of arboviruses in the city of Belém, state of Pará, Brazil. We investigated the presence of antibodies against the most prevalent arboviruses. This study was aimed at evaluating some clinical and physical parameters of domestic pigeons, including the presence of antibodies to Amazon-endemic arboviruses. Eighty-five healthy pigeons were captured in Mangal das Garças Park, in Belém, and were bled. Upon capture, the birds were subjected to a clinical examination in search of alterations that could indicate the presence of arboviruses. Blood samples were converted to serum and tested using the haemagglutination inhibition (HI) technique with a panel of 19 antigens of arboviruses circulating in the Amazon. The confirmation assay for the positive reactions to the viral species tested by HI was a neutralisation test in new-born Swiss albino mice (Mus musculus) [mouse neutralisation test (MNT)]. A total of 10 (11.8%) serum samples tested positive for antiflavivirus antibodies by HI. All the samples positive for the HI test were subjected to MNT for detection of viruses and yielded negative results (logarithmic neutralisation index < 1.7). The results represent the first serological detection of antiarbovirus antibodies in domestic pigeons as potential hosts of arboviruses in Brazil. The detection of haemagglutination-inhibiting antibodies against genus Flavivirus indicated that there was recent contact between the analysed domestic pigeons and these arboviruses. Further studies are needed to evaluate the role of free-living pigeons in the maintenance cycle and spread of arboviruses in the Amazon.

Clinical and serological tests for arboviruses in free-living domestic pigeons (Columba livia)

PubMed Central

Ramos, Bruna Alves; Chiang, Jannifer Oliveira; Martins, Lívia Carício; Chagas, Liliane Leal das; Silva, Franko de Arruda e; Ferreira, Milene Silveira; Freitas, Maria Nazaré Oliveira; de Alcantara, Bianca Nascimento; da Silva, Sandro Patroca; Miranda, Stefânia Araújo; Sepulvreda, Barbara Alves; Corrêa, Layna Thayssa Guimarães; Negrão, Andréa Maria Góes; Vasconcelos, Pedro Fernando da Costa; Casseb, Alexandre do Rosário

2017-01-01

BACKGROUND In this study, we evaluated the role of free-living domestic pigeons (Columba livia) as a reservoir of arboviruses in the city of Belém, state of Pará, Brazil. We investigated the presence of antibodies against the most prevalent arboviruses. OBJECTIVES This study was aimed at evaluating some clinical and physical parameters of domestic pigeons, including the presence of antibodies to Amazon-endemic arboviruses. METHODS Eighty-five healthy pigeons were captured in Mangal das Garças Park, in Belém, and were bled. Upon capture, the birds were subjected to a clinical examination in search of alterations that could indicate the presence of arboviruses. Blood samples were converted to serum and tested using the haemagglutination inhibition (HI) technique with a panel of 19 antigens of arboviruses circulating in the Amazon. The confirmation assay for the positive reactions to the viral species tested by HI was a neutralisation test in new-born Swiss albino mice (Mus musculus) [mouse neutralisation test (MNT)]. FINDINGS A total of 10 (11.8%) serum samples tested positive for antiflavivirus antibodies by HI. All the samples positive for the HI test were subjected to MNT for detection of viruses and yielded negative results (logarithmic neutralisation index < 1.7). MAIN CONCLUSION The results represent the first serological detection of antiarbovirus antibodies in domestic pigeons as potential hosts of arboviruses in Brazil. The detection of haemagglutination-inhibiting antibodies against genus Flavivirus indicated that there was recent contact between the analysed domestic pigeons and these arboviruses. Further studies are needed to evaluate the role of free-living pigeons in the maintenance cycle and spread of arboviruses in the Amazon. PMID:28767977

Implementation of maternal blood cell-free DNA testing in early screening for aneuploidies.

PubMed

Gil, M M; Quezada, M S; Bregant, B; Ferraro, M; Nicolaides, K H

2013-07-01

To explore the feasibility of routine maternal blood cell-free (cf) DNA testing in screening for trisomies 21, 18 and 13 at 10 weeks' gestation. In this prospective study, women attending The Fetal Medicine Centre in London, UK, between October 2012 and April 2013, with singleton pregnancy and live fetus with CRL 32-45 mm, were screened for trisomies 21, 18 and 13 by cfDNA testing at 10 weeks and the combined test at 12 weeks. cfDNA testing was performed in 1005 singleton pregnancies with a median maternal age of 37 (range, 20-49) years. Risks for trisomies were provided for 957 (95.2%) cases and in 98.0% these were available within 14 days from sampling. In 48 (4.8%) cases no result was provided due to problems with delivery to the laboratory, low fetal fraction or assay failure. Repeat sampling was performed in 40 cases and a result obtained in 27 (67.5%) of these. In 11 cases the risk score for trisomy 21 and in five cases that for trisomy 18 was > 99%, in one the risk for trisomy 13 was 34% and in 968 the risk for each of the three trisomies was < 0.01%. The suspected trisomies were confirmed by karyotyping after chorionic villus sampling (CVS), except in one case of trisomy 18 in which the karyotype was normal. On the basis of the maternal age distribution of the study population, the expected and observed numbers for each of the three trisomies were similar. Both cfDNA and combined testing detected all trisomies, but the estimated false-positive rates (FPR) were 0.1% and 3.4%, respectively. Routine screening for trisomies 21, 18 and 13 by cfDNA testing at 10 weeks is feasible and has a lower FPR than does combined testing, but abnormal results require confirmation by CVS. Copyright © 2013 ISUOG. Published by John Wiley & Sons, Ltd.

Maternal plasma DNA testing for aneuploidy in pregnancies achieved by assisted reproductive technologies.

PubMed

Lambert-Messerlian, Geralyn; Kloza, Edward M; Williams, John; Loucky, Jaroslav; O'Brien, Barbara; Wilkins-Haug, Louise; Mahoney, Maurice J; De Biasio, Pierangela; Borrell, Antoni; Ehrich, Mathias; van den Boom, Dirk; Bombard, Allan T; Deciu, Cosmin; Palomaki, Glenn E

2014-05-01

We sought to compare measurements of circulating cell-free DNA as well as Down syndrome test results in women with naturally conceived pregnancies with those conceived using assisted reproductive technologies. Data regarding assisted reproductive technologies were readily available from seven enrollment sites participating in an external clinical validation trial of nested case/control design. Measurements of circulating cell-free fetal and total DNA, fetal fraction (ratio of fetal to total DNA), chromosome-specific z-scores, and karyotype results were available for analysis. Analyses were restricted to 632 euploid (5.2% assisted reproductive technologies) and 73 Down syndrome (13.7% assisted reproductive technologies), including 16 twin pregnancies. No differences were found for fetal or total circulating cell-free DNA, or for the fetal fraction in euploid (P = 0.70) or Down syndrome (P = 0.58) pregnancies by method of conception. There appeared to be systematic z-score reductions for chromosomes 21, 18, and 13 in assisted reproductive technologies versus natural euploid pregnancies (P = 0.048, 0.0032, and 0.36, respectively). Assisted reproductive technologies and naturally conceived pregnancies contribute similar levels of circulating cell-free DNA into maternal circulation. Small differences in the z-scores of pregnancies achieved by assisted reproductive technologies were observed and do not appear to be test-related artifacts. However, the findings need confirmation before any consideration of changes to testing and reporting protocols.

Cell-free DNA testing in the maternal blood in high-risk pregnancies after first-trimester combined screening.

PubMed

Persico, Nicola; Boito, Simona; Ischia, Benedetta; Cordisco, Adalgisa; De Robertis, Valentina; Fabietti, Isabella; Periti, Enrico; Volpe, Paolo; Fedele, Luigi; Rembouskos, Georgios

2016-03-01

The objective of this study was to investigate a strategy for clinical implementation of cell-free DNA (cfDNA) testing in high-risk pregnancies after first-trimester combined screening. In 259 singleton pregnancies undergoing invasive testing after first-trimester combined screening, a maternal blood sample was sent to the laboratory Natera for cfDNA testing using a single-nucleotide polymorphism-based methodology. The cfDNA test provided a result in 249 (96.1%) pregnancies and, among these, identified as being at high risk 35 of 36 cases of trisomy 21, 13 of 13 with trisomy 18, five of five with trisomy 13 and three of four with sex chromosome aneuploidies. A policy of performing an invasive test in women with a combined risk of ≥1 in 10 or NT ≥4 mm and offering cfDNA testing to the remaining cases would detect all cases of trisomy 21, 18 or 13, 80% of sex aneuploidies and 62.5% of other defects and would avoid an invasive procedure in 82.4% of euploid fetuses. In high-risk pregnancies after combined screening, a policy of selecting a subgroup for invasive testing and another for cfDNA testing would substantially reduce the number of invasive procedures and retain the ability to diagnose most of the observed aneuploidies. © 2016 John Wiley & Sons, Ltd.

Test performance and acceptability of self- versus provider-collected swabs for high-risk HPV DNA testing in female-to-male trans masculine patients

PubMed Central

Deutsch, Madeline B.; Peitzmeier, Sarah M.; White Hughto, Jaclyn M.; Cavanaugh, Timothy P.; Pardee, Dana J.; McLean, Sarah A.; Panther, Lori A.; Gelman, Marcy; Mimiaga, Matthew J.; Potter, Jennifer E.

2018-01-01

Background High-risk human papillomavirus (hrHPV) causes virtually all cervical cancers. Trans masculine (TM) people (those assigned female at birth who identify with a gender other than female) have low uptake of conventional cervical cancer screening. Self-collected hrHPV DNA testing has high levels of acceptability among cisgender (non-transgender) females and may support increased cervical cancer screening uptake in TM individuals. Objective To assess the test performance and acceptability of self-collected vaginal specimens in comparison to provider-collected cervical swabs for hrHPV DNA detection in TM individuals ages 21–64 years. Methods Between March 2015-September 2016, 150 TM participants with a cervix (mean age = 27.5 years; SD = 5.7) completed a one-time study visit comprised of a self-report survey, self-collected vaginal HPV DNA swab, clinician-administered cervical HPV swab, and brief interview on acceptability of clinical procedures. Participants were randomized to complete either self- or provider-collection first to minimize ordering effects. Self- and provider-collected samples were tested for 13 hrHPV DNA types using a DNA Hybridization Assay. The primary outcome variable was the concordance (kappa statistic) and performance (sensitivity, specificity) of self-collected vaginal HPV DNA specimens versus provider-collected cervical HPV swabs as the gold standard. Results Of the 131 participants completing both the self- and provider-collected HPV tests, 21 cases of hrHPV were detected by the provider cervical swab (gold standard; 16.0% hrHPV prevalence); 15 of these cases were accurately detected by the self-collected vaginal swab (71.4% concordance) (Kappa = 0.75, 95% Confidence Interval [CI]: 0.59, 0.92; p<0.001). Compared to the provider-collected cervical hrHPV DNA sample (gold standard), the self-collected vaginal hrHPV DNA test demonstrated a sensitivity of 71.4% (95% CI: 0.52, 0.91; p = 0.0495) and specificity of 98.2% (95% CI: 0.96, 1

Test performance and acceptability of self- versus provider-collected swabs for high-risk HPV DNA testing in female-to-male trans masculine patients.

PubMed

Reisner, Sari L; Deutsch, Madeline B; Peitzmeier, Sarah M; White Hughto, Jaclyn M; Cavanaugh, Timothy P; Pardee, Dana J; McLean, Sarah A; Panther, Lori A; Gelman, Marcy; Mimiaga, Matthew J; Potter, Jennifer E

2018-01-01

High-risk human papillomavirus (hrHPV) causes virtually all cervical cancers. Trans masculine (TM) people (those assigned female at birth who identify with a gender other than female) have low uptake of conventional cervical cancer screening. Self-collected hrHPV DNA testing has high levels of acceptability among cisgender (non-transgender) females and may support increased cervical cancer screening uptake in TM individuals. To assess the test performance and acceptability of self-collected vaginal specimens in comparison to provider-collected cervical swabs for hrHPV DNA detection in TM individuals ages 21-64 years. Between March 2015-September 2016, 150 TM participants with a cervix (mean age = 27.5 years; SD = 5.7) completed a one-time study visit comprised of a self-report survey, self-collected vaginal HPV DNA swab, clinician-administered cervical HPV swab, and brief interview on acceptability of clinical procedures. Participants were randomized to complete either self- or provider-collection first to minimize ordering effects. Self- and provider-collected samples were tested for 13 hrHPV DNA types using a DNA Hybridization Assay. The primary outcome variable was the concordance (kappa statistic) and performance (sensitivity, specificity) of self-collected vaginal HPV DNA specimens versus provider-collected cervical HPV swabs as the gold standard. Of the 131 participants completing both the self- and provider-collected HPV tests, 21 cases of hrHPV were detected by the provider cervical swab (gold standard; 16.0% hrHPV prevalence); 15 of these cases were accurately detected by the self-collected vaginal swab (71.4% concordance) (Kappa = 0.75, 95% Confidence Interval [CI]: 0.59, 0.92; p<0.001). Compared to the provider-collected cervical hrHPV DNA sample (gold standard), the self-collected vaginal hrHPV DNA test demonstrated a sensitivity of 71.4% (95% CI: 0.52, 0.91; p = 0.0495) and specificity of 98.2% (95% CI: 0.96, 1.00; p<0.0001). Over 90% of participants

Opportunities for embryo transfer in the age of DNA testing

USDA-ARS?s Scientific Manuscript database

Embryo transfer (ET) has contributed to increasing selection intensity in cattle breeding for many years. Preimplantation DNA testing offers the opportunity to increase selection response further through increasing within-family selection intensity. Further increases in between-family selection inte...

Impact of DNA testing for early-onset familial Alzheimer disease and frontotemporal dementia.

PubMed

Steinbart, E J; Smith, C O; Poorkaj, P; Bird, T D

2001-11-01

DNA testing of persons at risk for hereditary, degenerative neurologic diseases is relatively new. Only anecdotal reports of such testing in familial Alzheimer disease (FAD) exist, and little is know about the personal and social impact of such testing. In a descriptive, observational study, individuals at 50% risk for autosomal dominant, early-onset FAD or frontotemporal dementia with parkinsonism linked to chromosome 17 underwent DNA testing for the genetic mutations previously identified in affected family members. Individuals were followed up for (1/2) to 3 years and were interviewed regarding attitudes toward the testing process and the impact of the results. Twenty-one (8.4%) of 251 persons at risk for FAD or frontotemporal dementia requested genetic testing. The most common reasons for requesting testing were concern about early symptoms of dementia, financial or family planning, and relief from anxiety. Twelve individuals had positive DNA test results, and 6 of these had early symptoms of dementia; 8 had negative results; and 1 has not yet received results. Of 14 asymptomatic individuals completing testing, 13 believed the testing was beneficial. Two persons reported moderate anxiety and 1 reported moderate depression. As expected, persons with negative test results had happier experiences overall, but even they had to deal with ongoing anxiety and depression. Thus far, there have been no psychiatric hospitalizations, suicide attempts, or denials of insurance. Genetic testing in early-onset FAD and frontotemporal dementia can be completed successfully. Most individuals demonstrate effective coping skills and find the testing to be beneficial, but long-term effects remain unknown.

Positive view and increased likely uptake of follow-up testing with analysis of cell-free fetal DNA as alternative to invasive testing among Danish pregnant women.

PubMed

Miltoft, Caroline B; Rode, Line; Tabor, Ann

2018-05-01

The aim of this study was to investigate the attitude (view, likely uptake and preferred strategy) towards cell-free fetal DNA (cfDNA) testing among pregnant women before a first-trimester risk assessment for trisomy 21 (unselected women) and after obtaining a high risk. Unselected and high-risk women attending first-trimester screening (Rigshospitalet, Copenhagen University Hospital) were invited to fill out the questionnaire Antenatal testing for Down syndrome as an online survey. The survey included 203 unselected and 50 high-risk women (response rates of 74.8% and 84.7%, respectively). Nearly all considered cfDNA testing a positive development in antenatal care, and 97.2% would like it to be offered. Offering cfDNA testing as an alternative to invasive testing would increase the uptake of follow-up testing compared with invasive testing alone (98.8% vs. 90.7%, p < 0.001). Women who would only accept follow up by cfDNA testing were more likely to continue an affected pregnancy (30.0% vs. 3.6%, p < 0.001) or have doubts about termination (50.0% vs. 32.1%, p < 0.001). Offering cfDNA testing would likely increase the uptake of follow-up testing without a corresponding rise in the termination rate of affected fetuses as some women test for information only. However, both unselected and high-risk women had overwhelmingly positive views underlining attention to avoid routinization. © 2018 Nordic Federation of Societies of Obstetrics and Gynecology.

Cell-free fetal DNA testing: a pilot study of obstetric healthcare provider attitudes toward clinical implementation.

PubMed

Sayres, Lauren C; Allyse, Megan; Norton, Mary E; Cho, Mildred K

2011-11-01

To provide a preliminary assessment of obstetric healthcare provider opinions surrounding implementation of cell-free fetal DNA testing. A 37-question pilot survey was used to address questions around the translation and use of non-invasive prenatal testing using cell-free fetal DNA. The survey was distributed and collected at a Continuing Medical Education course on obstetrics and gynecology. Of 62 survey respondents, 73% were female and 87% held MD/DO degrees. Respondents generally agreed that patients want prenatal diagnostic information to help make decisions about a pregnancy and that cell-free fetal DNA testing would encourage the testing of more patients for more conditions. However, there was an overall lack of knowledge or conviction about using this technology. Genetic counseling and professional society approval were deemed important to implementation, whereas the possibility of direct-to-consumer testing and government regulation produced mixed responses. Respondents indicated that they would be more likely to offer cell-free fetal DNA testing for chromosomal abnormalities and single-gene disorders, but would be cautious with respect to determination of sex and behavioral or late-onset conditions. Preliminary assessment indicates uncertainty among obstetric providers about the details of implementing cell-free fetal DNA testing and suggests expanded research on perspectives of this stakeholder group. Copyright © 2011 John Wiley & Sons, Ltd.

Cost-effectiveness of human papillomavirus DNA testing in the United Kingdom, The Netherlands, France, and Italy.

PubMed

Kim, Jane J; Wright, Thomas C; Goldie, Sue J

2005-06-15

European countries with established cytology-based screening programs for cervical cancer will soon face decisions about whether to incorporate human papillomavirus (HPV) DNA testing and what strategies will be most cost-effective. We assessed the cost-effectiveness of incorporating HPV DNA testing into existing cervical cancer screening programs in the United Kingdom, The Netherlands, France, and Italy. We created a computer-based model of the natural history of cervical carcinogenesis for each using country-specific data on cervical cancer risk and compared each country's current screening policy with two new strategies: 1) cytology throughout a woman's lifetime, using HPV DNA testing as a triage strategy for equivocal cytology results ("HPV triage"), as well as 2) cytology until age 30 years and HPV DNA testing in combination with cytology in women more than 30 years of age ("combination testing"). Outcomes included reduction in lifetime cervical cancer risk, increase in life expectancy, lifetime costs, and incremental cost-effectiveness ratios, expressed as cost per year of life saved. We explored alternative protocols and conducted sensitivity analysis on key parameters of the model over a relevant range of values to identify the most cost-effective options for each country. Both HPV DNA testing strategies, HPV triage and combination testing, were more effective than each country's status quo screening policy. Incremental cost-effectiveness ratios for HPV triage were less than $13,000 per year of life saved, whereas those for combination testing ranged from $9800 to $75,900 per year of life saved, depending on screening interval. We identified options that would be very cost-effective (i.e., cost-effectiveness ratio less than the gross domestic product per capita) in each of the four countries. HPV DNA testing has the potential to improve health benefits at a reasonable cost compared with current screening policies in four European countries.

Performance of high titre attenuated canine parvovirus vaccine in pups with maternally derived antibody.

PubMed

Burtonboy, S; Charlier, P; Hertoghs, J; Lobmann, M; Wiseman, A; Woods, S

1991-04-20

The performance of live, attenuated, homologous, canine parvovirus vaccines was studied in 140 puppies aged from four to 11 weeks. In the presence of maternally derived antibody the ability of the vaccines to elicit a serological response, as determined by the haemagglutination inhibition test and a standardised ELISA, was found to be dose (infectious titre) related. An experimental vaccine containing 10(7.0) TCID50 of virus induced seroconversion rates of 95, 89, 82 and 44 per cent in dogs with haemagglutination inhibition antibody titres of less than or equal to 8, 16, 32 and greater than 32, respectively. The standardised ELISA appeared to be better than the haemagglutination inhibition test with respect to variability and subjectivity, especially when titres were low.

A leukocyte activation test identifies food items which induce release of DNA by innate immune peripheral blood leucocytes.

PubMed

Garcia-Martinez, Irma; Weiss, Theresa R; Yousaf, Muhammad N; Ali, Ather; Mehal, Wajahat Z

2018-01-01

Leukocyte activation (LA) testing identifies food items that induce a patient specific cellular response in the immune system, and has recently been shown in a randomized double blinded prospective study to reduce symptoms in patients with irritable bowel syndrome (IBS). We hypothesized that test reactivity to particular food items, and the systemic immune response initiated by these food items, is due to the release of cellular DNA from blood immune cells. We tested this by quantifying total DNA concentration in the cellular supernatant of immune cells exposed to positive and negative foods from 20 healthy volunteers. To establish if the DNA release by positive samples is a specific phenomenon, we quantified myeloperoxidase (MPO) in cellular supernatants. We further assessed if a particular immune cell population (neutrophils, eosinophils, and basophils) was activated by the positive food items by flow cytometry analysis. To identify the signaling pathways that are required for DNA release we tested if specific inhibitors of key signaling pathways could block DNA release. Foods with a positive LA test result gave a higher supernatant DNA content when compared to foods with a negative result. This was specific as MPO levels were not increased by foods with a positive LA test. Protein kinase C (PKC) inhibitors resulted in inhibition of positive food stimulated DNA release. Positive foods resulted in CD63 levels greater than negative foods in eosinophils in 76.5% of tests. LA test identifies food items that result in release of DNA and activation of peripheral blood innate immune cells in a PKC dependent manner, suggesting that this LA test identifies food items that result in release of inflammatory markers and activation of innate immune cells. This may be the basis for the improvement in symptoms in IBS patients who followed an LA test guided diet.

Noninvasive Prenatal Paternity Testing (NIPAT) through Maternal Plasma DNA Sequencing: A Pilot Study.

PubMed

Jiang, Haojun; Xie, Yifan; Li, Xuchao; Ge, Huijuan; Deng, Yongqiang; Mu, Haofang; Feng, Xiaoli; Yin, Lu; Du, Zhou; Chen, Fang; He, Nongyue

2016-01-01

Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future.

Evaluation of six presumptive tests for blood, their specificity, sensitivity, and effect on high molecular-weight DNA.

PubMed

Tobe, Shanan S; Watson, Nigel; Daéid, Niamh Nic

2007-01-01

Luminol, leuchomalachite green, phenolphthalein, Hemastix, Hemident, and Bluestar are all used as presumptive tests for blood. In this study, the tests were subjected to dilute blood (from 1:10,000 to 1:10,000,000), many common household substance, and chemicals. Samples were tested for DNA to determine whether the presumptive tests damaged or destroyed DNA. The DNA loci tested were D2S1338 and D19S433. Leuchomalachite green had a sensitivity of 1:10,000, while the remaining tests were able to detect blood to a dilution of 1:100,000. Substances tested include saliva, semen, potato, tomato, tomato sauce, tomato sauce with meat, red onion, red kidney bean, horseradish, 0.1 M ascorbic acid, 5% bleach, 10% cupric sulfate, 10% ferric sulfate, and 10% nickel chloride. Of all the substances tested, not one of the household items reacted with every test; however, the chemicals did. DNA was recovered and amplified from luminol, phenolphthalein, Hemastix, and Bluestar, but not from leuchomalachite green or Hemident.

A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

PubMed

Thierry, Alain R

2016-01-01

Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics

Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

DTIC Science & Technology

2012-12-12

immunogenic hantavirus M gene-based DNA vaccines against the HFRS hantaviruses , we ini- tiated preclinical testing of these vaccines, delivered using a...Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR (S) 5d. PROJECT...Vaccination with pWRG/ PUU-M(s2) protected hamsters against infection with PUUV but not against infection by related HFRS-associated hantaviruses

A Neutrality Test for Detecting Selection on DNA Methylation Using Single Methylation Polymorphism Frequency Spectrum

PubMed Central

Wang, Jun; Fan, Chuanzhu

2015-01-01

Inheritable epigenetic mutations (epimutations) can contribute to transmittable phenotypic variation. Thus, epimutations can be subject to natural selection and impact the fitness and evolution of organisms. Based on the framework of the modified Tajima’s D test for DNA mutations, we developed a neutrality test with the statistic “Dm” to detect selection forces on DNA methylation mutations using single methylation polymorphisms. With computer simulation and empirical data analysis, we compared the Dm test with the original and modified Tajima’s D tests and demonstrated that the Dm test is suitable for detecting selection on epimutations and outperforms original/modified Tajima’s D tests. Due to the higher resetting rate of epimutations, the interpretation of Dm on epimutations and Tajima’s D test on DNA mutations could be different in inferring natural selection. Analyses using simulated and empirical genome-wide polymorphism data suggested that genes under genetic and epigenetic selections behaved differently. We applied the Dm test to recently originated Arabidopsis and human genes, and showed that newly evolved genes contain higher level of rare epialleles, suggesting that epimutation may play a role in origination and evolution of genes and genomes. Overall, we demonstrate the utility of the Dm test to detect whether the loci are under selection regarding DNA methylation. Our analytical metrics and methodology could contribute to our understanding of evolutionary processes of genes and genomes in the field of epigenetics. The Perl script for the “Dm” test is available at http://fanlab.wayne.edu/ (last accessed December 18, 2014). PMID:25539727

Utilisation of co-testing 
(human papillomavirus DNA testing and cervical cytology) after treatment of CIN: - a survey of GPs' awareness and knowledge.

PubMed

Munro, Aime; Codde, Jim; Semmens, James; Leung, Yee; Spilsbury, Katrina; Williams, Vincent; Steel, Nerida; Cohen, Paul; Pavicic, Heidi; Westoby, Vicki; O'Leary, Peter

2015-01-01

Patients have an increased risk of persistent/recurrent cervical disease if they received treatment for a high-grade squamous intraepithelial lesion (HSIL). Consequently, understanding whether co-testing (human papillomavirus [HPV] DNA testing and cervical cytology) is fully utilised by general practitioners (GPs) is paramount. After consultation with key stakeholders, an anonymous, self-completion questionnaire was developed and disseminated to GPs who had provided cervical cytology. Responses were received from 745 GPs (30.9% response rate). A significant number (34.3%) of GPs were unaware of the use of co-testing (HPV DNA testing and cervical cytology) for the management of patients after HSIL treatment. Additionally, the majority of GPs reported they did not 'always' receive a clear follow-up plan for patients after treatment of an HSIL. GPs require further support and education to ensure successful adoption of co-testing (HPV DNA testing and cervical cytology), specifically, for patients treated for an HSIL.

Interest of proviral HIV-1 DNA genotypic resistance testing in virologically suppressed patients candidate for maintenance therapy.

PubMed

Allavena, C; Rodallec, A; Leplat, A; Hall, N; Luco, C; Le Guen, L; Bernaud, C; Bouchez, S; André-Garnier, E; Boutoille, D; Ferré, V; Raffi, F

2018-01-01

Switch of antiretroviral therapy in virologically suppressed HIV-infected patients is frequent, to prevent toxicities, for simplification or convenience reasons. Pretherapeutic genotypic resistance testing on RNA can be lacking in some patients, which could enhance the risk of virologic failure, if resistance-associated mutations of the new regimen are not taken into account. Proviral DNA resistance testing in 69 virologically suppressed patients on antiretroviral treatment with no history of virological failure were pair-wised compared with pre-ART plasma RNA resistance testing. The median time between plasma (RNA testing) and whole blood (proviral DNA testing) was 47 months (IQR 29-63). A stop codon was evidenced in 23% (16/69) of proviral DNA sequences; these strains were considered as defective, non-replicative, and not taken into consideration. Within the non defective strains, concordance rate between plasma RNA and non-defective proviral DNA was high both on protease (194/220 concordant resistance-associated mutations=88%) and reverse transcriptase (28/37 concordant resistance-associated mutations=76%) genes. This study supports that proviral DNA testing might be an informative tool before switching antiretrovirals in virologically suppressed patients with no history of virological failure, but the interpretation should be restricted to non-defective viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

Estimation of the Proportion of Genetic Variation Accounted for by DNA Tests

USDA-ARS?s Scientific Manuscript database

An increasingly relevant question in evaluating commercial DNA tests is "What proportion of the additive genetic variation in the target trait is accounted for by the test?" Therefore, several estimators of this quantity were evaluated by simulation of a population of 1000 animals with 100 sires, ea...

First-trimester contingent screening for trisomies 21, 18 and 13 by biomarkers and maternal blood cell-free DNA testing.

PubMed

Nicolaides, K H; Syngelaki, A; Poon, L C; Gil, M M; Wright, D

2014-01-01

To examine potential performance of screening for trisomies by cell-free (cf) DNA testing in maternal blood contingent on results of first-line testing by combinations of fetal translucency thickness (NT), fetal heart rate (FHR), ductus venosus pulsatility index (DV PIV), and serum-free β-human chorionic gonadotropin (β-hCG), pregnancy-associated plasma protein-A (PAPP-A), placental growth factor (PLGF) and α-fetoprotein (AFP). Performance was estimated for firstly, screening by cfDNA in all pregnancies and secondly, cfDNA testing contingent on results of first-line testing by combinations of ultrasound and biochemical markers. In first-line screening by cfDNA testing, the detection rate for trisomy 21 and trisomies 18 or 13 would be 99 and 96%, respectively, after invasive testing in 1% of the population. In contingent screening, a detection rate of 98% for trisomy 21 and 96% for trisomy 18 or 13, at an invasive testing rate of 0.7%, can be achieved by carrying out cfDNA testing in about 35, 20 and 11% of cases identified by first-line screening with the combined test alone (age, NT, FHR, β-hCG, PAPP-A), the combined test plus PLGF and AFP and the combined test plus PLGF, AFP and DV PIV, respectively. Effective first-trimester screening for trisomies can be achieved by contingent screening incorporating biomarkers and cfDNA testing. © 2013 S. Karger AG, Basel.

Application of Optical Fibers to DNA’s Testing Program.

DTIC Science & Technology

1980-10-15

economic impact. In addition to benefitting UGT , advances in fiber optic technology can greatly impact other DNA activities such as hardening of military...components and simulation and testing in high radiation environments. Using the UGT environment as a test bed, optical fibers can be characterized in...OPTIC SYSTEMS 33 3-3.1 Active System Design 37 4 USE OF FIBERS IN UGT 47 4-1 ADVANTAGES OF FIBERS FOR UGT 47 4-2 DIAGNOSTIC APPLICATIONS 4-3 EFFECTS

Is maternal plasma DNA testing impacting serum-based screening for aneuploidy in the United States?

PubMed

Palomaki, Glenn E; Ashwood, Edward R; Best, Robert G; Lambert-Messerlian, Geralyn; Knight, George J

2015-11-01

We sought to determine whether tests for fetal aneuploidy based on next-generation sequencing of cell-free DNA in maternal circulation have had an impact on routine serum-based screening in the general pregnant population. We compared results from laboratory surveys in 2011 and 2014 that reported types of prenatal serum screening tests and numbers of tests performed. Testing records from two prenatal serum screening laboratories examined temporal trends in the proportion of screened women 35 years of age and older from 2008 (or 2009) to 2014. The 82 laboratory survey results available for comparison showed that 1.7 million women were screened in 2014, a 5% increase over 2011. In the two screening laboratories, the proportion of screened women age 35 and older increased for several years but then experienced reductions of 8 and 18% by mid-2014 when compared with the highest rates observed. As of 2014, maternal plasma DNA testing appears to have had only a minor impact on serum screening rates in the United States. Ongoing surveillance has the potential to determine if, and when, DNA testing begins to replace serum testing as a primary screen for Down syndrome in the United States.

A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA.

PubMed

Ramos, Andrea Estefanía; Muñoz, Marina; Cortés-Vecino, Jesús Alfredo; Barato, Paola; Patarroyo, Manuel Alfonso

2017-11-29

Neospora caninum is a cyst-forming, coccidian parasite which is known to cause neurological disorders in dogs and abortion and neonatal mortality in cows and other livestock. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay based on the Neospora caninum Nc-5 gene and compares its efficacy for detecting DNA to that of a semi-nested PCR test. Six primers were designed based on the Nc-5 repeat region of N. caninum. Specific LAMP primers led to successful amplification of N. caninum DNA at 63 °C in 30 min. The LAMP assay was highly specific (i.e. it did not reveal cross-reactivity with other parasite species) and had a low N. caninum plasmid DNA limit of detection (1 fg), which is ten times higher than that for the semi-nested PCR. LAMP applicability was evaluated using a set of naturally-infected samples (59 from canine faeces and five from bovine abortions). Thirty-nine percent (25/64) of the naturally-infected samples were positive for N. caninum DNA by LAMP and 36% (23/64) by semi-nested PCR. However, the LAMP assay is much faster to perform than semi-nested PCR and provides results in 30 min. The optimized reaction conditions described in this study resulted in a sensitive, specific and rapid technique for detecting N. caninum DNA. Considering the advantages of LAMP for detecting N. caninum DNA, further assays aimed at testing its usefulness on a wider range of field samples are recommended.

Blood free-circulating DNA testing by highly sensitive methylation assay to diagnose colorectal neoplasias.

PubMed

Suehiro, Yutaka; Hashimoto, Shinichi; Higaki, Shingo; Fujii, Ikuei; Suzuki, Chieko; Hoshida, Tomomi; Matsumoto, Toshihiko; Yamaoka, Yuko; Takami, Taro; Sakaida, Isao; Yamasaki, Takahiro

2018-03-30

Although methylated TWIST1 is a biomarker of colorectal neoplasia, its detection from serum samples is very difficult by conventional bisulfite-based methylation assays. Therefore, we have developed a new methylation assay that enables counting of even one copy of a methylated gene in a small DNA sample amount without DNA bisulfite treatment. We performed this study to evaluate the sensitivity and specificity of serum DNA testing by the new methylation assay in combination with and without the fecal immunochemical test for hemoglobin for the detection of colorectal neoplasia. This study comprised 113 patients with colorectal neoplasia and 25 control individuals. For the new methylation assay, DNA was treated in two stages with methylation-sensitive restriction enzymes, followed by measurement of copy numbers of hTERT and methylated TWIST1 by multiplex droplet digital PCR. The fecal immunochemical test had a sensitivity of 8.0% for non-advanced adenoma, 24.3% for advanced adenoma, and 44.4% for colorectal cancer, and a specificity of 88.0%. The new assay had a sensitivity of 36.0% for non-advanced adenoma, 30.0% for advanced adenoma, and 44.4% for colorectal cancer, and a specificity of 92.0%. Combination of the both tests increased the sensitivity to 40.0%, 45.7%, and 72.2% for the detection of non-advanced adenoma, advanced adenoma, and colorectal cancer, respectively, and resulted in a specificity of 84.0%. Combination of both tests may provide an alternative screening strategy for colorectal neoplasia including potentially precancerous lesions and colorectal cancer.

Feasibility of cell-free circulating tumor DNA testing for lung cancer.

PubMed

Santarpia, Mariacarmela; Karachaliou, Niki; González-Cao, Maria; Altavilla, Giuseppe; Giovannetti, Elisa; Rosell, Rafael

2016-01-01

Tumor tissue genotyping is used routinely for lung cancer to identify specific targetable oncogenic alterations, including EGFR mutations and ALK rearrangements. However, tumor tissue from a single biopsy is often insufficient for molecular testing, may offer a limited evaluation because of tumor heterogeneity and can be difficult to obtain. Cell-free circulating tumor DNA has been widely investigated as a potential surrogate for tissue biopsy for noninvasive assessment of tumor-related genomic alterations. New techniques have improved EGFR mutations detection in ctDNA, thus supporting the use of this liquid biopsy for predicting response to EGFR tyrosine kinase inhibitors (TKIs) and monitoring the emergence of resistance. The serial evaluation of ctDNA during treatment is feasible and can be used to track tumor changes in real time and for a wide range of clinically useful applications.

Testing the Use of Implicit Solvent in the Molecular Dynamics Modelling of DNA Flexibility

NASA Astrophysics Data System (ADS)

Mitchell, J.; Harris, S.

DNA flexibility controls packaging, looping and in some cases sequence specific protein binding. Molecular dynamics simulations carried out with a computationally efficient implicit solvent model are potentially a powerful tool for studying larger DNA molecules than can be currently simulated when water and counterions are represented explicitly. In this work we compare DNA flexibility at the base pair step level modelled using an implicit solvent model to that previously determined from explicit solvent simulations and database analysis. Although much of the sequence dependent behaviour is preserved in implicit solvent, the DNA is considerably more flexible when the approximate model is used. In addition we test the ability of the implicit solvent to model stress induced DNA disruptions by simulating a series of DNA minicircle topoisomers which vary in size and superhelical density. When compared with previously run explicit solvent simulations, we find that while the levels of DNA denaturation are similar using both computational methodologies, the specific structural form of the disruptions is different.

Putting it all behind: long-term psychological impact of an inconclusive DNA test result for breast cancer.

PubMed

van Dijk, Sandra; Otten, Wilma; Tollenaar, Rob A E M; van Asperen, Christi J; Tibben, Aad

2008-10-01

An inconclusive DNA-result for breast cancer may leave women with uncertainty that cannot be relieved. We assessed the influence of beliefs women held about their inconclusive DNA-result on psychological well-being and whether women had been able to put the period of DNA testing behind them. In total, 215 women completed a baseline and a follow-up questionnaire 2.5 till 7 years after DNA test disclosure. Within the group of 147 women who received an inconclusive result (either a personal result or the result of an affected family member) multiple regression analyses were applied to investigate the relevance of women's personal beliefs. Personal beliefs and ambivalence about an inconclusive DNA-result were associated with cancer-related worry and distress (P < 0.05). Moreover, these beliefs seemed to be an especially strong predictor of whether women had been able to leave the period of DNA testing behind them, even after controlling for all measures of psychological distress (P < 0.001). Psychological distress measures may provide an important but incomplete picture of how women make sense of an inconclusive DNA-result. These findings underscore the importance of discussing counselees' beliefs and expectations openly to enhance well-being and adaptation on the long term.

Forgotten evidence: A mixed methods study of why sexual assault kits (SAKs) are not submitted for DNA forensic testing.

PubMed

Campbell, Rebecca; Fehler-Cabral, Giannina; Bybee, Deborah; Shaw, Jessica

2017-10-01

Throughout the United States, hundreds of thousands of sexual assault kits (SAKs) (also termed "rape kits") have not been submitted by the police for forensic DNA testing. DNA evidence can help sexual assault investigations and prosecutions by identifying offenders, revealing serial offenders through DNA matches across cases, and exonerating those who have been wrongly accused. In this article, we describe a 5-year action research project conducted with 1 city that had large numbers of untested SAKs-Detroit, Michigan-and our examination into why thousands of rape kits in this city were never submitted for forensic DNA testing. This mixed methods study combined ethnographic observations and qualitative interviews to identify stakeholders' perspectives as to why rape kits were not routinely submitted for testing. Then, we quantitatively examined whether these factors may have affected police practices regarding SAK testing, as evidenced by predictable changes in SAK submission rates over time. Chronic resource scarcity only partially explained why the organizations that serve rape victims-the police, crime lab, prosecution, and victim advocacy-could not test all rape kits, investigate all reported sexual assaults, and support all rape survivors. SAK submission rates significantly increased once criminal justice professionals in this city had full access to the FBI DNA forensic database Combined DNA Index System (CODIS), but even then, most SAKs were still not submitted for DNA testing. Building crime laboratories' capacities for DNA testing and training police on the utility of forensic evidence and best practices in sexual assault investigations can help remedy, and possibly prevent, the problem of untested rape kits. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

A Prospective Blinded Evaluation of Urine-DNA Testing for Detection of Urothelial Bladder Carcinoma in Patients with Gross Hematuria.

PubMed

Dahmcke, Christina M; Steven, Kenneth E; Larsen, Louise K; Poulsen, Asger L; Abdul-Al, Ahmad; Dahl, Christina; Guldberg, Per

2016-12-01

Retrospective studies have provided proof of principle that bladder cancer can be detected by testing for the presence of tumor DNA in urine. We have conducted a prospective blinded study to determine whether a urine-based DNA test can replace flexible cystoscopy in the initial assessment of gross hematuria. A total of 475 consecutive patients underwent standard urological examination including flexible cystoscopy and computed tomography urography, and provided urine samples immediately before (n=461) and after (n=444) cystoscopy. Urine cells were collected using a filtration device and tested for eight DNA mutation and methylation biomarkers. Clinical evaluation identified 99 (20.8%) patients with urothelial bladder tumors. With this result as a reference and based on the analysis of all urine samples, the DNA test had a sensitivity of 97.0%, a specificity of 76.9%, a positive predictive value of 52.5%, and a negative predictive value of 99.0%. In three patients with a positive urine-DNA test without clinical evidence of cancer, a tumor was detected at repeat cystoscopy within 16 mo. Our results suggest that urine-DNA testing can be used to identify a large subgroup of patients with gross hematuria in whom cystoscopy is not required. We tested the possibility of using a urine-based DNA test to check for bladder cancer in patients with visible blood in the urine. Our results show that the test efficiently detects bladder cancer and therefore may be used to greatly reduce the number of patients who would need to undergo cystoscopy. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Clinico-laboratory aspects of anti-nuclear and anti-native DNA antibody tests.

PubMed

Webb, J

1978-01-01

Available techniques for detection of anti-nuclear antibodies are here briefly reviewed. The relatively insensitive LE cell test has been largely supplanted by the indirect immunofluorescent ANA test which should be reported in terms of titre and pattern. Specific measurement of nDNA antibodies is now a regular technique in SLE diagnosis and management.

Maternal plasma DNA testing: experience of women counseled at a prenatal diagnosis center.

PubMed

O'Brien, Barbara M; Kloza, Edward M; Halliday, Jacquelyn V; Lambert-Messerlian, Geralyn M; Palomaki, Glenn E

2014-10-01

To evaluate the early introduction of circulating cell-free (ccf) DNA testing in a prenatal diagnosis center serving a statewide population. A retrospective chart review of patients at high aneuploidy risk counseled during the two 10-week periods that documents indication, risk, maternal age, insurance coverage, decisions, and reasoning behind that decision. Among the 299 included women, indication was advanced maternal age (17% with and 56% without an additional indication), positive serum screen (15%), and abnormal ultrasound (12%). Uptake increased from 10% to 17%, as did patient awareness of the test (4% to 14%). Women with lower copayments were more likely to complete testing (23% vs. 5%, p<0.001). Most women completing testing (75%) wanted to avoid an invasive procedure, while those declining cited testing would not change anything (47%), preferred diagnostic testing (16%), negative follow-up testing (20%), and cost/insurance issues (9%). One of 42 tests was positive (trisomy 21). Individual patient follow-up allows us to document ccfDNA-related patient decision-making. Nearly half of the women did not want further testing and one in seven preferred immediate diagnostic testing. Patient costs were a barrier to testing that, if avoided, could increase test uptake by 50% or more.

Pitfalls of Establishing DNA Barcoding Systems in Protists: The Cryptophyceae as a Test Case

PubMed Central

Hoef-Emden, Kerstin

2012-01-01

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5′-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed. PMID:22970104

Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

PubMed

Hoef-Emden, Kerstin

2012-01-01

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5'-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

Screening for Sex Chromosome Aneuploidy by Cell-Free DNA Testing: Patient Choice and Performance.

PubMed

Bevilacqua, Elisa; Ordóñez, Elena; Hurtado, Ivan; Rueda, Laura; Mazzone, Eléonora; Cirigliano, Vincenzo; Jani, Jacques C

2017-08-23

To study patient choice regarding testing for sex chromosome aneuploidy (SCA) and the performance of cell-free DNA (cfDNA) screening for SCA. Patient choice regarding screening for SCA and factors influencing this choice were evaluated in a single center. In a subsequent two-center study, cases that screened positive for SCA were analyzed to determine the positive predictive value (PPV) for each SCA. In all, 1,957 (61.9%) of the 3,162 patients undergoing cfDNA testing opted for SCA screening. Regression analysis demonstrated that independent predictors of a patient's decision for SCA were earlier gestational age, spontaneous conception, and cfDNA chosen as a primary method of screening. A total of 161 cases screened positive for SCA and follow-up data were available for 118 (73.3%). Forty-six of the 61 cases of 45,X were false-positive results and 15 were concordant with the fetal karyotype (PPV = 24.6%). Seventeen of the 22 cases of 47,XXX were false positive and 5 concordant (PPV = 22.7%). Eleven of the 30 cases of 47,XXY were false positive and 19 concordant (PPV = 63.3%). All 5 cases of 47,XYY were correctly identified, thus yielding a PPV of 100%. More than half of the patients undergoing cfDNA aneuploidy screening also opted for SCA testing, but they were less likely to do so in the presence of an increased risk of trisomy. SCAs involving the X chromosome had a lower PPV than those involving the Y chromosome. © 2017 S. Karger AG, Basel.

DNA vaccination protects mice against Zika virus-induced damage to the testes

PubMed Central

Griffin, Bryan D.; Muthumani, Kar; Warner, Bryce M.; Majer, Anna; Hagan, Mable; Audet, Jonathan; Stein, Derek R.; Ranadheera, Charlene; Racine, Trina; De La Vega, Marc-Antoine; Piret, Jocelyne; Kucas, Stephanie; Tran, Kaylie N.; Frost, Kathy L.; De Graff, Christine; Soule, Geoff; Scharikow, Leanne; Scott, Jennifer; McTavish, Gordon; Smid, Valerie; Park, Young K.; Maslow, Joel N.; Sardesai, Niranjan Y.; Kim, J. Joseph; Yao, Xiao-jian; Bello, Alexander; Lindsay, Robbin; Boivin, Guy; Booth, Stephanie A.; Kobasa, Darwyn; Embury-Hyatt, Carissa; Safronetz, David; Weiner, David B.; Kobinger, Gary P.

2017-01-01

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract. PMID:28589934

Multitarget stool DNA tests increases colorectal cancer screening among previously noncompliant Medicare patients

PubMed Central

Prince, Mark; Lester, Lynn; Chiniwala, Rupal; Berger, Barry

2017-01-01

AIM To determine the uptake of noninvasive multitarget stool DNA (mt-sDNA) in a cohort of colorectal cancer (CRC) screening non-compliant average-risk Medicare patients. METHODS This cross sectional primary care office-based study examined mt-sDNA uptake in routine clinical practice among 393 colorectal cancer screening non-compliant Medicare patients ages 50-85 ordered by 77 physicians in a multispecialty group practice (USMD Physician Services, Dallas, TX) from October, 2014-September, 2015. Investigators performed a Health Insurance Portability and Accountability Act compliant retrospective review of electronic health records to identify mt-sDNA use in patients who were either > 10 years since last colonoscopy and/or > 1 year since last fecal occult blood test. Test positive patients were advised to get diagnostic colonoscopy and thereafter patients were characterized by the most clinically significant lesion documented on histopathology of biopsies or excisional tissue. Descriptive statistics were employed. Key outcome measures included mt-sDNA compliance and diagnostic colonoscopy compliance on positive cases. RESULTS Over 12 mo, 77 providers ordered 393 mt-sDNA studies with 347 completed (88.3% compliance). Patient mean age was 69.8 (50-85) and patients were 64% female. Mt-sDNA was negative in 85.3% (296/347) and positive in 14.7% (51/347). Follow-up colonoscopy was performed in 49 positive patients (96.1% colonoscopy compliance) with two patients lost to follow up. Index findings included: colon cancer (4/49, 8.2%), advanced adenomas (21/49, 42.9%), non-advanced adenomas (15/49, 30.6%), and negative results (9/49, 18.4%). The positive predictive value for advanced colorectal lesions was 51.0% and for any colorectal neoplasia was 81.6%. The mean age of patients with colorectal cancer was 70.3 and all CRC's were localized Stage I (2) and Stage II (2), three were located in the proximal colon and one was located in the distal colon. CONCLUSION Mt-sDNA provided

Multitarget stool DNA tests increases colorectal cancer screening among previously noncompliant Medicare patients.

PubMed

Prince, Mark; Lester, Lynn; Chiniwala, Rupal; Berger, Barry

2017-01-21

To determine the uptake of noninvasive multitarget stool DNA (mt-sDNA) in a cohort of colorectal cancer (CRC) screening non-compliant average-risk Medicare patients. This cross sectional primary care office-based study examined mt-sDNA uptake in routine clinical practice among 393 colorectal cancer screening non-compliant Medicare patients ages 50-85 ordered by 77 physicians in a multispecialty group practice (USMD Physician Services, Dallas, TX) from October, 2014-September, 2015. Investigators performed a Health Insurance Portability and Accountability Act compliant retrospective review of electronic health records to identify mt-sDNA use in patients who were either > 10 years since last colonoscopy and/or > 1 year since last fecal occult blood test. Test positive patients were advised to get diagnostic colonoscopy and thereafter patients were characterized by the most clinically significant lesion documented on histopathology of biopsies or excisional tissue. Descriptive statistics were employed. Key outcome measures included mt-sDNA compliance and diagnostic colonoscopy compliance on positive cases. Over 12 mo, 77 providers ordered 393 mt-sDNA studies with 347 completed (88.3% compliance). Patient mean age was 69.8 (50-85) and patients were 64% female. Mt-sDNA was negative in 85.3% (296/347) and positive in 14.7% (51/347). Follow-up colonoscopy was performed in 49 positive patients (96.1% colonoscopy compliance) with two patients lost to follow up. Index findings included: colon cancer (4/49, 8.2%), advanced adenomas (21/49, 42.9%), non-advanced adenomas (15/49, 30.6%), and negative results (9/49, 18.4%). The positive predictive value for advanced colorectal lesions was 51.0% and for any colorectal neoplasia was 81.6%. The mean age of patients with colorectal cancer was 70.3 and all CRC's were localized Stage I (2) and Stage II (2), three were located in the proximal colon and one was located in the distal colon. Mt-sDNA provided medical benefit to screening

Genetic counselors' experience with cell-free fetal DNA testing as a prenatal screening option for aneuploidy.

PubMed

Horsting, Julie M H; Dlouhy, Stephen R; Hanson, Katelyn; Quaid, Kimberly; Bai, Shaochun; Hines, Karrie A

2014-06-01

First identified in 1997, cell-free fetal DNA (cffDNA) has just recently been used to detect fetal aneuploidy of chromosomes 13, 18, and 21, showing its potential to revolutionize prenatal genetic testing as a non-invasive screening tool. Although this technological advancement is exciting and has certain medical applications, it has been unclear how it will be implemented in a clinical setting. Genetic counselors will likely be instrumental in answering that question, but to date, there is no published research regarding prenatal counselors' implementation of and experiences with cffDNA testing. We developed a 67 question survey to gather descriptive information from counselors regarding their personal opinions, experiences, thoughts, and concerns regarding the validity, usefulness, and implementation of this new technology. A total of 236 individuals completed a portion of the survey; not all respondents answered all questions. Qualitative questions complemented quantitative survey items, allowing respondents to voice their thoughts directly. Results indicate that counselors value cffDNA testing as a screening option but are concerned regarding how some obstetricians and patients make use of this testing. Further results, discussion, and practice implications are presented.

Guidelines for HPV-DNA Testing for Cervical Cancer Screening in Brazil.

PubMed

Zeferino, Luiz Carlos; Bastos, Joana Bragança; Vale, Diama Bhadra Andrade Peixoto do; Zanine, Rita Maria; Melo, Yara Lucia Mendes Furtado de; Primo, Walquíria Quida Salles Pereira; Corrêa, Flávia de Miranda; Val, Isabel Cristina Chulvis do; Russomano, Fábio

2018-06-06

Evidence-based clinical guidelines ensure best practice protocols are available in health care. There is a widespread use of human papillomavirus deoxyribonucleic acid (HPV-DNA) tests in Brazil, regardless of the lack of official guidelines. On behalf of the Brazilian Association for the Lower Genital Tract Pathology and Colposcopy (ABPTGIC, in the Portuguese acronym), a team of reviewers searched for published evidence and developed a set of recommendations for the use of HPV-DNA tests in cervical cancer screening in Brazil. The product of this process was debated and consensus was sought by the participants. One concern of the authors was the inclusion of these tests in the assessment of women with cytologic atypia and women treated for cervical intraepithelial neoplasia (CIN). Testing for HPV is recommended in an organized screening scenario to identify women with precursor lesions or asymptomatic cervical cancer older than 30 years of age, and it can be performed every 5 years. It also has value after the cytology showing atypical squamous cells of undetermined significance (ASC-US) or low-grade squamous intraepithelial lesions (LSILs) as a triage test for colposcopy, in the investigation of other cytological alterations when no abnormal findings are observed at colposcopy, seeking to exclude disease, or, further, after treatment of high-grade cervical intraepithelial neoplasia, to rule out residual disease. Thieme Revinter Publicações Ltda Rio de Janeiro, Brazil.

Effects of direct-to-consumer advertising and clinical guidelines on appropriate use of human papillomavirus DNA tests.

PubMed

Price, Rebecca Anhang; Frank, Richard G; Cleary, Paul D; Goldie, Sue J

2011-02-01

Both clinical guidelines and direct-to-consumer (DTC) advertising influence the use of new health care technologies, but little is known about their relative effects. The introduction of a cervical cancer screening test in 2000 offered a unique opportunity to assess the 2 strategies. To evaluate the effects of clinical guidelines and a targeted DTC advertising campaign on overall and appropriate use of human papillomavirus (HPV) DNA tests. Quasi-experimental study using difference-in-differences analysis. Data were MarketScan private insurance claims for 500,000 women aged 21 to 64 enrolled at least 12 consecutive months from January 2001 through December 2005. Both clinical guidelines and DTC advertising were associated with increases in overall HPV DNA test use. DTC advertising was associated with a statistically significant increase in HPV DNA test use in 2 groups of DTC cities (+5.57%, P < 0.0001; +2.54%, P < 0.0001). DTC advertising was associated with comparable increases in the probability of appropriate and inappropriate use of the HPV DNA test in primary screening. Clinical guideline releases from the American College of Obstetricians and Gynecologists, and by a cosponsored panel, were associated with greater increases in HPV DNA tests for appropriate primary screening than for inappropriate primary screening (β = 0.3347, P < 0.05 and β = 0.4175, P < 0.01). DTC advertising was associated with increased overall use of a cervical cancer screening test, whereas clinical guidelines were differentially associated with increased appropriate use. These findings suggest distinct influences of consumer marketing and professional guidelines on the use of health care products and services.

DNA methylome profiling of maternal peripheral blood and placentas reveal potential fetal DNA markers for non-invasive prenatal testing.

PubMed

Xiang, Yuqian; Zhang, Junyu; Li, Qiaoli; Zhou, Xinyao; Wang, Teng; Xu, Mingqing; Xia, Shihui; Xing, Qinghe; Wang, Lei; He, Lin; Zhao, Xinzhi

2014-09-01

Utilizing epigenetic (DNA methylation) differences to differentiate between maternal peripheral blood (PBL) and fetal (placental) DNA has been a promising strategy for non-invasive prenatal testing (NIPT). However, the differentially methylated regions (DMRs) have yet to be fully ascertained. In the present study, we performed genome-wide comparative methylome analysis between maternal PBL and placental DNA from pregnancies of first trimester by methylated DNA immunoprecipitation-sequencing (MeDIP-Seq) and Infinium HumanMethylation450 BeadChip assays. A total of 36 931 DMRs and 45 804 differentially methylated sites (DMSs) covering the whole genome, exclusive of the Y chromosome, were identified via MeDIP-Seq and Infinium 450k array, respectively, of which 3759 sites in 2188 regions were confirmed by both methods. Not only did we find the previously reported potential fetal DNA markers in our identified DMRs/DMSs but also we verified fully the identified DMRs/DMSs in the validation round by MassARRAY EpiTYPER. The screened potential fetal DNA markers may be used for NIPT on aneuploidies and other chromosomal diseases, such as cri du chat syndrome and velo-cardio-facial syndrome. In addition, these potential markers may have application in the early diagnosis of placental dysfunction, such as pre-eclampsia. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Canadian women's attitudes toward noninvasive prenatal testing of fetal DNA in maternal plasma (.).

PubMed

Pariente, Gali; Hasan, Lara; Gadot, Yifat; De Souza, Leanne R; Lebovic, Gerald; Berger, Howard

2016-12-01

To determine the perceptions and attitudes of Canadian women to Noninvasive Prenatal Testing of fetal DNA. A designed questionnaire was administered to women attending the outpatient antenatal clinic at a tertiary urban hospital. Attitudes to current and new prenatal screening modalities were assessed using a five-point Likert scale. Bowker's test of symmetry was used to compare individual responses regarding the two screening modalities. Changes in women's responses pre- and post-delivery were also compared. One hundred and twenty-nine women were enrolled in this study. 88% of women state that they would perform prenatal screening via fetal DNA in the maternal plasma if available. When compared to conventional screening, significantly less women believe that the NIPT should be available upon request for non-medical traits (36.4% versus 60.4%, p < 0.001). When compared to their answer before delivery, more women agreed that screening with fetal DNA in maternal plasma could be used in a negative way to select for desired non-medical traits such as gender. The use of fetal DNA in the maternal plasma is widely accepted in our Canadian population as a future method of noninvasive prenatal screening despite recognition of certain ethical concerns. This information can be used when implementing new genetic screening programs.

Forensic individual age estimation with DNA: From initial approaches to methylation tests.

PubMed

Freire-Aradas, A; Phillips, C; Lareu, M V

2017-07-01

Individual age estimation is a key factor in forensic science analysis that can provide very useful information applicable to criminal, legal, and anthropological investigations. Forensic age inference was initially based on morphological inspection or radiography and only later began to adopt molecular approaches. However, a lack of accuracy or technical problems hampered the introduction of these DNA-based methodologies in casework analysis. A turning point occurred when the epigenetic signature of DNA methylation was observed to gradually change during an individual´s lifespan. In the last four years, the number of publications reporting DNA methylation age-correlated changes has gradually risen and the forensic community now has a range of age methylation tests applicable to forensic casework. Most forensic age predictor models have been developed based on blood DNA samples, but additional tissues are now also being explored. This review assesses the most widely adopted genes harboring methylation sites, detection technologies, statistical age-predictive analyses, and potential causes of variation in age estimates. Despite the need for further work to improve predictive accuracy and establishing a broader range of tissues for which tests can analyze the most appropriate methylation sites, several forensic age predictors have now been reported that provide consistency in their prediction accuracies (predictive error of ±4 years); this makes them compelling tools with the potential to contribute key information to help guide criminal investigations. Copyright © 2017 Central Police University.

Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test ▿

PubMed Central

Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

2011-01-01

There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

Use of bDNA testing in the immunologically nonresponding patient who has a low or undetectable viral load by RT-PCR testing.

PubMed

Grimes, Richard M; Lewis, Stanley T; Visnegarwala, Fehmida; Goodly, Joseph; Sutton, Richard; Rodriguez-Barradas, Maria

2003-01-01

Studies have shown that reverse transcription-polymerase chain reaction (RT-PCR) technology underquantifies viral loads in patients with non-B clades of HIV-1. Testing with bDNA technology gave higher viral loads in these subtypes. A study was conducted to determine whether virologically responding patients on HAART who were not immunologically responding would have higher viral loads using bDNA technology and whether these differences were due to non-B clades. Forty-eight patients receiving HAART for more than 6 months who were having inappropriate immunologic responses in spite of undetectable or very low viral loads determined by RT-PCR (<3000 copies by Roche Amplicor 1.0) were studied. These patients had bDNA viral loads performed. All patients who had bDNA viral loads equivalent to >3000 by RT-PCR had clade and genotypic studies performed. Fifteen patients had viral loads by bDNA that were equivalent to >3000 copies by RT-PCR. Four of these were found to have non-B clades (one D clade and three AG clade). The D clade patient had multidrug resistance; none of the AG clade patients had resistance. Of the remaining 11 patients, virus could not be recovered from 2 and 9 had a B clade. Six of these nine had genotypic resistance to HAART drugs. bDNA testing may be useful in the immunologically nonresponding patient.

First-trimester contingent screening for trisomy 21 by biomarkers and maternal blood cell-free DNA testing.

PubMed

Nicolaides, K H; Wright, D; Poon, L C; Syngelaki, A; Gil, M M

2013-07-01

To define risk cut-offs with corresponding detection rates (DR) and false-positive rates (FPR) in screening for trisomy 21 using maternal age and combinations of first-trimester biomarkers in order to determine which women should undergo contingent maternal blood cell-free (cf) DNA testing. From singleton pregnancies undergoing screening for aneuploidies at three UK hospitals between March 2006 and May 2012, we analyzed prospectively collected data on the following biomarkers: fetal nuchal translucency thickness (NT) and ductus venosus pulsatility index for veins (DV-PIV) at 11 + 0 to 13 + 6 weeks' gestation and serum free β-human chorionic gonadotropin (β-hCG), pregnancy-associated plasma protein-A (PAPP-A), placental growth factor (PlGF) and alpha-fetoprotein (AFP) at 8 + 0 to 13 + 6 weeks. Estimates of risk cut-offs, DRs and FPRs were derived for combinations of biomarkers and these were used to define the best strategy for contingent cfDNA testing. In contingent screening, detection of 98% of fetuses with trisomy 21 at an overall invasive testing rate < 0.5% can be potentially achieved by offering cfDNA testing to about 36%, 21% and 11% of cases identified by first-line screening using the combined test alone, using the combined test with the addition of serum PlGF and AFP and using the combined test with the addition of PlGF, AFP and DV-PIV, respectively. Effective first-trimester screening for trisomy 21, with DR of 98% and invasive testing rate < 0.5%, can be potentially achieved by contingent screening incorporating biomarkers and cfDNA testing. Copyright © 2013 ISUOG. Published by John Wiley & Sons, Ltd.

Screening for trisomies by cell-free DNA testing of maternal blood: consequences of a failed result.

PubMed

Revello, R; Sarno, L; Ispas, A; Akolekar, R; Nicolaides, K H

2016-06-01

First, to report the distribution of the fetal fraction of cell-free (cf) DNA and the rate of a failed cfDNA test result in trisomies 21, 18 and 13, by comparison with pregnancies unaffected by these trisomies, second, to examine the possible effects of maternal and fetal characteristics on the fetal fraction, and third, to consider the options for further management of pregnancies with a failed result. This was a cohort study of 10 698 singleton pregnancies undergoing screening for fetal trisomies 21, 18 and 13 by cfDNA testing at 10-14 weeks' gestation. There were 160 cases of trisomy 21, 50 of trisomy 18, 16 of trisomy 13 and 10 472 were unaffected by these trisomies. Multivariate regression analysis was used to determine significant predictors of fetal fraction and a failed cfDNA test result amongst maternal and fetal characteristics. Fetal fraction decreased with increasing body mass index and maternal age, was lower in women of South Asian racial origin than in Caucasians and in assisted compared to natural conceptions. It increased with fetal crown-rump length and higher levels of serum pregnancy-associated plasma protein-A and free β-human chorionic gonadotropin. The median fetal fraction was 11.0% (interquartile range (IQR), 8.3-14.4%) in the unaffected group, 10.7% (IQR, 7.8-14.3%) in trisomy 21, 8.6% (IQR, 5.0-10.2%) in trisomy 18 and 7.0% (IQR, 6.0-9.4%) in trisomy 13. There was a failed result from cfDNA testing after first sampling in 2.9% of the unaffected group, 1.9% of trisomy 21, 8.0% of trisomy 18 and 6.3% of trisomy 13. In the cases with a failed result, 7% of women had invasive testing, mainly because of high risk from the combined test and/or presence of sonographic features suggestive of trisomies 18 and 13. All cases of trisomies were detected prenatally. In cases of a failed cfDNA test, the rate of trisomies 18 and 13, but not trisomy 21, is higher than in those with a successful test. In the management of such cases, the decision in favor

Effects of Direct-to-Consumer Advertising and Clinical Guidelines on Appropriate Use of Human Papillomavirus DNA Tests

PubMed Central

2011-01-01

Background Both clinical guidelines and direct-to-consumer (DTC) advertising influence use of new health care technologies, but little is known about their relative effects. The introduction of a cervical cancer screening test in 2000 offered a unique opportunity to assess the two strategies. Objective To evaluate the effects of clinical guidelines and a targeted DTC advertising campaign on overall and appropriate use of human papillomavirus (HPV) DNA tests. Research Design Quasi-experimental study using difference-in-differences analysis. Data were MarketScan private insurance claims for 500,000 women ages 21 to 64 enrolled at least 12 consecutive months from January 2001 through December 2005. Results Both clinical guidelines and DTC advertising were associated with increases in overall HPV DNA test use. DTC advertising was associated with a statistically significant increase in HPV DNA test use in two groups of DTC cities (+5.57 percent, p<0.0001; +2.54 percent, p<0.0001). DTC advertising was associated with comparable increases in the probability of appropriate and inappropriate use of the HPV DNA test in primary screening. Clinical guideline releases from the American College of Obstetricians and Gynecologists, and by a co-sponsored panel, were associated with greater increases in HPV DNA tests for appropriate primary screening than for inappropriate primary screening (β=0.3347, p<0.05 and β=0.4175, p<0.01). Conclusions DTC advertising was associated with increased overall use of a cervical cancer screening test, while clinical guidelines were differentially associated with increased appropriate use. These findings suggest distinct influences of consumer marketing and professional guidelines on the use of health care products and services. PMID:21150798

WHAT DNA CAN AND CANNOT SAY: PERSPECTIVES OF IMMIGRANT FAMILIES ABOUT THE USE OF GENETIC TESTING IN IMMIGRATION

PubMed Central

Barata, Llilda P.; Starks, Helene; Kelley, Maureen; Kuszler, Patricia; Burke, Wylie

2016-01-01

Genetic technologies are being implemented in areas that extend beyond the field of medicine to address social and legal problems. An emerging example is the implementation of genetic testing in the family petitioning process in immigration policy. This use of genetic testing offers the potential benefits of reducing immigration fraud and making the process more efficient and accessible for immigrants, especially those without documentation. However, little is known about the positive or negative impacts of such testing on immigrant families and their communities. This study collected empirical data through family interviews to understand the experiences and attitudes of individuals who have taken a DNA test to prove a family relationship for immigration purposes. Based on study results, we present a set of recommendations to improve the processes with which DNA testing is applied to immigration cases. We argue that DNA testing might serve as a useful tool for families who lack documentary evidence of a family relationship. However, testing might also reveal sensitive information, such as misattributed parentage, that can damage relationships and cause serious harm to beneficiaries, especially children. Petitioners should be provided with adequate information to form an understanding of the DNA test and its implementation as well as the positive and negative consequences from using it, in order to carefully assess whether DNA testing will help their case. We recommend that additional protections be put in place to safeguard children from the potential impacts of misattributed parentage or disclosure of hidden social adoptions. This research provides empirical evidence to inform policy related to the use of genetic testing in immigration. PMID:26855553

A test of the transcription model for biased inheritance of yeast mitochondrial DNA.

PubMed

Lorimer, H E; Brewer, B J; Fangman, W L

1995-09-01

Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA) replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]). These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-] cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold. Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-] mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41. Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results, and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA replication initiation.

Cost-Effectiveness of High-Risk Human Papillomavirus Testing With Messenger RNA Versus DNA Under United States Guidelines for Cervical Cancer Screening.

PubMed

Ting, Jie; Smith, Jennifer S; Myers, Evan R

2015-10-01

To compare the cost-effectiveness of high-risk human papillomavirus (hrHPV) testing using a hrHPV DNA and a hrHPV messenger RNA (mRNA) assay under current US cervical cancer screening guidelines. We constructed a Markov model for stochastic cost-effectiveness analysis using published data. We compared screening efficiency using DNA and mRNA testing for the following: (1) cotesting with cytology in women 30 to 65 years, and (2) triage of women with mild cervical cytological abnormalities (atypical squamous cells of undetermined significance [ASC-US]) in the United States. Screening end point is histologically confirmed high-grade lesions (cervical intraepithelial neoplasia grade 2, 3, or invasive cancer). Sensitivity and specificity estimates of DNA and mRNA testing to detect cervical intraepithelial neoplasia grade 2, 3, or invasive cancer were obtained from 2 published trials: the US Clinical Evaluation of APTIMA mRNA (CLEAR) study for ASC-US triage and the French APTIMA Screening Evaluation (FASE) study for cotesting. Costs of DNA and mRNA testing were assumed identical. Costs of screening, diagnosis, and treatment of cervical neoplasia and cancer were from previously published estimates, adjusted to 2012 US dollars. Inputs were modeled as distributions for Monte Carlo probabilistic sensitivity analysis. Model outcomes were costs per life-year saved for each strategy, discounted at 3% annually. For both cotesting and ASC-US triage, mRNA testing cost less than DNA testing, whereas life expectancies were widely overlapping. There was a 100% probability that DNA testing was not cost-effective at $100,000/life-year saved threshold for ASC-US triage and a 55% probability that DNA testing was not cost-effective at the same threshold for cotesting. Based on the available evidence, mRNA testing for cotesting or ASC-US triage is likely to be more efficient than DNA testing under current US cervical cancer screening guidelines.

Selecting accurate post-elimination monitoring tools to prevent reemergence of urogenital schistosomiasis in Morocco: a pilot study.

PubMed

Balahbib, Abdelaali; Amarir, Fatima; Corstjens, Paul L A M; de Dood, Claudia J; van Dam, Govert J; Hajli, Amina; Belhaddad, Meryem; El Mansouri, Bouchra; Sadak, Abderrahim; Rhajaoui, Mohamed; Adlaoui, El Bachir

2017-04-06

After alleged stop of transmission of schistosomiasis and further down the line in post elimination settings, sensitive tools are required to monitor infection status to prevent potential re-emergence. In Rahala, where transmission cycle of Schistosoma haematobium is interrupted since 2004 but where 30% of snails are still infected by S. bovis, potential human S. bovis infection can't be excluded. As methods based on egg-counts do not provide the required sensitivity, antibody or antigen assays are envisaged as the most appropriate tools for this type of monitoring. In this pilot study, the performances of three assays were compared: two commercially available antibody tests (ELISA and haemagglutination format) indicating exposure, and an antigen test (lateral flow strip format) demonstrating active infection. All 37 recruited study participants resided in Rahala (Akka, province Tata, Morocco). Participants had been diagnosed and cured from schistosomiasis in the period between 1983 and 2003. In 2015 these asymptomatic participants provided fresh clinical samples (blood and urine) for analysis with the aforementioned diagnostics tests. No eggs were identified in the urine of the 37 participants. The haemagglutination test indicated 6 antibody positives whereas the ELISA indicated 28 antibody positives, one indecisive and one false positive. ELISA and haemagglutination results matched for 18 individuals, amongst which 5 out of 6 haemagglutination positives. With the antigen test (performed on paired serum and urine samples), serum from two participants (cured 21 and 32 years ago) indicated the presence of low levels of the highly specific Schistosoma circulating anodic antigen (CAA), demonstrating low worm level infections (less than 5 pg/ml corresponding to probably single worm pair). One tested also CAA positive with urine. ELISA indicated the presence of human anti-Schistosoma antibodies in these two CAA positive cases, haemagglutination results were negative

Cell-Free Fetal DNA Testing for Prenatal Diagnosis.

PubMed

Drury, S; Hill, M; Chitty, L S

Prenatal diagnosis and screening have undergone rapid development in recent years, with advances in molecular technology driving the change. Noninvasive prenatal testing (NIPT) for Down syndrome as a highly sensitive screening test is now available worldwide through the commercial sector with many countries moving toward implementation into their publically funded maternity systems. Noninvasive prenatal diagnosis (NIPD) can now be performed for definitive diagnosis of some recessive and X-linked conditions, rather than just paternally inherited dominant and de novo conditions. NIPD/T offers pregnant couples greater choice during their pregnancy as these safer methods avoid the risk of miscarriage associated with invasive testing. As the cost of sequencing falls and technology develops further, there may well be potential for whole exome and whole genome sequencing of the unborn fetus using cell-free DNA in the maternal plasma. How such assays can or should be implemented into the clinical setting remain an area of significant debate, but it is clear that the progress made to date for safer prenatal testing has been welcomed by expectant couples and their healthcare professionals. © 2016 Elsevier Inc. All rights reserved.

[Comparison of the CMV antigenemia test and CMV-DNA PCR results in solid organ transplant recipients].

PubMed

Özkarata, Emre; Özkarataş, Emre; Özbek, Ö Alpay; Avkan Oğuz, Vildan; Sayıner, A Arzu

2016-01-01

Cytomegalovirus (CMV) infection is among the most common important viral infections in solid organ transplant (SOT) recipients. Diagnostic tests for detecting CMV replication are widely used for this group of patients, however there is no clear agreement on the cut-off levels for interpretation of clinical decisions especially when the low level of viral load is detected. In this study, CMV pp65 antigenemia test results were compared with plasma CMV-DNA levels detected by quantitative real-time polymerase chain reaction (qPCR) in samples of kidney and liver transplant recipients in the Central Laboratory of Dokuz Eylul University Hospital between 2011 and 2013, and the correlation between these two tests and viral load equivalent to antigenemia positivity were determined. In the study, pp65 antigenemia and CMV-DNA qPCR results were evaluated retrospectively. The samples from the same patients were included if the time between antigenemia and CMV-DNA qPCR tests were less than 48 hours. SPSS v15.0 was used for correlation, regression and ROC curve analysis. The results of the 217 samples collected from 100 patients (59 male, 41 female; age range: 16-71, mean age: 46 ± 13 years), 36 liver and 64 kidney recipients were evaluated in the study. Of the patients 80% were CMV IgM negative, IgG positive; 1% was CMV IgG and IgM positive; 2% were CMV IgM and IgG negative, while for 17 patients serological results could not be reached. CMV pp65 antigenemia and CMV-DNA were both negative in 102 (47%) samples, while both were positive in 37 (17%) samples. The single sample from a case with CMV IgM and IgG positivity yielded negative results for both antigenemia and CMV-DNA tests. In 78 samples antigenemia were negative and CMV-DNA qPCR were positive, while there were no samples with antigenemia positive and qPCR negative. Mean values of antigenemia and qPCR tests were 23 positive cells/200.000 leukocytes (range: 1 to 230 positive cells) and 12.595 copies/ml (range: 180 to 106

Integrating prior knowledge in multiple testing under dependence with applications to detecting differential DNA methylation.

PubMed

Kuan, Pei Fen; Chiang, Derek Y

2012-09-01

DNA methylation has emerged as an important hallmark of epigenetics. Numerous platforms including tiling arrays and next generation sequencing, and experimental protocols are available for profiling DNA methylation. Similar to other tiling array data, DNA methylation data shares the characteristics of inherent correlation structure among nearby probes. However, unlike gene expression or protein DNA binding data, the varying CpG density which gives rise to CpG island, shore and shelf definition provides exogenous information in detecting differential methylation. This article aims to introduce a robust testing and probe ranking procedure based on a nonhomogeneous hidden Markov model that incorporates the above-mentioned features for detecting differential methylation. We revisit the seminal work of Sun and Cai (2009, Journal of the Royal Statistical Society: Series B (Statistical Methodology)71, 393-424) and propose modeling the nonnull using a nonparametric symmetric distribution in two-sided hypothesis testing. We show that this model improves probe ranking and is robust to model misspecification based on extensive simulation studies. We further illustrate that our proposed framework achieves good operating characteristics as compared to commonly used methods in real DNA methylation data that aims to detect differential methylation sites. © 2012, The International Biometric Society.

Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production

PubMed Central

Wallenhammar, Ann-Charlotte; Gunnarson, Albin; Hansson, Fredrik; Jonsson, Anders

2016-01-01

Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR) in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil−1) in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g−1 soil) in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20%) showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g−1 soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g−1 soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of prevention of

Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production.

PubMed

Wallenhammar, Ann-Charlotte; Gunnarson, Albin; Hansson, Fredrik; Jonsson, Anders

2016-04-22

Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR) in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil(-1)) in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g(-1) soil) in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20%) showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g(-1) soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g(-1) soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of prevention of

Accuracy and Cost-Effectiveness of Cervical Cancer Screening by High-Risk HPV DNA Testing of Self-Collected Vaginal Samples

PubMed Central

Balasubramanian, Akhila; Kulasingam, Shalini L.; Baer, Atar; Hughes, James P.; Myers, Evan R.; Mao, Constance; Kiviat, Nancy B.; Koutsky, Laura A.

2010-01-01

Objective Estimate the accuracy and cost-effectiveness of cervical cancer screening strategies based on high-risk HPV DNA testing of self-collected vaginal samples. Materials and Methods A subset of 1,665 women (18-50 years of age) participating in a cervical cancer screening study were screened by liquid-based cytology and by high-risk HPV DNA testing of both self-collected vaginal swab samples and clinician-collected cervical samples. Women with positive/abnormal screening test results and a subset of women with negative screening test results were triaged to colposcopy. Based on individual and combined test results, five screening strategies were defined. Estimates of sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or worse were calculated and a Markov model was used to estimate the incremental cost-effectiveness ratios (ICERs) for each strategy. Results Compared to cytology-based screening, high-risk HPV DNA testing of self-collected vaginal samples was more sensitive (68%, 95%CI=58%-78% versus 85%, 95%CI=76%-94%) but less specific (89%, 95%CI=86%-91% versus 73%, 95%CI=67%-79%). A strategy of high-risk HPV DNA testing of self-collected vaginal samples followed by cytology triage of HPV positive women, was comparably sensitive (75%, 95%CI=64%-86%) and specific (88%, 95%CI=85%-92%) to cytology-based screening. In-home self-collection for high-risk HPV DNA detection followed by in-clinic cytology triage had a slightly lower lifetime cost and a slightly higher quality-adjusted life expectancy than did cytology-based screening (ICER of triennial screening compared to no screening was $9,871/QALY and $12,878/QALY, respectively). Conclusions Triennial screening by high-risk HPV DNA testing of in-home, self-collected vaginal samples followed by in-clinic cytology triage was cost-effective. PMID:20592553

[Research status and prospects of DNA test on difficult specimens].

PubMed

Dang, Hua-Wei; Mao, Jiong; Wang, Hui; Huang, Jiang-Ping; Bai, Xiao-Gang

2012-02-01

This paper reviews the advances of DNA detection on three types of difficult biological specimens including degraded samples, trace evidences and mixed samples. The source of different samples, processing methods and announcements were analyzed. New methods such as mitochondrial test system, changing the original experimental conditions, low-volume PCR amplification and new technologies such as whole genome amplification techniques, laser capture micro-dissection, and mini-STR technology in recent years are introduced.

Comparison of human papillomavirus DNA tests, liquid-based cytology and conventional cytology for the early detection of cervix uteri cancer.

PubMed

Girianelli, Vania R; Thuler, Luiz Claudio S; Szklo, Moyses; Donato, Alexandre; Zardo, Lucilia M G; Lozana, José A; Almeida Neto, Olimpio F; Carvalho, Aurenice C L; Matos, Jorge H; Figueiredo, Valeska

2006-12-01

To compare the performance of human papillomavirus DNA tests (samples collected by a healthcare professional and self-collected) and liquid-based cytology with conventional cytology in the detection of cervix uteri cancer and its precursor lesions. A cross-sectional study was carried out in 1777 women living in poor communities in Rio de Janeiro State, Brazil. Eligibility criteria included ages 25-59 years and not having had a Papanicolau test within at least 3 years prior to the study. Cytology (conventional or liquid-based) and human papillomavirus DNA (collected by a healthcare professional or self-collected) tests were performed using samples collected in a single visit. Women with abnormalities in at least one test and a systematic sample of 70 women with negative test results were referred to a colposcopic examination. Test readings were double-masked, and the outcome of interest was high-grade squamous intraepithelial lesion or worse. The pathology report was used as the gold standard. The prevalence of high-grade squamous intraepithelial lesion or worse was 2.0%. Human papillomavirus DNA test collected by a health professional alone or combined with conventional cytology had the highest sensitivity (91.4 and 97.1%, respectively). The highest specificity was found for conventional cytology (91.6%) and for a human papillomavirus DNA test collected by a healthcare professional (90.2%). On the basis of only test performance, the use of human papillomavirus DNA tests, alone or combined with cytology, would seem to be recommended. Its population-wide implementation, however, is conditional on a cost-effectiveness analysis.

A test strip platform based on DNA-functionalized gold nanoparticles for on-site detection of mercury (II) ions.

PubMed

Guo, Zhiyong; Duan, Jing; Yang, Fei; Li, Min; Hao, Tingting; Wang, Sui; Wei, Danyi

2012-05-15

A test strip, based on DNA-functionalized gold nanoparticles for Hg(2+) detection, has been developed, optimized and validated. The developed colorimetric mercury sensor system exhibited a highly sensitive and selective response to mercury. The measurement principle is based on thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination chemistry and streptavidin-biotin interaction. A biotin-labeled and thiolated DNA was immobilized on the gold nanoparticles (AuNPs) surface through a self-assembling method. Another thymine-rich DNA, which was introduced to form DNA duplexes on the AuNPs surface with thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination in the presence of Hg(2+), was immobilized on the nitrocellulose membrane as the test zone. When Hg(2+) ions were introduced into this system, they induced the two strands of DNA to intertwist by forming T-Hg(2+)-T bonds resulting in a red line at the test zone. The biotin-labeled and thiolated DNA-functionalized AuNPs could be captured by streptavidin which was immobilized on the nitrocellulose membrane as the control zone. Under optimized conditions, the detection limit for Hg(2+) was 3 nM, which is lower than the 10nM, maximum contaminant limit defined by the US Environmental Protection Agency (EPA) for drinking water. A parallel analysis of Hg(2+) in pool water samples using cold vapor atomic absorption spectrometry showed comparable results to those obtained from the strip test. Therefore, the results obtained in this study could be used as basic research for the development of Hg(2+) detection, and the method developed could be a potential on-site screening tool for the rapid detection of Hg(2+) in different water samples without special instrumentation. All experimental variables that influence the test strip response were optimized and reported. Copyright © 2012 Elsevier B.V. All rights reserved.

The price of performance: a cost and performance analysis of the implementation of cell-free fetal DNA testing for Down syndrome in Ontario, Canada.

PubMed

Okun, N; Teitelbaum, M; Huang, T; Dewa, C S; Hoch, J S

2014-04-01

To examine the cost and performance implications of introducing cell-free fetal DNA (cffDNA) testing within modeled scenarios in a publicly funded Canadian provincial Down syndrome (DS) prenatal screening program. Two clinical algorithms were created: the first to represent the current screening program and the second to represent one that incorporates cffDNA testing. From these algorithms, eight distinct scenarios were modeled to examine: (1) the current program (no cffDNA), (2) the current program with first trimester screening (FTS) as the nuchal translucency-based primary screen (no cffDNA), (3) a program substituting current screening with primary cffDNA, (4) contingent cffDNA with current FTS performance, (5) contingent cffDNA at a fixed price to result in overall cost neutrality,(6) contingent cffDNA with an improved detection rate (DR) of FTS, (7) contingent cffDNA with higher uptake of FTS, and (8) contingent cffDNA with optimized FTS (higher uptake and improved DR). This modeling study demonstrates that introducing contingent cffDNA testing improves performance by increasing the number of cases of DS detected prenatally, and reducing the number of amniocenteses performed and concomitant iatrogenic pregnancy loss of pregnancies not affected by DS. Costs are modestly increased, although the cost per case of DS detected is decreased with contingent cffDNA testing. Contingent models of cffDNA testing can improve overall screening performance while maintaining the provision of an 11- to 13-week scan. Costs are modestly increased, but cost per prenatally detected case of DS is decreased. © 2013 John Wiley & Sons, Ltd.

Comparative clinical utility of tumor genomic testing and cell-free DNA in metastatic breast cancer.

PubMed

Maxwell, Kara N; Soucier-Ernst, Danielle; Tahirovic, Emin; Troxel, Andrea B; Clark, Candace; Feldman, Michael; Colameco, Christopher; Kakrecha, Bijal; Langer, Melissa; Lieberman, David; Morrissette, Jennifer J D; Paul, Matt R; Pan, Tien-Chi; Yee, Stephanie; Shih, Natalie; Carpenter, Erica; Chodosh, Lewis A; DeMichele, Angela

2017-08-01

Breast cancer metastases differ biologically from primary disease; therefore, metastatic biopsies may assist in treatment decision making. Commercial genomic testing of both tumor and circulating tumor DNA have become available clinically, but utility of these tests in breast cancer management remains unclear. Patients undergoing a clinically indicated metastatic tumor biopsy were consented to the ongoing METAMORPH registry. Tumor and blood were collected at the time of disease progression before subsequent therapy, and patients were followed for response on subsequent treatment. Tumor testing (n = 53) and concurrent cell-free DNA (n = 32) in a subset of patients was performed using CLIA-approved assays. The proportion of patients with a genomic alteration was lower in tumor than in blood (69 vs. 91%; p = 0.06). After restricting analysis to alterations covered on both platforms, 83% of tumor alterations were detected in blood, while 90% of blood alterations were detected in tumor. Mutational load specific for the panel genes was calculated for both tumor and blood. Time to progression on subsequent treatment was significantly shorter for patients whose tumors had high panel-specific mutational load (HR 0.31, 95% CI 0.12-0.78) or a TP53 mutation (HR 0.35, 95% CI 0.20-0.79), after adjusting for stage at presentation, hormone receptor status, prior treatment type, and number of lines of metastatic treatment. Treating oncologists must distinguish platform differences from true biological heterogeneity when comparing tumor and cfDNA genomic testing results. Tumor and concurrent cfDNA contribute unique genomic information in metastatic breast cancer patients, providing potentially useful biomarkers for aggressive metastatic disease.

Combination of DNA-based and conventional methods to detect human leukocyte antigen polymorphism and its use for paternity testing.

PubMed

Kereszturya, László; Rajczya, Katalin; Lászikb, András; Gyódia, Eva; Pénzes, Mária; Falus, András; Petrányia, Gyõzõ G

2002-03-01

In cases of disputed paternity, the scientific goal is to promote either the exclusion of a falsely accused man or the affiliation of the alleged father. Until now, in addition to anthropologic characteristics, the determination of genetic markers included human leukocyte antigen gene variants; erythrocyte antigens and serum proteins were used for that reason. Recombinant DNA techniques provided a new set of highly variable genetic markers based on DNA nucleotide sequence polymorphism. From the practical standpoint, the application of these techniques to paternity testing provides greater versatility than do conventional genetic marker systems. The use of methods to detect the polymorphism of human leukocyte antigen loci significantly increases the chance of validation of ambiguous results in paternity testing. The outcome of 2384 paternity cases investigated by serologic and/or DNA-based human leukocyte antigen typing was statistically analyzed. Different cases solved by DNA typing are presented involving cases with one or two accused men, exclusions and nonexclusions, and tests of the paternity of a deceased man. The results provide evidence for the advantage of the combined application of various techniques in forensic diagnostics and emphasizes the outstanding possibilities of DNA-based assays. Representative examples demonstrate the strength of combined techniques in paternity testing.

Potential of environmental DNA to evaluate Northern pike (Esox lucius) eradication efforts: An experimental test and case study

USGS Publications Warehouse

Dunker, Kristine J.; Sepulveda, Adam; Massengill, Robert L.; Olsen, Jeffrey B.; Russ, Ora L.; Wenburg, John K.; Antonovich, Anton

2016-01-01

Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling

Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study

PubMed Central

Dunker, Kristine J.; Sepulveda, Adam J.; Massengill, Robert L.; Olsen, Jeffrey B.; Russ, Ora L.; Wenburg, John K.; Antonovich, Anton

2016-01-01

Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling

Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study.

PubMed

Dunker, Kristine J; Sepulveda, Adam J; Massengill, Robert L; Olsen, Jeffrey B; Russ, Ora L; Wenburg, John K; Antonovich, Anton

2016-01-01

Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling

Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

PubMed Central

Peter, Harald; Berggrav, Kathrine; Thomas, Peter; Pfeifer, Yvonne; Witte, Wolfgang; Templeton, Kate

2012-01-01

Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to antibiotic therapy, as they confer resistance to carbapenems, which are used to treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we describe the development and evaluation of a DNA microarray for the detection and genotyping of KPC genes (blaKPC) within a 5-h period. To test the whole assay procedure (DNA extraction plus a DNA microarray assay) directly from clinical specimens, we compared two commercial DNA extraction kits (the QIAprep Spin miniprep kit [Qiagen] and the urine bacterial DNA isolation kit [Norgen]) for the direct DNA extraction from urine samples (dilution series spiked in human urine). Reliable single nucleotide polymorphism (SNP) typing was demonstrated using 1 × 105 CFU/ml urine for Escherichia coli (Qiagen and Norgen) and 80 CFU/ml urine, on average, for K. pneumoniae (Norgen). This study presents, for the first time, the combination of a new KPC microarray with commercial sample preparation for detecting and genotyping microbial pathogens directly from clinical specimens; this paves the way toward tests providing epidemiological and diagnostic data, enabling better antimicrobial stewardship. PMID:23035190

Enhanced GSH synthesis by Bisphenol A exposure promoted DNA methylation process in the testes of adult rare minnow Gobiocypris rarus.

PubMed

Yuan, Cong; Zhang, Yingying; Liu, Yan; Zhang, Ting; Wang, Zaizhao

2016-09-01

DNA methylation is a commonly studied epigenetic modification. The mechanism of BPA on DNA methylation is poorly understood. The present study aims to explore whether GSH synthesis affects DNA methylation in the testes of adult male rare minnow Gobiocypris rarus in response to Bisphenol A (BPA). Male G. rarus was exposed to 1, 15 and 225μgL(-1) BPA for 7 days. The levels of global DNA methylation, hydrogen peroxide (H2O2) and glutathione (GSH) in the testes were analyzed. Meanwhile, the levels of enzymes involved in DNA methylation and de novo GSH synthesis, and the substrate contents for GSH production were measured. Furthermore, gene expression profiles of the corresponding genes of all studied enzymes were analyzed. Results indicated that BPA at 15 and 225μgL(-1) caused hypermethylation of global DNA in the testes. The 15μgL(-1) BPA resulted in significant decrease of ten-eleven translocation proteins (TETs) while 225μgL(-1) BPA caused significant increase of DNA methyltransferase proteins (DNMTs). Moreover, 225μgL(-1) BPA caused significant increase of H2O2 and GSH levels, and the de novo GSH synthesis was enhanced. These results indicated that the significant decrease of the level of TETs may be sufficient to cause the DNA hypermethylation by 15μgL(-1) BPA. However, the significantly increased of DNMTs contributed to the significant increase of DNA methylation levels by 225μgL(-1) BPA. Moreover, the elevated de novo GSH synthesis may promote the DNA methylation process. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA banking and DNA databanking by academic and commercial laboratories

DOE Office of Scientific and Technical Information (OSTI.GOV)

McEwen, J.E.; Reilly, P.R.

The advent of DNA-based testing is giving rise to DNA banking (the long-term storage of cells, transformed cell lines, or extracted DNA for subsequent retrieval and analysis) and DNA data banking (the indefinite storage of information derived from DNA analysis). Large scale acquisition and storage of DNA and DNA data has important implications for the privacy rights of individuals. A survey of 148 academically based and commercial DNA diagnostic laboratories was conducted to determine: (1) the extent of their DNA banking activities; (2) their policies and experiences regarding access to DNA samples and data; (3) the quality assurance measures theymore » employ; and (4) whether they have written policies and/or depositor`s agreements addressing specific issues. These issues include: (1) who may have access to DNA samples and data; (2) whether scientists may have access to anonymous samples or data for research use; (3) whether they have plans to contact depositors or retest samples if improved tests for a disorder become available; (4) disposition of samples at the end of the contract period if the laboratory ceases operations, if storage fees are unpaid, or after a death or divorce; (5) the consequence of unauthorized release, loss, or accidental destruction of samples; and (6) whether depositors may share in profits from the commercialization of tests or treatments developed in part from studies of stored DNA. The results suggest that many laboratories are banking DNA, that many have already amassed a large number of samples, and that a significant number plan to further develop DNA banking as a laboratory service over the next two years. Few laboratories have developed written policies governing DNA banking, and fewer still have drafted documents that define the rights and obligations of the parties. There may be a need for increased regulation of DNA banking and DNA data banking and for better defined policies with respect to protecting individual privacy.« less

Tests of spool models for DNA packaging in phage lambda.

PubMed

Widom, J; Baldwin, R L

1983-12-25

Experiments are reported which bear on two spool models proposed for packaging the DNA of phage lambda. Both spool models fill an assumed spherical cavity with DNA wrapped in cylindrical or quasi-cylindrical layers composed of adjacent circular turns. In the curved-spool model, a single continuous segment of DNA, about 20% of the DNA length and probably located near the left end of the DNA, is in contact with the coat protein of the phage capsid. In the straight spool model, there are several DNA segments in contact with the capsid; they are concentrated in one half (probably the left half) of lambda DNA. We have identified the loci on the DNA which are in contact with the capsid by chemical crosslinking, induced by ultraviolet-irradiation of phage containing 5-bromodeoxyuridine in place of thymine. In an electron microscope experiment, phage are first lysed with EDTA, and then spread in a cytochrome c film by the formamide method. The disrupted capsid, which has the appearance of a phage ghost, serves as a marker showing where the DNA is crosslinked to the coat. The left end of the DNA is not distinguished from the right end, and so the map of DNA-capsid contacts is folded over on itself. Contacts are found nearly randomly over the entire map. In a second experiment, DNA from lysed, crosslinked phage is cut either with EcoRI or HindIII restriction endonucleases and the cut restriction fragments are labeled at their ends with 32P. Density centrifugation in a CsCl gradient separates free DNA from restriction fragments crosslinked to protein. After digestion with proteinase k, the DNA fragments previously crosslinked to protein are identified by size after agarose gel electrophoresis. DNA fragments from all parts of the genome are found. These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spool model is unlikely. Alternatively, the manner of packaging the DNA may vary from one

The Diagnostic Performance of Stool DNA Testing for Colorectal Cancer: A Systematic Review and Meta-Analysis.

PubMed

Zhai, Rong-Lin; Xu, Fei; Zhang, Pei; Zhang, Wan-Li; Wang, Hui; Wang, Ji-Liang; Cai, Kai-Lin; Long, Yue-Ping; Lu, Xiao-Ming; Tao, Kai-Xiong; Wang, Guo-Bin

2016-02-01

This meta-analysis was designed to evaluate the diagnostic performance of stool DNA testing for colorectal cancer (CRC) and compare the performance between single-gene and multiple-gene tests.MEDLINE, Cochrane, EMBASE databases were searched using keywords colorectal cancers, stool/fecal, sensitivity, specificity, DNA, and screening. Sensitivity analysis, quality assessments, and performance bias were performed for the included studies.Fifty-three studies were included in the analysis with a total sample size of 7524 patients. The studies were heterogeneous with regard to the genes being analyzed for fecal genetic biomarkers of CRC, as well as the laboratory methods being used for each assay. The sensitivity of the different assays ranged from 2% to 100% and the specificity ranged from 81% to 100%. The meta-analysis found that the pooled sensitivities for single- and multigene assays were 48.0% and 77.8%, respectively, while the pooled specificities were 97.0% and 92.7%. Receiver operator curves and diagnostic odds ratios showed no significant difference between both tests with regard to sensitivity or specificity.This meta-analysis revealed that using assays that evaluated multiple genes compared with single-gene assays did not increase the sensitivity or specificity of stool DNA testing in detecting CRC.

Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine-needle aspirate samples.

PubMed

Dyhdalo, Kathryn; Macnamara, Stephen; Brainard, Jennifer; Underwood, Dawn; Tubbs, Raymond; Yang, Bin

2014-02-01

BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples. This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 10+ cells on a ThinPrep slide: 1+ (1-5 groups), 2+ (6-10 groups), 3+ (11-20 groups), and 4+ (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis. Approximately 49% of thyroid FNA samples had low cellularity (1-2+). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 1+ to 4+ cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 10+ cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells. This study demonstrates that by using 6 groups of 10+ follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens. Cancer (Cancer Cytopathol) 2014;122:114-22 © 2013 American Cancer Society. © 2013 American Cancer Society.

Screening and identification of RhD antigen mimic epitopes from a phage display random peptide library for the serodiagnosis of haemolytic disease of the foetus and newborn.

PubMed

Wang, Jiao; Song, Jingjing; Zhou, Shuimei; Fu, Yourong; Bailey, Jeffrey A; Shen, Changxin

2018-01-16

Identification of RhD antigen epitopes is a key component in understanding the pathogenesis of haemolytic disease of the foetus and newborn. Research has indicated that phage display libraries are useful tools for identifying novel mimic epitopes (mimotopes) which may help to determine antigen specificity. We selected the mimotopes of blood group RhD antigen by affinity panning a phage display library using monoclonal anti-D. After three rounds of biopanning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and then sent for sequencing and peptides synthesis. Next, competitive ELISA and erythrocyte haemagglutination inhibition tests were carried out to confirm the inhibitory activity of the synthetic peptide. To evaluate the diagnostic performance of the synthetic peptide, a diagnostic ELISA was examined. Fourteen of 35 phage clones that were chosen randomly from the titering plate were considered to be positive. Following DNA sequencing and translation, 11 phage clones were found to represent the same peptide - RMKMLMMLMRRK (P4) - whereas each of the other three clones represented a unique peptide. Through the competitive ELISA and erythrocyte haemagglutination inhibition tests, the peptide (P4) was verified to have the ability to mimic the RhD antigen. The diagnostic ELISA for P4 proved to be sensitive (82.61%) and specific (88.57%). This study reveals that the P4 peptide can mimic RhD antigen and paves the way for the development of promising targeted diagnostic and therapeutic platforms for haemolytic disease of the foetus and newborn.

Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

PubMed

Janku, Filip; Zhang, Shile; Waters, Jill; Liu, Li; Huang, Helen J; Subbiah, Vivek; Hong, David S; Karp, Daniel D; Fu, Siqing; Cai, Xuyu; Ramzanali, Nishma M; Madwani, Kiran; Cabrilo, Goran; Andrews, Debra L; Zhao, Yue; Javle, Milind; Kopetz, E Scott; Luthra, Rajyalakshmi; Kim, Hyunsung J; Gnerre, Sante; Satya, Ravi Vijaya; Chuang, Han-Yu; Kruglyak, Kristina M; Toung, Jonathan; Zhao, Chen; Shen, Richard; Heymach, John V; Meric-Bernstam, Funda; Mills, Gordon B; Fan, Jian-Bing; Salathia, Neeraj S

2017-09-15

Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy. Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did ( P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF ( P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03). Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR . ©2017 American Association for Cancer Research.

Replacing the combined test by cell-free DNA testing in screening for trisomies 21, 18 and 13: impact on the diagnosis of other chromosomal abnormalities.

PubMed

Syngelaki, Argyro; Pergament, Eugene; Homfray, Tessa; Akolekar, Ranjit; Nicolaides, Kypros H

2014-01-01

To estimate the proportion of other chromosomal abnormalities that could be missed if combined testing was replaced by cell-free (cf) DNA testing as the method of screening for trisomies 21, 18 and 13. The prevalence of trisomies 21, 18 or 13, sex chromosome aneuploidies, triploidy and other chromosomal abnormalities was examined in pregnancies undergoing first-trimester combined screening and chorionic villus sampling (CVS). In 1,831 clinically significant chromosomal abnormalities in pregnancies with combined risk for trisomies 21, 18 and 13≥1:100, the contribution of trisomies 21, 18 or 13, sex chromosome aneuploidies, triploidy and other chromosomal abnormalities at high risk of adverse outcome was 82.9, 8.2, 3.9 and 5.0%, respectively. Combined screening followed by CVS for risk≥1:10 and cfDNA testing for risk 1:11-1:2,500 could detect 97% of trisomy 21 and 98% of trisomies 18 and 13. Additionally, 86% of monosomy X, half of 47,XXY, 47,XYY or 47,XXX, half of other chromosomal abnormalities and one third of triploidies, which are currently detected by combined screening and CVS for risk≥1:100, could be detected. Screening by cfDNA testing, contingent on results of combined testing, improves detection of trisomies, but misses a few of the other chromosomal abnormalities detected by screening with the combined test. © 2014 S. Karger AG, Basel.

Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

NASA Astrophysics Data System (ADS)

Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

1992-04-01

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

Current Status of Testing for Microdeletion Syndromes and Rare Autosomal Trisomies Using Cell-Free DNA Technology.

PubMed

Yaron, Yuval; Jani, Jacques; Schmid, Maximilian; Oepkes, Dick

2015-11-01

Noninvasive prenatal testing using cell-free DNA in maternal blood for trisomy 21 was introduced in 2011. This technology has continuously evolved with the addition of screening for trisomy 18 and trisomy 13 followed by the inclusion of sex chromosome aneuploidies. Expanded noninvasive prenatal test panels have recently become available, which enable screening for microdeletion syndromes such as the 22q11.2 deletion (associated with the velocardiofacial syndrome) and others. However, the performance data for these microdeletion syndromes are derived from a small number of samples, mostly generated in vitro. Rigorous performance evaluation, as was done at least for trisomy 21 testing using cell-free DNA analysis, is difficult to perform given the rarity of each condition. In addition, detection rates may vary considerably depending on deletion size. Importantly, positive predictive values (PPVs), strongly influenced by the low prevalence, are expected to be significantly lower than 10% for most conditions. Thus, screening in an average-risk population is likely to have many more false-positives than affected cases detected. Conversely, testing in a high-risk population such as fetuses with cardiac anomalies may have higher PPVs, but a negative result needs to be considered carefully as a result of uncertain information about detection rates and a significant residual risk for other copy number variants and single gene disorders. This article integrates current knowledge on cell-free DNA testing for microdeletions with the aim to assist clinicians and policymakers in designing optimal programs for screening in pregnancy.

Method of quantitating dsDNA

DOEpatents

Stark, Peter C.; Kuske, Cheryl R.; Mullen, Kenneth I.

2002-01-01

A method for quantitating dsDNA in an aqueous sample solution containing an unknown amount of dsDNA. A first aqueous test solution containing a known amount of a fluorescent dye-dsDNA complex and at least one fluorescence-attenutating contaminant is prepared. The fluorescence intensity of the test solution is measured. The first test solution is diluted by a known amount to provide a second test solution having a known concentration of dsDNA. The fluorescence intensity of the second test solution is measured. Additional diluted test solutions are similarly prepared until a sufficiently dilute test solution having a known amount of dsDNA is prepared that has a fluorescence intensity that is not attenuated upon further dilution. The value of the maximum absorbance of this solution between 200-900 nanometers (nm), referred to herein as the threshold absorbance, is measured. A sample solution having an unknown amount of dsDNA and an absorbance identical to that of the sufficiently dilute test solution at the same chosen wavelength is prepared. Dye is then added to the sample solution to form the fluorescent dye-dsDNA-complex, after which the fluorescence intensity of the sample solution is measured and the quantity of dsDNA in the sample solution is determined. Once the threshold absorbance of a sample solution obtained from a particular environment has been determined, any similarly prepared sample solution taken from a similar environment and having the same value for the threshold absorbance can be quantified for dsDNA by adding a large excess of dye to the sample solution and measuring its fluorescence intensity.

Field testing of Schistosoma japonicum DNA vaccines in cattle in China.

PubMed

Shi, Fuhui; Zhang, Yaobi; Lin, Jiaojiao; Zuo, Xin; Shen, Wei; Cai, Yiumin; Ye, Ping; Bickle, Quentin D; Taylor, Martin G

2002-11-01

Vaccines are needed to reduce the zoonotic reservoir of Schistosoma japonicum infection in bovines in China. We have developed two experimental DNA vaccines and have already shown these to be capable of inducing partial protection in water buffalo naturally exposed to the risk of S. japonicum infection in the field. We now report a similar field trial in cattle, the other major bovine reservoir host species in China. Groups of cattle were vaccinated with the VRSj28 vaccine or the VRSj23 vaccine, or, to test whether protection could be enhanced by combination vaccination, with both these DNA vaccines together. After vaccination, the cattle were exposed to natural infection in the field for a period of 54 days. Worm and egg counts carried out at the end of the experiment showed that each of the vaccine groups showed partial resistance, and that combined vaccination was not more effective than vaccination with the individual plasmids.

Comparison of the cobas Human Papillomavirus (HPV) Test with the Hybrid Capture 2 and Linear Array HPV DNA Tests

PubMed Central

Sadorra, Mark; LaMere, Brandon J.; Kail, Randi; Aldrich, Carrie; Kinney, Walter; Fetterman, Barbara; Lorey, Thomas; Schiffman, Mark; Castle, Philip E.

2012-01-01

The cobas human papillomavirus (HPV) test (cobas) was recently approved by the U.S. Food and Drug Administration (FDA) and identifies HPV16 and HPV18 separately as well as detecting a pool of 11 HR-HPV genotypes (HPV31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68) and also HPV66. We compared cobas, Linear Array (LA), and Hybrid Capture 2 (HC2) assays for detection of carcinogenic HPV DNA, and cobas and LA for detection of HPV16 and HPV18 DNA, among the first 1,852 women enrolled in the HPV Persistence and Progression Cohort (PaP Cohort) study. Specimens were tested by all 3 assays 1 year after an HC2-positive result. In 1,824 specimens with cobas results, cobas had an 85.9% agreement with HC2 and 91.0% agreement with LA for carcinogenic HPV detection. When results between cobas and HC2 disagreed, cobas tended to call more women HPV positive (P < 0.01). Categorizing cobas and LA results hierarchically according to cancer risk (HPV16, HPV18, other carcinogenic HPV genotypes, or carcinogen negative), there was a 90% agreement for all categories of HPV (n = 1,824). We found good agreement between the two U.S. FDA-approved HPV tests, with discrepancies between the two assays due to specific characteristics of the individual assays. Additional studies are needed to compare HC2 and cobas for detecting and predicting CIN3 to understand the clinical implications of the discrepant test results between the two tests. PMID:22075592

Optimizing blood collection, transport and storage conditions for cell free DNA increases access to prenatal testing.

PubMed

Wong, David; Moturi, Sharmili; Angkachatchai, Vach; Mueller, Reinhold; DeSantis, Grace; van den Boom, Dirk; Ehrich, Mathias

2013-08-01

Fetal mutations and fetal chromosomal abnormalities can be detected by molecular analysis of circulating cell free fetal DNA (ccffDNA) from maternal plasma. This comprehensive study was aimed to investigate and verify blood collection and blood shipping conditions that enable Noninvasive Prenatal Testing. Specifically, the impact of shipping and storage on the stability and concentration of circulating cell-free DNA (ccfDNA) in Streck® Cell-Free DNA™ Blood Collection Tubes (Streck BCTs, Streck, Omaha NE). These BCTs were designed to minimize cellular degradation, and thus effectively prevent dilution of fetal ccf DNA by maternal genomic DNA, was evaluated. Peripheral venous maternal blood was collected into Streck BCTs to investigate four aspects of handling and processing conditions: (1) time from blood draw to plasma processing; (2) storage temperature; (3) mechanical stress; and (4) lot-to-lot tube variations. Maternal blood stored in Streck BCTs for up to 7 days at ambient temperature provides stable concentrations of ccffDNA. The amount of fetal DNA did not change over a broad range of storage temperatures (4°C, 23°C, 37°C, 40°C), but the amount of total (largely maternal) DNA increased in samples stored at 23°C and above, indicating maternal cell degradation and genomic DNA release at elevated temperatures. Shipping maternal blood in Streck BCTs, did not affect sample quality. Maternal plasma DNA stabilized for 0 to 7 days in Streck BCTs can be used for non-invasive prenatal molecular applications, when temperatures are maintained within the broad parameters assessed in this study. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Initial experience with non-invasive prenatal testing of cell-free DNA for major chromosomal anomalies in a clinical setting.

PubMed

Comas, Carmina; Echevarria, Mónica; Rodríguez, M Angeles; Prats, Pilar; Rodríguez, Ignacio; Serra, Bernat

2015-07-01

To evaluate non-invasive prenatal testing (NIPT) of cell-free DNA (cfDNA) as a screening method for major chromosomal anomalies (CA) in a clinical setting. From January to December 2013, Panorama™ test or Harmony™ prenatal test were offered as advanced NIPT, in addition to first-trimester combined screening in singleton pregnancies. The cohort included 333 pregnant women with a mean maternal age (MA) of 37 years who underwent testing at a mean gestational age of 14.6 weeks. Eighty-four percent were low-risk pregnancies. Results were provided in 97.3% of patients at a mean reporting time of 12.9 calendar days. Repeat sampling was performed in six cases and results were obtained in five of them. No results were provided in four cases. Four cases of Down syndrome were detected and there was one discordant result of Turner syndrome. We found no statistical differences between commercial tests except in reporting time, fetal fraction and MA. The cfDNA fraction was statistically associated with test type, maternal weight, BMI and log βhCG levels. NIPT has the potential to be a highly effective screening method for major CA in a clinical setting.

Clinical utility of sperm DNA fragmentation testing: practice recommendations based on clinical scenarios

PubMed Central

Majzoub, Ahmad; Esteves, Sandro C.; Ko, Edmund; Ramasamy, Ranjith; Zini, Armand

2016-01-01

Sperm DNA fragmentation (SDF) has been generally acknowledged as a valuable tool for male fertility evaluation. While its detrimental implications on sperm function were extensively investigated, little is known about the actual indications for performing SDF analysis. This review delivers practice based recommendations on commonly encountered scenarios in the clinic. An illustrative description of the different SDF measurement techniques is presented. SDF testing is recommended in patients with clinical varicocele and borderline to normal semen parameters as it can better select varicocelectomy candidates. High SDF is also linked with recurrent spontaneous abortion (RSA) and can influence outcomes of different assisted reproductive techniques. Several studies have shown some benefit in using testicular sperm rather than ejaculated sperm in men with high SDF, oligozoospermia or recurrent in vitro fertilization (IVF) failure. Infertile men with evidence of exposure to pollutants can benefit from sperm DNA testing as it can help reinforce the importance of lifestyle modification (e.g., cessation of cigarette smoking, antioxidant therapy), predict fertility and monitor the patient’s response to intervention. PMID:28078226

DnaSAM: Software to perform neutrality testing for large datasets with complex null models.

PubMed

Eckert, Andrew J; Liechty, John D; Tearse, Brandon R; Pande, Barnaly; Neale, David B

2010-05-01

Patterns of DNA sequence polymorphisms can be used to understand the processes of demography and adaptation within natural populations. High-throughput generation of DNA sequence data has historically been the bottleneck with respect to data processing and experimental inference. Advances in marker technologies have largely solved this problem. Currently, the limiting step is computational, with most molecular population genetic software allowing a gene-by-gene analysis through a graphical user interface. An easy-to-use analysis program that allows both high-throughput processing of multiple sequence alignments along with the flexibility to simulate data under complex demographic scenarios is currently lacking. We introduce a new program, named DnaSAM, which allows high-throughput estimation of DNA sequence diversity and neutrality statistics from experimental data along with the ability to test those statistics via Monte Carlo coalescent simulations. These simulations are conducted using the ms program, which is able to incorporate several genetic parameters (e.g. recombination) and demographic scenarios (e.g. population bottlenecks). The output is a set of diversity and neutrality statistics with associated probability values under a user-specified null model that are stored in easy to manipulate text file. © 2009 Blackwell Publishing Ltd.

Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case.

PubMed

Avin, Farhat A; Subha, Bhassu; Tan, Yee-Shin; Braukmann, Thomas W A; Vikineswary, Sabaratnam; Hebert, Paul D N

2017-09-01

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648-bp segment near the 5' terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus , the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5' region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus . Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.

The Society for Translational Medicine: clinical practice guidelines for sperm DNA fragmentation testing in male infertility

PubMed Central

Cho, Chak-Lam; Majzoub, Ahmad; Esteves, Sandro C.

2017-01-01

Sperm DNA fragmentation (SDF) testing has been emerging as a valuable tool for male fertility evaluation. While the essential role of sperm DNA integrity in human reproduction was extensively studied, the clinical indication of SDF testing is less clear. This clinical practice guideline provides recommendations of clinical utility of the test supported by evidence. It is intended to serve as a reference for fertility specialists in identifying the circumstances in which SDF testing should be of greatest clinical value. SDF testing is recommended in patients with clinical varicocele and borderline to normal semen parameters as it can better select varicocelectomy candidates. Outcomes of natural pregnancy and assisted reproductive techniques (ART) can be predicted by result of SDF tests. High SDF is also linked with recurrent pregnancy loss (RPL) and failure of ART. Result of SDF testing may change the management decision by selecting the most appropriate ART with the highest success rate for infertile couples. Several studies have demonstrated the benefit in using testicular instead of ejaculated sperm in men with high SDF, oligozoospermia or recurrent in vitro fertilization (IVF) failure. Infertile men with modifiable lifestyle factor may benefit from SDF testing by reinforcing risk factor modification and monitoring patient’s progress to intervention. PMID:29082206

DNA book.

PubMed

Kawai, Jun; Hayashizaki, Yoshihide

2003-06-01

We propose herein a new method of DNA distribution, whereby DNA clones or PCR products are printed directly onto the pages of books and delivered to users along with relevant scientific information. DNA sheets, comprising water-soluble paper onto which DNA is spotted, can be bound into books. Readers can easily extract the DNA fragments from DNA sheets and amplify them using PCR. We show that DNA sheets can withstand various conditions that may be experienced during bookbinding and delivery, such as high temperatures and humidity. Almost all genes (95%-100% of randomly selected RIKEN mouse cDNA clones) were recovered successfully by use of PCR. Readers can start their experiments after a 2-h PCR amplification without waiting for the delivery of DNA clones. The DNA Book thus provides a novel method for delivering DNA in a timely and cost-effective manner. A sample DNA sheet (carrying RIKEN mouse cDNA clones encoding genes of enzymes for the TCA cycle) is included in this issue for field-testing. We would greatly appreciate it if readers could attempt to extract DNA and report the results and whether the DNA sheet was shipped to readers in good condition.

A multi‐centre evaluation of nine rapid, point‐of‐care syphilis tests using archived sera

PubMed Central

Herring, A J; Ballard, R C; Pope, V; Adegbola, R A; Changalucha, J; Fitzgerald, D W; Hook, E W; Kubanova, A; Mananwatte, S; Pape, J W; Sturm, A W; West, B; Yin, Y P; Peeling, R W

2006-01-01

Objectives To evaluate nine rapid syphilis tests at eight geographically diverse laboratory sites for their performance and operational characteristics. Methods Tests were compared “head to head” using locally assembled panels of 100 archived (50 positive and 50 negative) sera at each site using as reference standards the Treponema pallidum haemagglutination or the T pallidum particle agglutination test. In addition inter‐site variation, result stability, test reproducibility and test operational characteristics were assessed. Results All nine tests gave good performance relative to the reference standard with sensitivities ranging from 84.5–97.7% and specificities from 84.5–98%. Result stability was variable if result reading was delayed past the recommended period. All the tests were found to be easy to use, especially the lateral flow tests. Conclusions All the tests evaluated have acceptable performance characteristics and could make an impact on the control of syphilis. Tests that can use whole blood and do not require refrigeration were selected for further evaluation in field settings. PMID:17118953

Diagnostic value of stool DNA testing for multiple markers of colorectal cancer and advanced adenoma: a meta-analysis.

PubMed

Yang, Hua; Xia, Bing-Qing; Jiang, Bo; Wang, Guozhen; Yang, Yi-Peng; Chen, Hao; Li, Bing-Sheng; Xu, An-Gao; Huang, Yun-Bo; Wang, Xin-Ying

2013-08-01

The diagnostic value of stool DNA (sDNA) testing for colorectal neoplasms remains controversial. To compensate for the lack of large-scale unbiased population studies, a meta-analysis was performed to evaluate the diagnostic value of sDNA testing for multiple markers of colorectal cancer (CRC) and advanced adenoma. The PubMed, Science Direct, Biosis Review, Cochrane Library and Embase databases were systematically searched in January 2012 without time restriction. Meta-analysis was performed using a random-effects model using sensitivity, specificity, diagnostic OR (DOR), summary ROC curves, area under the curve (AUC), and 95% CIs as effect measures. Heterogeneity was measured using the χ(2) test and Q statistic; subgroup analysis was also conducted. A total of 20 studies comprising 5876 individuals were eligible. There was no heterogeneity for CRC, but adenoma and advanced adenoma harboured considerable heterogeneity influenced by risk classification and various detection markers. Stratification analysis according to risk classification showed that multiple markers had a high DOR for the high-risk subgroups of both CRC (sensitivity 0.759 [95% CI 0.711 to 0.804]; specificity 0.883 [95% CI 0.846 to 0.913]; AUC 0.906) and advanced adenoma (sensitivity 0.683 [95% CI 0.584 to 0.771]; specificity 0.918 [95% CI 0.866 to 0.954]; AUC 0.946) but not for the average-risk subgroups of either. In the methylation subgroup, sDNA testing had significantly higher DOR for CRC (sensitivity 0.753 [95% CI 0.685 to 0.812]; specificity 0.913 [95% CI 0.860 to 0.950]; AUC 0.918) and advanced adenoma (sensitivity 0.623 [95% CI 0.527 to 0.712]; specificity 0.926 [95% CI 0.882 to 0.958]; AUC 0.910) compared with the mutation subgroup. There was no significant heterogeneity among studies for subgroup analysis. sDNA testing for multiple markers had strong diagnostic significance for CRC and advanced adenoma in high-risk subjects. Methylation makers had more diagnostic value than mutation

Six consecutive false positive cases from cell-free fetal DNA testing in a single referring centre

PubMed Central

Dugo, Nella; Padula, Francesco; Mobili, Luisa; Brizzi, Cristiana; D’Emidio, Laura; Cignini, Pietro; Mesoraca, Alvaro; Bizzoco, Domenico; Cima, Antonella; Giorlandino, Claudio

2014-01-01

Introduction recent studies have proposed the introduction of cell-free fetal DNA testing (NIPT-Non Invasive Prenatal Testing) in routine clinical practice emphasizing its high sensibility and specificity. In any case, false positive and false negative findings may result from placental mosaicism, because cell-free fetal DNA originates mainly from placenta. Case we report six cases of women who underwent chorionic villus sampling (CVS) or amniocentesis to confirm the results from NIPT: two Turner syndromes, two Triple X, one Patau syndrome, one Edward syndrome. Results using classic cytogenetic analysis and, also, Array - Comparative Genomic Hybridization (Array CGH) the karyotype of all 5 fetuses was found to be normal. Conclusion results from NIPT must always be confirmed by invasive prenatal diagnosis. It is mandatory to inform the patient that the CVS and amniocentesis still represent the only form of prenatal diagnostic test available. PMID:25332757

Genomic typing of canine parvovirus circulating in the State of Rio de Janeiro, Brazil from 1995 to 2001 using polymerase chain reaction assay.

PubMed

Costa, A P; Leite, J P G; Labarthe, N V; Garcia, R C N Cubel

2005-11-01

In this study, the genomic types of canine parvovirus (CPV) circulating in the State of Rio de Janeiro, Brazil, from 1995 to 2001, were investigated using the polymerase chain reaction assay (PCR). A total of 78 faecal samples from gastroenteritic puppies, confirmed as positive for canine parvovirus by haemagglutination/haemagglutination inhibition tests or virus isolation in cell culture (MDCK), were examined. The viral DNA was extracted from faecal samples using a combination of phenol- chloroform and silica-guanidine thiocyanate methods. PCR was carried out with differential pairs of primers to distinguish the old (CPV-2) and new types of virus (CPv-2a or CPV-2b). Specific amplicons were observed for all samples using the primer pair P2ab, which detects CPV-2a and CPV-2b. Seventy-six from a total of 78 samples (97%) were considered as CPV-2b because of their reaction with the primer pair P2b. Thirty samples (30/78) were from previously vaccinated puppies and in 15 of them the enteritis symptoms began from 1 to 12 days after vaccination. PCR confirmed the infection by wild virus (CPV-2b) in 5 of these 15 puppies who had received old-type vaccines. Our results show that CPV-2b was the prevalent type circulating in the State of Rio de Janeiro from 1995 to 2001.

Non-invasive, serum DNA pregnancy testing leading to incidental discovery of cancer: a good thing?

PubMed

Prasad, Vinay

2015-11-01

Cell-free DNA for perinatal screening is a growing industry. Non-invasive prenatal testing (NIPT) is based on the premise that foetal DNA is able to cross the placental barrier and enter the mother's circulation, where it can be examined for chromosomal abnormalities, such as trisomy 13, 18 or 21. Such tests are expected to be widely used by pregnant women, with the annual market expected to surpass $1 billion. Recently, a number of case reports have emerged in the haematology-oncology literature. The routine use of NIPT has led to the discovery of maternal neoplasms. Most writers have concluded that this is yet another benefit of the test; however, a closer examination of the cases reveals that this incidental detection may not improve patient outcomes. In some cases, early detection provides lead time bias, but does not change the ultimate clinical outcome, and in other cases, detection constitutes earlier knowledge of a cancer whose natural history cannot be altered. Here, we explore in detail cases where cancer was incidentally discovered among women undergoing routine non-invasive pregnancy testing, and investigate whether or not these women were benefitted by the discovery. Published by Elsevier Ltd.

Prospective first-trimester screening for trisomies by cell-free DNA testing of maternal blood in twin pregnancy.

PubMed

Sarno, L; Revello, R; Hanson, E; Akolekar, R; Nicolaides, K H

2016-06-01

First, to examine in twin pregnancies the performance of first-trimester screening for fetal trisomies 21, 18 and 13 by cell-free (cf) DNA testing of maternal blood and, second, to compare twin and singleton pregnancies regarding the distribution of fetal fraction of cfDNA and rate of failure to obtain a result. This was a prospective study in 438 twin and 10 698 singleton pregnancies undergoing screening for fetal trisomies by cfDNA testing at 10 + 0 to 13 + 6 weeks' gestation. Chromosome-selective sequencing of cfDNA was used and, in twin pregnancies, an algorithm was applied that relies on the lower fetal fraction contributed by the two fetuses. Multivariate regression analysis was used to determine significant predictors of fetal fraction and a failed result. In twin pregnancies, the median fetal fraction was lower (8.0% (interquartile range (IQR), 6.0-10.4%) vs 11.0% (IQR, 8.3-14.4%); P < 0.0001) and failure rate after first sampling was higher (9.4% vs 2.9%; P < 0.0001) compared to in singletons. Multivariate logistic regression analysis demonstrated that the risk of test failure increased with increasing maternal age and body mass index and decreased with fetal crown-rump length. The risk was increased in women of South Asian racial origin and in pregnancies conceived by in-vitro fertilization (IVF). The main contributor to the higher rate of failure in twins was conception by IVF which was observed in 9.5% of singletons and 56.2% of twins. In the 417 twin pregnancies with a cfDNA result after first or second sampling, the detection rate was 100% (8/8) for trisomy 21 and 60% (3/5) for trisomies 18 or 13, at a false-positive rate (FPR) of 0.25% (1/404). In the 10 530 singleton pregnancies with a cfDNA result after first or second sampling, the detection rate was 98.7% (156/158) for trisomy 21 and 80.3% (49/61) for trisomies 18 or 13, at a FPR of 0.22% (23/10 311). In twin pregnancies undergoing first-trimester screening for trisomies by cfDNA testing, the

Estimation Of The Proportion Of Variation Accounted For By DNA Tests. II: Phenotypic Variance

USDA-ARS?s Scientific Manuscript database

The proportion of phenotypic variation accounted for (Rp2) is an important characteristic of a DNA test. Therefore, several estimators of this quantity were evaluated by simulation of 500 replicates of a population of 1000 progeny of 100 sires (3 levels of narrow sense heritability and 4 levels of ...

Testing the Efficacy of DNA Barcodes for Identifying the Vascular Plants of Canada.

PubMed

Braukmann, Thomas W A; Kuzmina, Maria L; Sills, Jesse; Zakharov, Evgeny V; Hebert, Paul D N

2017-01-01

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers (rbcL, matK, ITS2). Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. To test identification success, we employed a DNA barcode reference library with sequence records for 96% of the 5108 vascular plant species known from Canada, but coverage varied from 94% for rbcL to 60% for ITS2 and 39% for matK. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance (MPD) and mean nearest taxon distance (MNTD) were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. All three genes performed strongly in assigning the taxa present in local floras to the correct genus with values ranging from 91% for rbcL to 97% for ITS2 and 98% for matK. However, matK delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and rbcL (~44%). Despite the low number of plant taxa in the Canadian Arctic, DNA barcodes had the least success in discriminating species from this biogeographic region with resolution ranging from 36% with rbcL to 69% with matK. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for rbcL and 87% for matK. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is

Testing the Efficacy of DNA Barcodes for Identifying the Vascular Plants of Canada

PubMed Central

Kuzmina, Maria L.; Sills, Jesse; Zakharov, Evgeny V.; Hebert, Paul D. N.

2017-01-01

Their relatively slow rates of molecular evolution, as well as frequent exposure to hybridization and introgression, often make it difficult to discriminate species of vascular plants with the standard barcode markers (rbcL, matK, ITS2). Previous studies have examined these constraints in narrow geographic or taxonomic contexts, but the present investigation expands analysis to consider the performance of these gene regions in discriminating the species in local floras at sites across Canada. To test identification success, we employed a DNA barcode reference library with sequence records for 96% of the 5108 vascular plant species known from Canada, but coverage varied from 94% for rbcL to 60% for ITS2 and 39% for matK. Using plant lists from 27 national parks and one scientific reserve, we tested the efficacy of DNA barcodes in identifying the plants in simulated species assemblages from six biogeographic regions of Canada using BLAST and mothur. Mean pairwise distance (MPD) and mean nearest taxon distance (MNTD) were strong predictors of barcode performance for different plant families and genera, and both metrics supported ITS2 as possessing the highest genetic diversity. All three genes performed strongly in assigning the taxa present in local floras to the correct genus with values ranging from 91% for rbcL to 97% for ITS2 and 98% for matK. However, matK delivered the highest species discrimination (~81%) followed by ITS2 (~72%) and rbcL (~44%). Despite the low number of plant taxa in the Canadian Arctic, DNA barcodes had the least success in discriminating species from this biogeographic region with resolution ranging from 36% with rbcL to 69% with matK. Species resolution was higher in the other settings, peaking in the Woodland region at 52% for rbcL and 87% for matK. Our results indicate that DNA barcoding is very effective in identifying Canadian plants to a genus, and that it performs well in discriminating species in regions where floristic diversity is

DNA Book

PubMed Central

Kawai, Jun; Hayashizaki, Yoshihide

2003-01-01

We propose herein a new method of DNA distribution, whereby DNA clones or PCR products are printed directly onto the pages of books and delivered to users along with relevant scientific information. DNA sheets, comprising water-soluble paper onto which DNA is spotted, can be bound into books. Readers can easily extract the DNA fragments from DNA sheets and amplify them using PCR. We show that DNA sheets can withstand various conditions that may be experienced during bookbinding and delivery, such as high temperatures and humidity. Almost all genes (95%–100% of randomly selected RIKEN mouse cDNA clones) were recovered successfully by use of PCR. Readers can start their experiments after a 2-h PCR amplification without waiting for the delivery of DNA clones. The DNA Book thus provides a novel method for delivering DNA in a timely and cost-effective manner. A sample DNA sheet (carrying RIKEN mouse cDNA clones encoding genes of enzymes for the TCA cycle) is included in this issue for field-testing. We would greatly appreciate it if readers could attempt to extract DNA and report the results and whether the DNA sheet was shipped to readers in good condition. PMID:12819147

Quantitative analysis of genomic DNA degradation in whole blood under various storage conditions for molecular diagnostic testing.

PubMed

Permenter, Jessalyn; Ishwar, Arjun; Rounsavall, Angie; Smith, Maddie; Faske, Jennifer; Sailey, Charles J; Alfaro, Maria P

2015-12-01

Proper storage of whole blood is crucial for isolating nucleic acids from leukocytes and to ensure adequate performance of downstream assays in the molecular diagnostic laboratory. Short-term and long-term storage recommendations are lacking for successful isolation of genomic DNA (gDNA). Container type (EDTA or heparin), temperature (4 °C and room temperature) and time (1-130 days) were assessed as criterion for sample acceptance policies. The percentage of integrated area (%Ti) between 150 and 10,000 bp from the 2200 TapeStation electropherogram was calculated to measure gDNA degradation. Refrigerated EDTA samples yielded gDNA with low %Ti (high quality). Heparinized samples stored at room temperature yielded gDNA of worst quality. Downstream analysis demonstrated that the quality of the gDNA correlated with the quality of the data; samples with high %Ti generated significantly lower levels of high molecular weight amplicons. Recommendations from these analyses include storing blood samples intended for nucleic acid isolation in EDTA tubes at 4 °C for long term storage (>10 days). gDNA should be extracted within 3 days when blood is stored at room temperature regardless of the container. Finally, refrigerated heparinized samples should not be stored longer than 9 days if expecting high quality gDNA isolates. Laboratories should consider many factors, in addition to the results obtained herein, to update their policies for sample acceptance for gDNA extraction intended for molecular genetic testing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Highly Effective DNA Extraction Method for Nuclear Short Tandem Repeat Testing of Skeletal Remains from Mass Graves

PubMed Central

Davoren, Jon; Vanek, Daniel; Konjhodzić, Rijad; Crews, John; Huffine, Edwin; Parsons, Thomas J.

2007-01-01

Aim To quantitatively compare a silica extraction method with a commonly used phenol/chloroform extraction method for DNA analysis of specimens exhumed from mass graves. Methods DNA was extracted from twenty randomly chosen femur samples, using the International Commission on Missing Persons (ICMP) silica method, based on Qiagen Blood Maxi Kit, and compared with the DNA extracted by the standard phenol/chloroform-based method. The efficacy of extraction methods was compared by real time polymerase chain reaction (PCR) to measure DNA quantity and the presence of inhibitors and by amplification with the PowerPlex 16 (PP16) multiplex nuclear short tandem repeat (STR) kit. Results DNA quantification results showed that the silica-based method extracted on average 1.94 ng of DNA per gram of bone (range 0.25-9.58 ng/g), compared with only 0.68 ng/g by the organic method extracted (range 0.0016-4.4880 ng/g). Inhibition tests showed that there were on average significantly lower levels of PCR inhibitors in DNA isolated by the organic method. When amplified with PP16, all samples extracted by silica-based method produced 16 full loci profiles, while only 75% of the DNA extracts obtained by organic technique amplified 16 loci profiles. Conclusions The silica-based extraction method showed better results in nuclear STR typing from degraded bone samples than a commonly used phenol/chloroform method. PMID:17696302

Patient perceptions of stool DNA testing for pan-digestive cancer screening: A survey questionnaire

PubMed Central

Yang, Dennis; Hillman, Shauna L; Harris, Ann M; Sinicrope, Pamela S; Devens, Mary E; Ahlquist, David A

2014-01-01

AIM: To explore patient interest in a potential multi-organ stool-DNA test (MUST) for pan-digestive cancer screening. METHODS: A questionnaire was designed and mailed to 1200 randomly-selected patients from the Mayo Clinic registry. The 29-item survey questionnaire included items related to demographics, knowledge of digestive cancers, personal and family history of cancer, personal concern of cancer, colorectal cancer (CRC) screening behavior, interest in MUST, importance of test features in a cancer screening tool, and comparison of MUST with available CRC screening tests. All responses were summarized descriptively. χ2 and Rank Sum Test were used for categorical and continuous variables, respectively. RESULTS: Completed surveys were returned by 434 (29% aged 50-59, 37% 60-69, 34% 70-79, 52% women). Most participants (98%) responded they would use MUST. In order of importance, respondents rated multi-cancer detection, absence of bowel preparation, safety and noninvasiveness as most attractive characteristics. For CRC screening, MUST was preferred over colorectal-only stool-DNA testing (53%), occult blood testing (75%), colonoscopy (84%), sigmoidoscopy (91%), and barium enema (95%), P < 0.0001 for each. Among those not previously screened, most (96%) indicated they would use MUST if available. Respondents were confident in their ability to follow instructions to perform MUST (98%). Only 9% of respondents indicated that fear of finding cancer was a concern with MUST, and only 3% indicated unpleasantness of stool sampling as a potential barrier. CONCLUSION: Patients are receptive to the concept of MUST, preferred MUST over conventional CRC screening modalities and valued its potential feature of multi-cancer detection. PMID:24803808

Noninvasive prenatal testing of trisomies 21 and 18 by massively parallel sequencing of maternal plasma DNA in twin pregnancies.

PubMed

Huang, Xuan; Zheng, Jing; Chen, Min; Zhao, Yangyu; Zhang, Chunlei; Liu, Lifu; Xie, Weiwei; Shi, Shuqiong; Wei, Yuan; Lei, Dongzhu; Xu, Chenming; Wu, Qichang; Guo, Xiaoling; Shi, Xiaomei; Zhou, Yi; Liu, Qiufang; Gao, Ya; Jiang, Fuman; Zhang, Hongyun; Su, Fengxia; Ge, Huijuan; Li, Xuchao; Pan, Xiaoyu; Chen, Shengpei; Chen, Fang; Fang, Qun; Jiang, Hui; Lau, Tze Kin; Wang, Wei

2014-04-01

The objective of this study is to assess the performance of noninvasive prenatal testing for trisomies 21 and 18 on the basis of massively parallel sequencing of cell-free DNA from maternal plasma in twin pregnancies. A double-blind study was performed over 12 months. A total of 189 pregnant women carrying twins were recruited from seven hospitals. Maternal plasma DNA sequencing was performed to detect trisomies 21 and 18. The fetal karyotype was used as gold standard to estimate the sensitivity and specificity of sequencing-based noninvasive prenatal test. There were nine cases of trisomy 21 and two cases of trisomy 18 confirmed by karyotyping. Plasma DNA sequencing correctly identified nine cases of trisomy 21 and one case of trisomy 18. The discordant case of trisomy 18 was an unusual case of monozygotic twin with discordant fetal karyotype (one normal and the other trisomy 18). The sensitivity and specificity of maternal plasma DNA sequencing for fetal trisomy 21 were both 100% and for fetal trisomy 18 were 50% and 100%, respectively. Our study further supported that sequencing-based noninvasive prenatal testing of trisomy 21 in twin pregnancies could be achieved with a high accuracy, which could effectively avoid almost 95% of invasive prenatal diagnosis procedures. © 2013 John Wiley & Sons, Ltd.

Test of Cure for Anogenital Gonorrhoea Using Modern RNA-Based and DNA-Based Nucleic Acid Amplification Tests: A Prospective Cohort Study.

PubMed

Wind, Carolien M; Schim van der Loeff, Maarten F; Unemo, Magnus; Schuurman, Rob; van Dam, Alje P; de Vries, Henry J C

2016-06-01

The use of nucleic acid amplification tests (NAATs) to diagnose Neisseria gonorrhoeae infections complicates the performance of a test of cure (TOC) to monitor treatment failure, if this is indicated. As evidence for the timing of TOC using modern NAATs is limited, we performed a prospective cohort study to assess time to clearance when using modern RNA- and DNA-based NAATs. We included patients with anogenital gonorrhoea visiting the Sexually Transmitted Infection Clinic Amsterdam from March through October 2014. After treatment with ceftriaxone mono- or dual therapy (with azithromycin or doxycycline), anal, vaginal, or urine samples were self-collected during 28 consecutive days, and analyzed using an RNA-based NAAT (Aptima Combo 2) and a DNA-based NAAT (Cobas 4800). Clearance was defined as 3 consecutive negative results, and blips as isolated positive results following clearance. We included 77 patients; 5 self-cleared gonorrhoea before treatment and 10 were lost to follow-up. Clearance rate of the remaining 62 patients was 100%. Median time to clearance was 2 days, with a range of 1-7 days for RNA-based NAAT and 1-15 days for DNA-based NAAT. The risk of finding a blip after clearance was 0.8% and 1.5%, respectively. One patient had a reinfection. If indicated, we recommend that TOC be performed for anogenital gonorrhoea at least 7 or 14 days after administering therapy, when using modern RNA- or DNA-based NAATs, respectively. When interpreting TOC results for possible treatment failure, both the occurrence of blips and a possible reinfection need to be taken into account. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Comparative analysis of Campylobacter isolates from wild birds and chickens using MALDI-TOF MS, biochemical testing, and DNA sequencing.

PubMed

Lawton, Samantha J; Weis, Allison M; Byrne, Barbara A; Fritz, Heather; Taff, Conor C; Townsend, Andrea K; Weimer, Bart C; Mete, Aslı; Wheeler, Sarah; Boyce, Walter M

2018-05-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared to conventional biochemical testing methods and nucleic acid analyses (16S rDNA sequencing, hippurate hydrolysis gene testing, whole genome sequencing [WGS]) for species identification of Campylobacter isolates obtained from chickens ( Gallus gallus domesticus, n = 8), American crows ( Corvus brachyrhynchos, n = 17), a mallard duck ( Anas platyrhynchos, n = 1), and a western scrub-jay ( Aphelocoma californica, n = 1). The test results for all 27 isolates were in 100% agreement between MALDI-TOF MS, the combined results of 16S rDNA sequencing, and the hippurate hydrolysis gene PCR ( p = 0.0027, kappa = 1). Likewise, the identifications derived from WGS from a subset of 14 isolates were in 100% agreement with the MALDI-TOF MS identification. In contrast, biochemical testing misclassified 5 isolates of C. jejuni as C. coli, and 16S rDNA sequencing alone was not able to differentiate between C. coli and C. jejuni for 11 sequences ( p = 0.1573, kappa = 0.0857) when compared to MALDI-TOF MS and WGS. No agreement was observed between MALDI-TOF MS dendrograms and the phylogenetic relationships revealed by rDNA sequencing or WGS. Our results confirm that MALDI-TOF MS is a fast and reliable method for identifying Campylobacter isolates to the species level from wild birds and chickens, but not for elucidating phylogenetic relationships among Campylobacter isolates.

The use of Hemastix and the subsequent lack of DNA recovery using the Promega DNA IQ system.

PubMed

Poon, Hiron; Elliott, Jim; Modler, Jeff; Frégeau, Chantal

2009-11-01

Following implementation of our automated process incorporating the Promega DNA IQ system as a DNA extraction method, a large number of blood-containing exhibits failed to produce DNA. These exhibits had been tested with the Hemastix reagent strip, commonly used by police investigators and forensic laboratories as a screening test for blood. Some exhibits were even tainted green following transfer of the presumptive test reagents onto the samples. A series of experiments were carried out to examine the effect of the Hemastix chemistries on the DNA IQ system. Our results indicate that one or more chemicals imbedded in the Hemastix reagent strip severely reduce the ability to recover DNA from any suspected stain using the DNA IQ magnetic bead technology. The 3,3',5,5'-tetramethylbenzidine (TMB) used as the reporting dye appears to interact with the magnetic beads to prevent DNA recovery. Hydrogen peroxide does not seem to be involved. The Hemastix chemistries do not interfere in any way with DNA extraction performed using phenol-chloroform. The incompatibility of the Hemastix chemistries on the DNA IQ system forced us to adopt an indirect approach using filter paper to carry out the presumptive test.

Concordance study between the ParaDNA® Intelligence Test, a rapid DNA profiling assay, and a conventional STR typing kit (AmpFlSTR® SGM Plus®).

PubMed

Ball, G; Dawnay, N; Stafford-Allen, B; Panasiuk, M; Rendell, P; Blackman, S; Duxbury, N; Wells, S

2015-05-01

The ParaDNA® Intelligence Test enables STR profiling directly from human biological samples and evidence items collected from crime scene in 75min. Designed for non-expert use this system allows DNA information to be available to investigators before it would typically be available from a laboratory. The ParaDNA Intelligence Test system amplifies D3S1358, D8S119, D16S539, D18S1358 and TH01 STR loci and the gender typing locus amelogenin and detects the alleles present with HyBeacon® probes. Individual DNA samples from 381 UK Caucasian individuals were analysed using AmpFlSTR® SGM Plus® and the ParaDNA Intelligence Test with the derived STR profiles compared. Here we describe the high level of concordance demonstrated between the two systems and discuss this with reference to allele frequencies and the discriminatory power offered by the ParaDNA Intelligence Test. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Estimation of the Proportion of Variation Accounted for by DNA Tests. I: Genetic Variance

USDA-ARS?s Scientific Manuscript database

The proportion of genetic variation accounted for (Rg2) is an important characteristic of a DNA test. For each of 3 levels of narrow sense heritability of the observed trait (h2gy) and 4 levels of Rg2, 500 independent replicates of an observed trait and a molecular breeding value (MBV) for 1000 offs...

Non-invasive prenatal testing using massively parallel sequencing of maternal plasma DNA: from molecular karyotyping to fetal whole-genome sequencing.

PubMed

Lo, Y M Dennis

2013-12-01

The discovery of cell-free fetal DNA in maternal plasma in 1997 has stimulated a rapid development of non-invasive prenatal testing. The recent advent of massively parallel sequencing has allowed the analysis of circulating cell-free fetal DNA to be performed with unprecedented sensitivity and precision. Fetal trisomies 21, 18 and 13 are now robustly detectable in maternal plasma and such analyses have been available clinically since 2011. Fetal genome-wide molecular karyotyping and whole-genome sequencing have now been demonstrated in a number of proof-of-concept studies. Genome-wide and targeted sequencing of maternal plasma has been shown to allow the non-invasive prenatal testing of β-thalassaemia and can potentially be generalized to other monogenic diseases. It is thus expected that plasma DNA-based non-invasive prenatal testing will play an increasingly important role in future obstetric care. It is thus timely and important that the ethical, social and legal issues of non-invasive prenatal testing be discussed actively by all parties involved in prenatal care. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Analysis of fingerprint samples, testing various conditions, for forensic DNA identification.

PubMed

Ostojic, Lana; Wurmbach, Elisa

2017-01-01

Fingerprints can be of tremendous value for forensic biology, since they can be collected from a wide variety of evident types, such as handles of weapons, tools collected in criminal cases, and objects with no apparent staining. DNA obtained from fingerprints varies greatly in quality and quantity, which ultimately affects the quality of the resulting STR profiles. Additional difficulties can arise when fingerprint samples show mixed STR profiles due to the handling of multiple persons. After applying a tested protocol for sample collection (swabbing with 5% Triton X-100), DNA extraction (using an enzyme that works at elevated temperatures), and PCR amplification (AmpFlSTR® Identifiler® using 31cycles) extensive analysis was performed to better understand the challenges inherent to fingerprint samples, with the ultimate goal of developing valuable profiles (≥50% complete). The impact of time on deposited fingerprints was investigated, revealing that while the quality of profiles deteriorated, full STR profiles could still be obtained from samples after 40days of storage at room temperature. By comparing the STR profiles from fingerprints of the dominant versus the non-dominant hand, we found a slightly better quality from the non-dominant hand, which was not always significant. Substrates seem to have greater effects on fingerprints. Tests on glass, plastic, paper and metal (US Quarter dollar, made of Cu and Ni), common substrates in offices and homes, showed best results for glass, followed by plastic and paper, while almost no profiles were obtained from a Quarter dollar. Important for forensic casework, we also assessed three-person mixtures of touched fingerprint samples. Unlike routinely used approaches for sampling evidence, the surface of an object (bottle) was sectioned into six equal parts and separate samples were taken from each section. The samples were processed separately for DNA extraction and STR amplification. The results included a few single

Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study.

PubMed

Gianaroli, Luca; Magli, M Cristina; Pomante, Alessandra; Crivello, Anna M; Cafueri, Giulia; Valerio, Marzia; Ferraretti, Anna P

2014-12-01

To investigate the presence of DNA in blastocyst fluids (BFs) and to estimate whether the chromosomal status predicted by its analysis corresponds with the ploidy condition in trophectoderm (TE) cells, the whole embryo, and that predicted by polar bodies (PBs) or blastomeres. Prospective study. In vitro fertilization unit. Seventeen couples undergoing preimplantation genetic screening with the use of array comparative genomic hybridization on PBs (n = 12) or blastomeres (n = 5). BFs and TE cells were retrieved from 51 blastocysts for separate chromosomal analysis. Presence of DNA in BFs and assessment of the corresponding chromosome condition; correlation with the results in TE cells and those predicted by the analysis done at earlier stages. DNA was detected in 39 BFs (76.5%). In 38 of 39 cases (97.4%) the ploidy condition of BFs was confirmed in TE cells, and the rate of concordance per single chromosome was 96.6% (904/936). In relation to the whole embryo, the ploidy condition corresponded in all cases with a per-chromosome concordance of 98.1%. The testing of PBs and blastomeres had 93.3% and 100% prediction of BF ploidy condition with a concordance per chromosome of 93.5% and 94%, respectively. Blastocentesis could represent an alternative source of material for chromosomal testing, because the BF is highly predictive of the embryo ploidy condition and chromosome content. Our data confirm the relevance of the oocyte and of the early-cleavage embryo in determining the ploidy condition of the resulting blastocyst. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Salmon redd identification using environmental DNA (eDNA)

USGS Publications Warehouse

Pilliod, David S.; Laramie, Matthew B.

2016-06-10

IntroductionThe purpose of this project was to develop a technique to use environmental DNA (eDNA) to distinguish between redds made by Chinook salmon (Oncorhynchus tshawytscha) and redds made by Coho salmon (O. kisutch) and to distinguish utilized redds from test/abandoned redds or scours that have the appearance of redds. The project had two phases:Phase 1. Develop, test, and optimize a molecular assay for detecting and identifying Coho salmon DNA and differentiating it from Chinook salmon DNA.Phase 2. Demonstrate the efficacy of the technique.Collect and preserve water samples from the interstitial spaces of 10 known redds (as identified by expert observers) of each species and 10 gravel patches that do not include a redd of either species.Collect control samples from the water column adjacent to each redd to establish background eDNA levels.Analyze the samples using the developed molecular assays for Coho salmon (phase I) and Chinook salmon (Laramie and others, 2015).Evaluate whether samples collected from Chinook and Coho redds have significantly higher levels of eDNA of the respective species than background levels (that is, from gravel, water column).Evaluate whether samples collected from the interstitial spaces of gravel patches that are not redds are similar to background eDNA levels.The Sandy River is a large tributary of the Columbia River. The Sandy River meets the Columbia River approximately 23 km upstream of Portland, Oregon. The Sandy River Basin provides overlapping spawning habitat for both Chinook and Coho salmon.Samples provided by Portland Water Bureau for analysis were collected from the Bull Run River, Sixes Creek, Still Creek, Arrah Wanna Side Channel, and Side Channel 18.

Evaluation of the COBAS AMPLICOR CMV MONITOR test for detection of viral DNA in specimens taken from patients after liver transplantation.

PubMed

Sia, I G; Wilson, J A; Espy, M J; Paya, C V; Smith, T F

2000-02-01

Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients.

Evaluation of the COBAS AMPLICOR CMV MONITOR Test for Detection of Viral DNA in Specimens Taken from Patients after Liver Transplantation

PubMed Central

Sia, Irene G.; Wilson, Jennie A.; Espy, Mark J.; Paya, Carlos V.; Smith, Thomas F.

2000-01-01

Detection of cytomegalovirus (CMV) DNA in blood by PCR is a sensitive method for the detection of infection in patients posttransplantation. The test, however, has low specificity for the identification of overt CMV disease. Quantitative CMV PCR has been shown to overcome this shortcoming. The COBAS AMPLICOR CMV MONITOR test was evaluated by using consecutive serum and peripheral blood mononuclear cell (PBMN) samples from liver transplant patients. Twenty-five patients had CMV viremia (by shell vial cell culture assay) and/or tissue-invasive disease (by biopsy); 20 had no active infection. A total of 262 serum and 62 PBMN specimens were tested. Of 159 serum specimens from patients with overt CMV infection, the COBAS assay detected CMV DNA in 21 patients (sensitivity, 84%). Only 1 of 103 samples from patients with no evidence of active infection had detectable CMV DNA (341 copies/ml). By comparison of 62 matching serum and PBMN samples by the same assay, 12 PBMN samples were exclusively positive, whereas only 2 serum samples were exclusively positive (P < 0.05). At the time of clinical CMV infection, viral copy numbers were higher in PBMNs than serum from four of five patients. The COBAS AMPLICOR CMV MONITOR test is a sensitive and specific test for the quantitative detection of CMV DNA in blood. Clinical applications of the assay will require further validation with samples from a larger population of transplant patients. PMID:10655353

Myth and reality: practical test system for the measurement of anti-DNA antibodies in the diagnosis of systemic lupus erythematosus (SLE).

PubMed

McCloskey, Laura J; Christner, Paul; Jacobs-Kosmin, Dana; Jaskowski, Troy D; Hill, Harry R; Lakos, Gabriella; Teodorescu, Marius

2010-01-01

The myth persists that only the labor intensive Farr radioimmunoassay and Crithidia luciliae immunofluorescence (CL-IFA) are systemic lupus erythematosus (SLE)-specific tests. We compared them to ELISA with bacteriophage lambda DNA (EL-dsDNA) and denatured calf thymus DNA (EL-ssDNA). By percentile ranking, the specificity cut-off level was set both out of clinical context (SOCC) on 100 blood bank donors, and in clinical context (SICC) on 100 patients with either rheumatoid arthritis or scleroderma (50/50). Clinical sensitivity was calculated on 100 random SLE patients. At 95% SICC, the sensitivity of Farr, CL-IFA, EL-dsDNA, and EL-ssDNA was similar (95%CI): 76% (66-84), 76% (66-84), 63% (53-72), and 75% (65-83), respectively; 87% of the patients were positive by at least one method and 55%by all methods. At 99% SICC, the sensitivity was also similar (95% CI): 57% (47-67), 47% (37-57), 58% (47-67), and 43% (33-53), respectively. The areas under ROC curve were similar (95% CI) when patients were used as controls for specificity. At 99% SOCC, EL-ssDNA identified 89% positive, 2 negative but positive by another method at 95% SICC, and 9 negative (i.e. 89/2/9), followed by CL-IFA (80/6/14), Farr (76/12/12), and EL-dsDNA (64/23/13). Thus, at relatively low cost and easy automation, under the same conditions of specificity, the two ELISA tests combined were at least as good, if not superior, to CL-IFA or Farr: they showed similar clinical sensitivity and also identified more patients with anti-DNA antibodies. (c) 2010 Wiley-Liss, Inc.

Development of a dual test procedure for DNA typing and methamphetamine detection using a trace amount of stimulant-containing blood.

PubMed

Irii, Toshiaki; Maebashi, Kyoko; Fukui, Kenji; Sohma, Ryoko; Matsumoto, Sari; Takasu, Shojiro; Iwadate, Kimiharu

2016-05-01

Investigation of drug-related crimes, such as violation of the Stimulant Drug Control Law, requires identifying the used drug (mainly stimulant drugs, methamphetamine hydrochloride) from a drug solution and the DNA type of the drug user from a trace of blood left in the syringe used to inject the drug. In current standard test procedures, DNA typing and methamphetamine detection are performed as independent tests that use two separate portions of a precious sample. The sample can be entirely used up by either analysis. Therefore, we developed a new procedure involving partial lysis of a stimulant-containing blood sample followed by separation of the lysate into a precipitate for DNA typing and a liquid-phase fraction for methamphetamine detection. The method enables these two tests to be run in parallel using a single portion of sample. Samples were prepared by adding methamphetamine hydrochloride water solution to blood. Samples were lysed with Proteinase K in PBS at 56°C for 20min, cooled at -20°C after adding methanol, and then centrifuged at 15,000rpm. Based on the biopolymer-precipitating ability of alcohol, the precipitate was used for DNA typing and the liquid-phase fraction for methamphetamine detection. For DNA typing, the precipitate was dissolved and DNA was extracted, quantified, and subjected to STR analysis using the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. For methamphetamine detection, the liquid-phase fraction was evaporated with N2 gas after adding 20μL acetic acid and passed through an extraction column; the substances captured in the column were eluted with a solvent, derivatized, and quantitatively detected using gas chromatograph/mass spectrometry. This method was simple and could be completed in approximately 2h. Both DNA typing and methamphetamine detection were possible, which suggests that this method may be valuable for use in criminal investigations. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A review of testing used in seroprevalence studies on measles and rubella.

PubMed

Dimech, Wayne; Mulders, Mick N

2016-07-29

Seroprevalence studies are an essential tool to monitor the efficacy of vaccination programmes, to understand population immunity and to identify populations at higher risk of infection. An overarching review of all aspects of seroprevalence studies for measles and rubella published between 1998 and June 2014 was undertaken and the findings reported elsewhere. This paper details the considerable variation in the testing formats identified in the review. Apart from serum/plasma samples, testing of oral fluid, breast milk, dry blood spots and capillary whole blood were reported. Numerous different commercial assays were employed, including microtitre plate assays, automated immunoassays and classical haemagglutination inhibition and neutralisation assays. A total of 29 of the 68 (43%) measles and 14 of the 58 (24%) rubella studies reported qualitative test results. Very little information on the testing environment, including quality assurance mechanisms used, was provided. Due to the large numbers of testing systems, the diversity of sample types used and the difficulties in accurate quantification of antibody levels, the results reported in individual studies were not necessarily comparable. Further efforts to standardise seroprevalence studies may overcome this deficiency. Copyright © 2016 Elsevier Ltd. All rights reserved.

Impact of DNA twist accumulation on progressive helical wrapping of torsionally constrained DNA.

PubMed

Li, Wei; Wang, Peng-Ye; Yan, Jie; Li, Ming

2012-11-21

DNA wrapping is an important mechanism for chromosomal DNA packaging in cells and viruses. Previous studies of DNA wrapping have been performed mostly on torsionally unconstrained DNA, while in vivo DNA is often under torsional constraint. In this study, we extend a previously proposed theoretical model for wrapping of torsionally unconstrained DNA to a new model including the contribution of DNA twist energy, which influences DNA wrapping drastically. In particular, due to accumulation of twist energy during DNA wrapping, it predicts a finite amount of DNA that can be wrapped on a helical spool. The predictions of the new model are tested by single-molecule study of DNA wrapping under torsional constraint using magnetic tweezers. The theoretical predictions and the experimental results are consistent with each other and their implications are discussed.

Structural Transformation of Wireframe DNA Origami via DNA Polymerase Assisted Gap-Filling.

PubMed

Agarwal, Nayan P; Matthies, Michael; Joffroy, Bastian; Schmidt, Thorsten L

2018-03-27

The programmability of DNA enables constructing nanostructures with almost any arbitrary shape, which can be decorated with many functional materials. Moreover, dynamic structures can be realized such as molecular motors and walkers. In this work, we have explored the possibility to synthesize the complementary sequences to single-stranded gap regions in the DNA origami scaffold cost effectively by a DNA polymerase rather than by a DNA synthesizer. For this purpose, four different wireframe DNA origami structures were designed to have single-stranded gap regions. This reduced the number of staple strands needed to determine the shape and size of the final structure after gap filling. For this, several DNA polymerases and single-stranded binding (SSB) proteins were tested, with T4 DNA polymerase being the best fit. The structures could be folded in as little as 6 min, and the subsequent optimized gap-filling reaction was completed in less than 3 min. The introduction of flexible gap regions results in fully collapsed or partially bent structures due to entropic spring effects. Finally, we demonstrated structural transformations of such deformed wireframe DNA origami structures with DNA polymerases including the expansion of collapsed structures and the straightening of curved tubes. We anticipate that this approach will become a powerful tool to build DNA wireframe structures more material-efficiently, and to quickly prototype and test new wireframe designs that can be expanded, rigidified, or mechanically switched. Mechanical force generation and structural transitions will enable applications in structural DNA nanotechnology, plasmonics, or single-molecule biophysics.

Comparison of clinical application of the Abbott HBV PCR kit and the VERSANT HBV DNA 3.0 test to measure serum hepatitis B virus DNA in Taiwanese patients.

PubMed

Yang, Jeng-Fu; Lin, Ya-Yun; Huang, Jee-Fu; Liu, Shu-Fen; Chu, Pei-Yu; Hsieh, Ming-Yen; Lin, Zu-Yau; Chen, Shinn-Cherng; Wang, Liang-Yen; Dai, Chia-Yen; Chuang, Wan-Long; Yu, Ming-Lung

2009-08-01

With an estimated 350-400 million people worldwide chronically infected with hepatitis B virus (HBV), and the subsequent serious complications caused by liver damage including cirrhosis, liver failure, and hepatocellular carcinoma, HBV infection remains a global health issue, particularly in Taiwan, an HBV-hyperendemic area. Sensitive and accurate quantification of HBV DNA is necessary to monitor patients with chronic hepatitis B who are receiving antiviral therapy to determine treatment response and adapt therapy. We evaluated and compared the clinical performance of two HBV DNA assays based on different technologies: the RealArt HBV PCR Kit (Abbott HBV DNA PCR kit, real-time polymerase chain reaction assay, detection limit: 27 IU/mL) and the VERSANT bDNA 3.0 assay (Bayer, branched DNA signal amplification assay, detection limit: 357 IU/mL). Serum levels of HBV DNA in 173 chronic HBV carriers were determined using both the RealArt HBV PCR Kit and the VERSANT bDNA 3.0 test. Of the 173 samples analyzed for baseline viral load detection, HBV DNA was quantifiable in 147 patients (82.1%) by the RealArt HBV PCR Kit, which was significantly higher than the 92 (53.2%) samples quantified by the VERSANT bDNA 3.0 assay. A total of 86 (49.7%) samples were quantifiable by both assays, whereas 25 (14.5%) were below the detection limit of both assays. The HBV DNA quantification values measured by the RealArt HBV PCR Kit and the VERSANT bDNA 3.0 assay were positively correlated (Spearman's rank correlation coefficient r = 0.932, p < 0.001). On average, the results derived from the RealArt HBV PCR Kit were 0.67 log lower than those of the VERSANT bDNA 3.0 assay. HBV DNA concentrations were significantly higher in 63 HBV e antigen (HBeAg)-seropositive patients than in 110 HBeAg-seronegative patients (5.42 +/- 2.34 logs vs. 3.21 +/- 2.27 logs, p < 0.001). The RealArt HBV PCR Kit is more sensitive and has a wider dynamic range than the VERSANT bDNA 3.0 assay in the clinical setting

"I think we've got too many tests!": Prenatal providers' reflections on ethical and clinical challenges in the practice integration of cell-free DNA screening.

PubMed

Gammon, B L; Kraft, S A; Michie, M; Allyse, M

2016-01-01

The recent introduction of cell-free DNA-based non-invasive prenatal screening (cfDNA screening) into clinical practice was expected to revolutionize prenatal testing. cfDNA screening for fetal aneuploidy has demonstrated higher test sensitivity and specificity for some conditions than conventional serum screening and can be conducted early in the pregnancy. However, it is not clear whether and how clinical practices are assimilating this new type of testing into their informed consent and counselling processes. Since the introduction of cfDNA screening into practice in 2011, the uptake and scope have increased dramatically. Prenatal care providers are under pressure to stay up to date with rapidly changing cfDNA screening panels, manage increasing patient demands, and keep up with changing test costs, all while attempting to use the technology responsibly and ethically. While clinical literature on cfDNA screening has shown benefits for specific patient populations, it has also identified significant misunderstandings among providers and patients alike about the power of the technology. The unique features of cfDNA screening, in comparison to established prenatal testing technologies, have implications for informed decision-making and genetic counselling that must be addressed to ensure ethical practice. This study explored the experiences of prenatal care providers at the forefront of non-invasive genetic screening in the United States to understand how this testing changes the practice of prenatal medicine. We aimed to learn how the experience of providing and offering this testing differs from established prenatal testing methodologies. These differences may necessitate changes to patient education and consent procedures to maintain ethical practice. We used the online American Congress of Obstetricians and Gynecologists Physician Directory to identify a systematic sample of five prenatal care providers in each U.S. state and the District of Columbia. Beginning

Value of high-risk HPV-DNA testing in the triage of ASCUS.

PubMed

Silverloo, Iréne; Andrae, Bengt; Wilander, Erik

2009-01-01

OBJECTIVE. Atypical squamous cells of undetermined significance (ASCUS) cells, occurring in organized cytological screening, may be either high-risk human papillomavirus (HPV) positive or negative. To refine the assessment of women with ASCUS, a high-risk HPV-DNA test is recommended as triage in Sweden. A total of 197 consecutive women (mean age 39 years, range 21-60) with a diagnosis of ASCUS from the primary screening were selected for triage. Their cervical smears were collected and evaluated by using conventional cytological examination in combination with a high-risk HPV-DNA test (hybrid capture 2). The women were categorized into four groups: Group A, Cytology + /HPV + ; Group B, Cytology-/HPV + ; Group C, Cytology + /HPV-; and Group D, Cytology-/ HPV-. Women within Groups A-C were admitted for colposcopy and cervical biopsy. The women in Group D were considered as a low-risk group for tumor development, and were re-examined after three years in the next round of the organized screening. In women in Group A (n=58) the prevalence of histological verified CIN2-3 was 41%, in Group B (n=41) 20%, and in Group C (n=9) 0%. In Group D (n=89), repeated primary screening three years later revealed CIN2-3 in two biopsies from 74 women studied (<3%). The prevalence of a high-risk HPV infection decreased with age in women with ASCUS. It was 74% in women <30 years and 19% in women > or =50 years. Adding a high-risk HPV test in secondary screening increased the identification of women with CIN2-3 lesions by 33% in comparison with repeat cytology (p=0.01). The clinical significance of the ASCUS diagnosis varied with age of the women.

What is in your cup of tea? DNA Verity Test to characterize black and green commercial teas

PubMed Central

Comparone, Maria; Di Maio, Antonietta; Del Guacchio, Emanuele; Menale, Bruno; Troisi, Jacopo; Aliberti, Francesco

2017-01-01

In this study, we used several molecular techniques to develop a fast and reliable protocol (DNA Verity Test, DVT) for the characterization and confirmation of the species or taxa present in herbal infusions. As a model plant for this protocol, Camellia sinensis, a traditional tea plant, was selected due to the following reasons: its historical popularity as a (healthy) beverage, its high selling value, the importation of barely recognizable raw product (i.e., crushed), and the scarcity of studies concerning adulterants or contamination. The DNA Verity Test includes both the sequencing of DNA barcoding markers and genotyping of labeled-PCR DNA barcoding fragments for each sample analyzed. This protocol (DVT) was successively applied to verify the authenticity of 32 commercial teas (simple or admixture), and the main results can be summarized as follows: (1) the DVT protocol is suitable to detect adulteration in tea matrices (contaminations or absence of certified ingredients), and the method can be exported for the study of other similar systems; (2) based on the BLAST analysis of the sequences of rbcL+matK±rps7-trnV(GAC) chloroplast markers, C. sinensis can be taxonomically characterized; (3) rps7-trnV(GAC) can be employed to discriminate C. sinensis from C. pubicosta; (4) ITS2 is not an ideal DNA barcode for tea samples, reflecting potential incomplete lineage sorting and hybridization/introgression phenomena in C. sinensis taxa; (5) the genotyping approach is an easy, inexpensive and rapid pre-screening method to detect anomalies in the tea templates using the trnH(GUG)-psbA barcoding marker; (6) two herbal companies provided no authentic products with a contaminant or without some of the listed ingredients; and (7) the leaf matrices present in some teabags could be constituted using an admixture of different C. sinensis haplotypes and/or allied species (C. pubicosta). PMID:28542606

Initial clinical laboratory experience in noninvasive prenatal testing for fetal aneuploidy from maternal plasma DNA samples.

PubMed

Futch, Tracy; Spinosa, John; Bhatt, Sucheta; de Feo, Eileen; Rava, Richard P; Sehnert, Amy J

2013-06-01

The aim of this study is to report the experience of noninvasive prenatal DNA testing using massively parallel sequencing in an accredited clinical laboratory. Laboratory information was examined for blood samples received for testing between February and November 2012 for chromosome 21 (Chr21), Chr18, and Chr13. Monosomy X (MX) testing was available from July 2012 for cystic hygroma indication. Outcomes were collected from providers on samples with positive results. There were 5974 samples tested, and results were issued within an average of 5.1 business days. Aneuploidy was detected in 284 (4.8%) samples (155 Chr21, 66 Chr18, 19 Chr13, 40 MX, and four double aneuploidy). Follow-ups are available for 245/284 (86%), and 77/284 (27.1%) are confirmed, including one double-aneuploidy case concordant with cytogenetics from maternal malignancy. Fourteen (0.2%) discordant (putative false-positive) results (one Chr21, six Chr18, three Chr13, three MX, and one Chr21/13) have been identified. Five (0.08%) false-negative cases are reported (two trisomy 21, two trisomy 18, and one MX). In 170 (2.8%) cases, the result for a single chromosome was indefinite. This report suggests that clinical testing of maternal cell-free DNA for fetal aneuploidy operates within performance parameters established in validation studies. Noninvasive prenatal testing is sensitive to biological contributions from placental and maternal sources. ©2013 Verinata Health, Inc. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

High sensitive detection of double-stranded DNA autoantibodies by a modified Crithidia luciliae immunofluorescence test.

PubMed

Conrad, Karsten; Ittenson, Annelore; Reinhold, Dirk; Fischer, Richard; Roggenbuck, Dirk; Büttner, Thomas; Bosselmann, Hans-Peter; Steinbach, Jörg; Schössler, Werner

2009-09-01

Anti-double-stranded (ds)DNA antibodies are serological markers of systemic lupus erythematosus (SLE). Of all anti-dsDNA antibody detection methods, the Crithidia luciliae immunofluorescence test (CLIFT) is thought to have the highest specificity for SLE. However, the clinical application is hampered by the low diagnostic sensitivity. A CLIFT with modified assay buffer (mCLIFT) was developed and compared with conventional CLIFT, using sera from 110 patients with SLE, 89 anti-dsDNA ELISA-positive patients with other diseases (non-SLE group A), 157 non-SLE patients with undetectable anti-dsDNA antibodies by ELISA (non-SLE group B), 77 disease controls (non-SLE group C), and 50 healthy blood donors. Out of the 110 anti-dsDNA antibody ELISA-positive SLE patients, 84 (76.4%) demonstrated a positive kinetoplast staining, using the mCLIFT, compared to only 42.3%, using the conventional CLIFT. The diagnostic specificity of mCLIFT was 100% with healthy blood donors and 98.1% with the non-SLE group C (anti-nuclear antibodies negative; no signs or symptoms of an autoimmune disease) included. In the non-SLE groups A and B with various other autoimmune diseases or symptoms of a possible autoimmune disease, positive mCLIFT results were obtained in 33.7% and 3.2%, respectively. In conclusion, by modification of the assay buffer, a significant increase in sensitivity of the CLIFT could be observed while retaining the high specificity for SLE. Further investigation is required to check whether the CLIFT-positive non-SLE patients develop SLE and whether anti-dsDNA antibodies detected by the mCLIFT represent a pathogenetic and diagnostic subgroup of autoantibodies that may improve the early diagnosis of SLE or SLE-overlap syndromes.

Performance of the cobas Hepatitis B virus (HBV) test using the cobas 4800 system and comparison of HBV DNA quantification ability between the COBAS AmpliPrep/COBAS TaqMan HBV test version 2.0 and cobas HBV test.

PubMed

Shin, Kyung-Hwa; Lee, Hyun-Ji; Chang, Chulhun L; Kim, Hyung-Hoi

2018-04-01

Hepatitis B virus (HBV) DNA levels are used to predict the response to therapy, determine therapy initiation, monitor resistance to therapy, and establish treatment success. To verify the performance of the cobas HBV test using the cobas 4800 system for HBV DNA quantification and to compare the HBV DNA quantification ability between the cobas HBV test and COBAS AmpliPrep/COBAS TaqMan HBV version 2.0 (CAP/CTM v2.0). The precision, linearity, and limit of detection of the cobas HBV test were evaluated using the 4th World Health Organization International Standard material and plasma samples. Clinical samples that yielded quantitative results using the CAP/CTM v2.0 and cobas HBV tests were subjected to correlational analysis. Three hundred forty-nine samples were subjected to correlational analysis, among which 114 samples showed results above the lower limit of quantification. Comparable results were obtained ([cobas HBV test] = 1.038 × [CAP/CTM v2.0]-0.173, r = 0.914) in 114 samples, which yielded values above the lower limit of quantification. The results for 86.8% of the samples obtained using the cobas HBV test were within 0.5 log 10 IU/mL of the CAP/CTM v2.0 results. The total precision values against the low and high positive controls were 1.4% (mean level: 2.25 log 10 IU/mL) and 3.2% (mean level: 6.23 log 10 IU/mL), respectively. The cobas HBV test demonstrated linearity (1.15-6.75 log 10 IU/mL, y = 0.95 × 6 + 0.17, r 2  = 0.994). The cobas HBV test showed good correlation with CAP/CTM v2.0, and had good precision and an acceptable limit of detection. The cobas HBV test using the cobas 4800 is a reliable method for quantifying HBV DNA levels in the clinical setting. Copyright © 2018. Published by Elsevier B.V.

Motivations for Undertaking DNA Sequencing-Based Non-Invasive Prenatal Testing for Fetal Aneuploidy: A Qualitative Study with Early Adopter Patients in Hong Kong

PubMed Central

Yi, Huso; Hallowell, Nina; Griffiths, Sian; Yeung Leung, Tak

2013-01-01

Background A newly introduced cell-free fetal DNA sequencing based non-invasive prenatal testing (DNA-NIPT) detects Down syndrome with sensitivity of 99% at early gestational stage without risk of miscarriage. Attention has been given to its public health implications; little is known from consumer perspectives. This qualitative study aimed to explore women’s motivations for using, and perceptions of, DNA-NIPT in Hong Kong. Methods and Findings In-depth interviews were conducted with 45 women who had undertaken DNA-NIPT recruited by purposive sampling based on socio-demographic and clinical characteristics. The sample included 31 women identified as high-risk from serum and ultrasound based Down syndrome screening (SU-DSS). Thematic narrative analysis examined informed-decision making of the test and identified the benefits and needs. Women outlined a number of reasons for accessing DNA-NIPT: reducing the uncertainty associated with risk probability-based results from SU-DSS, undertaking DNA-NIPT as a comprehensive measure to counteract risk from childbearing especially at advanced age, perceived predictive accuracy and absence of risk of harm to fetus. Accounts of women deemed high-risk or not high-risk are distinctive in a number of respects. High-risk women accessed DNA-NIPT to get a clearer idea of their risk. This group perceived SU-DSS as an unnecessary and confusing procedure because of its varying, protocol-dependent detection rates. Those women not deemed high-risk, in contrast, undertook DNA-NIPT for psychological assurance and to reduce anxiety even after receiving the negative result from SU-DSS. Conclusions DNA-NIPT was regarded positively by women who chose this method of screening over the routine, less expensive testing options. Given its perceived utility, health providers need to consider whether DNA-NIPT should be offered as part of universal routine care to women at high-risk for fetal aneuploidy. If this is the case, then further development

Development of an Automated DNA Detection System Using an Electrochemical DNA Chip Technology

NASA Astrophysics Data System (ADS)

Hongo, Sadato; Okada, Jun; Hashimoto, Koji; Tsuji, Koichi; Nikaido, Masaru; Gemma, Nobuhiro

A new compact automated DNA detection system Genelyzer™ has been developed. After injecting a sample solution into a cassette with a built-in electrochemical DNA chip, processes from hybridization reaction to detection and analysis are all operated fully automatically. In order to detect a sample DNA, electrical currents from electrodes due to an oxidization reaction of electrochemically active intercalator molecules bound to hybridized DNAs are detected. The intercalator is supplied as a reagent solution by a fluid supply unit of the system. The feasibility test proved that the simultaneous typing of six single nucleotide polymorphisms (SNPs) associated with a rheumatoid arthritis (RA) was carried out within two hours and that all the results were consistent with those by conventional typing methods. It is expected that this system opens a new way to a DNA testing such as a test for infectious diseases, a personalized medicine, a food inspection, a forensic application and any other applications.

DNA: The Strand that Connects Us All

ScienceCinema

Kaplan, Matt [Univ. of Arizona, Tucson, AZ (United States). Genetics Core Facility

2018-04-26

Learn how the methods and discoveries of human population genetics are applied for personal genealogical reconstruction and anthropological testing. Dr. Kaplan starts with a short general review of human genetics and the biology behind this form of DNA testing. He looks at how DNA testing is performed and how samples are processed in the University of Arizona laboratory. He also examines examples of personal genealogical results from Family Tree DNA and personal anthropological results from the Genographic Project. Finally, he describes the newest project in the UA laboratory, the DNA Shoah Project.

Unlocking biodiversity and conservation studies in high diversity environments using environmental DNA (eDNA): a test with Guianese freshwater fishes.

PubMed

Cilleros, Kévin; Valentini, Alice; Allard, Luc; Dejean, Tony; Etienne, Roselyne; Grenouillet, Gaël; Iribar, Amaia; Taberlet, Pierre; Vigouroux, Régis; Brosse, Sébastien

2018-05-16

Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and non-destructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large-scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA.

PubMed

Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takahashi, Shirushi; Kurosu, Akira; Takada, Aya; Saito, Kazuyuki

2016-05-01

A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real-time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples. © 2016 American Academy of Forensic Sciences.

DNA Barcoding in the Cycadales: Testing the Potential of Proposed Barcoding Markers for Species Identification of Cycads

PubMed Central

Sass, Chodon; Little, Damon P.; Stevenson, Dennis Wm.; Specht, Chelsea D.

2007-01-01

Barcodes are short segments of DNA that can be used to uniquely identify an unknown specimen to species, particularly when diagnostic morphological features are absent. These sequences could offer a new forensic tool in plant and animal conservation—especially for endangered species such as members of the Cycadales. Ideally, barcodes could be used to positively identify illegally obtained material even in cases where diagnostic features have been purposefully removed or to release confiscated organisms into the proper breeding population. In order to be useful, a DNA barcode sequence must not only easily PCR amplify with universal or near-universal reaction conditions and primers, but also contain enough variation to generate unique identifiers at either the species or population levels. Chloroplast regions suggested by the Plant Working Group of the Consortium for the Barcode of Life (CBoL), and two alternatives, the chloroplast psbA-trnH intergenic spacer and the nuclear ribosomal internal transcribed spacer (nrITS), were tested for their utility in generating unique identifiers for members of the Cycadales. Ease of amplification and sequence generation with universal primers and reaction conditions was determined for each of the seven proposed markers. While none of the proposed markers provided unique identifiers for all species tested, nrITS showed the most promise in terms of variability, although sequencing difficulties remain a drawback. We suggest a workflow for DNA barcoding, including database generation and management, which will ultimately be necessary if we are to succeed in establishing a universal DNA barcode for plants. PMID:17987130

Validation and Estimation of Additive Genetic Variation Associated with DNA Tests for Quantitative Beef Cattle Traits

USDA-ARS?s Scientific Manuscript database

The U.S. National Beef Cattle Evaluation Consortium (NBCEC) has been involved in the validation of commercial DNA tests for quantitative beef quality traits since their first appearance on the U.S. market in the early 2000s. The NBCEC Advisory Council initially requested that the NBCEC set up a syst...

USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

EPA Science Inventory

USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION
IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

John C. Rockett1, J. Christopher Luft1, J. Brian Garges1, M. Stacey Ricci2, Pasquale Patrizio2, Norman B. Hecht2 and David J. Dix1
Reproductive Toxicology Divisio...

Metastatic site location influences the diagnostic accuracy of ctDNA EGFR- mutation testing in NSCLC patients: a pooled analysis.

PubMed

Passiglia, Francesco; Rizzo, Sergio; Rolfo, Christian; Galvano, Antonio; Bronte, Enrico; Incorvaia, Lorena; Listi, Angela; Barraco, Nadia; Castiglia, Marta; Calo, Valentina; Bazan, Viviana; Russo, Antonio

2018-03-08

Recent studies evaluated the diagnostic accuracy of circulating tumor DNA (ctDNA) in the detection of epidermal growth factor receptor (EGFR) mutations from plasma of NSCLC patients, overall showing a high concordance as compared to standard tissue genotyping. However it is less clear if the location of metastatic site may influence the ability to identify EGFR mutations in plasma. This pooled analysis aims to evaluate the association between the metastatic site location and the sensitivity of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Data from all published studies, evaluating the sensitivity of plasma-based EGFR-mutation testing, stratified by metastatic site location (extrathoracic (M1b) vs intrathoracic (M1a)) were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the ctDNA analysis sensitivity, according to metastatic site location. A total of ten studies, with 1425 patients, were eligible. Pooled analysis showed that the sensitivity of ctDNA-based EGFR-mutation testing is significantly higher in patients with M1b vs M1a disease (OR: 5.09; 95% CIs: 2.93 - 8.84). A significant association was observed for both EGFR-activating (OR: 4.30, 95% CI: 2.35-7.88) and resistant T790M mutations (OR: 11.89, 95% CI: 1.45-97.22), regardless of the use of digital-PCR (OR: 5.85, 95% CI: 3.56-9.60) or non-digital PCR technologies (OR: 2.96, 95% CI: 2.24-3.91). These data suggest that the location of metastatic sites significantly influences the diagnostic accuracy of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Incorporating DNA Sequencing into Current Prenatal Screening Practice for Down's Syndrome

PubMed Central

Wald, Nicholas J.; Bestwick, Jonathan P.

2013-01-01

Background Prenatal screening for Down's syndrome is performed using biochemical and ultrasound markers measured in early pregnancy such as the Integrated test using first and second trimester markers. Recently, DNA sequencing methods have been introduced on free DNA in maternal plasma, yielding a high screening performance. These methods are expensive and there is a test failure rate. We determined the screening performance of merging the Integrated test with the newer DNA techniques in a protocol that substantially reduces the cost compared with universal DNA testing and still achieves high screening performance with no test failures. Methods Published data were used to model screening performance of a protocol in which all women receive the first stage of the Integrated test at about 11 weeks of pregnancy. On the basis of this higher risk women have reflex DNA testing and lower risk women as well as those with a failed DNA test complete the Integrated test at about 15 weeks. Results The overall detection rate was 95% with a 0.1% false-positive rate if 20% of women were selected to receive DNA testing. If all women had DNA testing the detection rate would be 3 to 4 percentage points higher with a false-positive rate 30 times greater if women with failed tests were treated as positive and offered a diagnostic amniocentesis, or 3 times greater if they had a second trimester screening test (Quadruple test) and treated as positive only if this were positive. The cost per women screened would be about one-fifth, compared with universal DNA testing, if the DNA test were 20 times the cost of the Integrated test. Conclusion The proposed screening protocol achieves a high screening performance without programme test failures and at a substantially lower cost than offering all women DNA testing. PMID:23527014

Effect of DNA type on response of DNA biosensor for carcinogens

NASA Astrophysics Data System (ADS)

Sani, Nor Diyana bt. Md.; Heng, Lee Yook; Surif, Salmijah; Lazim, Azwani Mat

2013-11-01

Carcinogens are cancer causing chemicals that can bind to DNA and cause damage to the DNA. These chemicals are available everywhere including in water, air, soil and food. Therefore, a sensor that can detect the presence of these chemicals will be a very useful tool. Since carcinogens bind to DNA, DNA can be used as the biological element in a biosensor. This study has utilized different types of DNA in a biosensor for carcinogen detection. The DNAs include double stranded calf thymus DNA, single stranded calf thymus DNA and guanine rich single stranded DNA. The modified SPE was exposed to a carcinogen followed by interaction with methylene blue which acts as the electroactive indicator. The SPE was then analysed using differential pulse voltammetry (DPV). Optimization studies were conducted for MB concentration and accumulation time, DNA concentration, as well as effect of buffer concentration, buffer pH and ionic strength. The performance of the biosensor was tested on a group 1 carcinogen, formaldehyde. The results indicated that the usage of guanine rich single stranded DNA also gives higher response as carcinogens prefer to bind with guanine compared to other bases.

Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction.

PubMed

Janku, F; Huang, H J; Fujii, T; Shelton, D N; Madwani, K; Fu, S; Tsimberidou, A M; Piha-Paul, S A; Wheler, J J; Zinner, R G; Naing, A; Hong, D S; Karp, D D; Cabrilo, G; Kopetz, E S; Subbiah, V; Luthra, R; Kee, B K; Eng, C; Morris, V K; Karlin-Neumann, G A; Meric-Bernstam, F

2017-03-01

Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy. Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care. Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001). Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Recruitment of DNA methyltransferase I to DNA repair sites.

PubMed

Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M Cristina; Leonhardt, Heinrich

2005-06-21

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair.

Prevalence of human papillomavirus infection among Iranian women using COBAS HPV DNA testing.

PubMed

Jamdar, Farzane; Farzaneh, Farah; Navidpour, Fariba; Younesi, Sarang; Balvayeh, Payam; Hosseini, Maryamsadat; Ghodssi-Ghasemabadi, Robabeh

2018-01-01

Persistent infection with High Risk Human Papillomavirus (HR HPV) typesplaysamajor role in the development of cervical cancer. Therefore, the detection of HR HPV types is an essential part of cervical cancer screening. The aim of this study was to estimate the prevalence of HR HPV infection among healthy women undergoing routine cervical cancer screening in Iran. In this cross-sectional study,the results of HPV DNA typing in 2453 normal Iranian womenwhowere referred for routine cervical cancer screening from September 2015 to March 2017 were analyzed. Participants were screened using COBAS assay for HPV DNA typing and liquid based cytology. A total of 2453 healthy sexually active women were included in this study. The mean age was 35.1 ± 8.08 years. The overall prevalence of HR HPV infection was 10.3%. HPV16 was found in 73 (3%) women. The prevalence of HPV18 and other HR HPV typeswere 16(0.7%) and166 (8.2%),respectively. Approximately, 5% of the study population had an abnormal cervical cytology (ASCUS or worse), of whom 34% were infected by HR HPV. The prevalence of HR HPV infection among Iranian women has increased in the recent years which indicates the need for public education and health planning toprevent this cancer through vaccination and early diagnosis using screening tests.HPV DNA typing, diagnosisand the distribution of prevalent genotypes should be considered in the development of comprehensive cervical cancer prevention programs in Iran.

DNA methylation profiling for a confirmatory test for blood, saliva, semen, vaginal fluid and menstrual blood.

PubMed

Lee, Hwan Young; Jung, Sang-Eun; Lee, Eun Hee; Yang, Woo Ick; Shin, Kyoung-Jin

2016-09-01

The ability to predict the type of tissues or cells from molecular profiles of crime scene samples has important practical implications in forensics. A previously reported multiplex assay using DNA methylation markers could only discriminate between 4 types of body fluids: blood, saliva, semen, and the body fluid which originates from female reproductive organ. In the present study, we selected 15 menstrual blood-specific CpG marker candidates based on analysis of 12 genome-wide DNA methylation profiles of vaginal fluid and menstrual blood. The menstrual blood-specificity of the candidate markers was confirmed by comparison with HumanMethylation450 BeadChip array data obtained for 58 samples including 12 blood, 12 saliva, 12 semen, 3 vaginal fluid, and 19 skin epidermis samples. Among 15CpG marker candidates, 3 were located in the promoter region of the SLC26A10 gene, and 2 of them (cg09696411 and cg18069290) showed high menstrual blood specificity. DNA methylation at the 2CpG markers was further tested by targeted bisulfite sequencing of 461 additional samples including 49 blood, 52 saliva, 34 semen, 125 vaginal fluid, and 201 menstrual blood. Because the 2 markers showed menstrual blood-specific methylation patterns, we modified our previous multiplex methylation SNaPshot reaction to include these 2 markers. In addition, a blood marker cg01543184 with cross reactivity to semen was replaced with cg08792630, and a semen-specific unmethylation marker cg17621389 was removed. The resultant multiplex methylation SNaPshot allowed positive identification of blood, saliva, semen, vaginal fluid and menstrual blood using the 9CpG markers which show a methylation signal only in the target body fluids. Because of the complexity in cell composition, menstrual bloods produced DNA methylation profiles that vary with menstrual cycle and sample collection methods, which are expected to provide more insight into forensic menstrual blood test. Moreover, because the developed

Triage of HR-HPV positive women with minor cytological abnormalities: a comparison of mRNA testing, HPV DNA testing, and repeat cytology using a 4-year follow-up of a population-based study.

PubMed

Persson, Maria; Elfström, K Miriam; Brismar Wendel, Sophia; Weiderpass, Elisabete; Andersson, Sonia

2014-01-01

Expression of the viral E6/E7 oncogenes of high-risk human papillomaviruses (HR-HPV) is necessary for malignant conversion and maintenance in cervical tissue. In order to determine whether HR-HPV E6/E7 mRNA testing more effectively predicts precancerous lesions and invasive cervical cancer than HR-HPV DNA testing, we aimed to compare triage using HR-HPV E6/E7 mRNA testing by APTIMA HPV Assay (APTIMA) to HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology. Liquid-based (PreservCyt) cell samples were obtained from HR-HPV-positive women diagnosed with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) within the framework of the population-based cervical cancer screening program in Stockholm, Sweden. Samples were tested for HR-HPV E6/E7 mRNA by APTIMA (Gene-Probe Inc., San Diego, CA, USA). Women were followed up for 4 years after the index cytology via medical and laboratory records, and the Stockholm Oncology Center. Nine of 25 (36%) women in the ASCUS group, and 64 of 180 (36%) women in the LSIL group developed cervical intraepithelial neoplasia (CIN) grade 2 or worse during 4 years of follow-up. 162 (74%) women were APTIMA-positive, and APTIMA had the highest sensitivity to predict CIN2 or worse and CIN3 or worse in the ASCUS (77.8% and 100%) and LSIL (78.1 and 75.8%) groups, although specificity was insufficient (<50%). HPV16 DNA testing and repeat cytology were more specific than APTIMA. The results of this population-based study with comprehensive follow-up support the use of APTIMA as a triage test for women with ASCUS. More focused investigation is required for women with LSIL.

Triage of HR-HPV Positive Women with Minor Cytological Abnormalities: A Comparison of mRNA Testing, HPV DNA Testing, and Repeat Cytology Using a 4-Year Follow-Up of a Population-Based Study

PubMed Central

Persson, Maria; Elfström, K. Miriam; Brismar Wendel, Sophia; Weiderpass, Elisabete; Andersson, Sonia

2014-01-01

Objective Expression of the viral E6/E7 oncogenes of high-risk human papillomaviruses (HR-HPV) is necessary for malignant conversion and maintenance in cervical tissue. In order to determine whether HR-HPV E6/E7 mRNA testing more effectively predicts precancerous lesions and invasive cervical cancer than HR-HPV DNA testing, we aimed to compare triage using HR-HPV E6/E7 mRNA testing by APTIMA HPV Assay (APTIMA) to HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology. Methods Liquid-based (PreservCyt) cell samples were obtained from HR-HPV-positive women diagnosed with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) within the framework of the population-based cervical cancer screening program in Stockholm, Sweden. Samples were tested for HR-HPV E6/E7 mRNA by APTIMA (Gene-Probe Inc., San Diego, CA, USA). Women were followed up for 4 years after the index cytology via medical and laboratory records, and the Stockholm Oncology Center. Results Nine of 25 (36%) women in the ASCUS group, and 64 of 180 (36%) women in the LSIL group developed cervical intraepithelial neoplasia (CIN) grade 2 or worse during 4 years of follow-up. 162 (74%) women were APTIMA-positive, and APTIMA had the highest sensitivity to predict CIN2 or worse and CIN3 or worse in the ASCUS (77.8% and 100%) and LSIL (78.1 and 75.8%) groups, although specificity was insufficient (<50%). HPV16 DNA testing and repeat cytology were more specific than APTIMA. Conclusion The results of this population-based study with comprehensive follow-up support the use of APTIMA as a triage test for women with ASCUS. More focused investigation is required for women with LSIL. PMID:24587193

Human papillomavirus mRNA and DNA testing in women with atypical squamous cells of undetermined significance: A prospective cohort study.

PubMed

Thomsen, Louise T; Dehlendorff, Christian; Junge, Jette; Waldstrøm, Marianne; Schledermann, Doris; Frederiksen, Kirsten; Kjaer, Susanne K

2016-10-15

In this prospective cohort study, we compared the performance of human papillomavirus (HPV) mRNA and DNA testing of women with atypical squamous cells of undetermined significance (ASC-US) during cervical cancer screening. Using a nationwide Danish pathology register, we identified women aged 30-65 years with ASC-US during 2005-2011 who were tested for HPV16/18/31/33/45 mRNA using PreTect HPV-Proofer (n = 3,226) or for high-risk HPV (hrHPV) DNA using Hybrid Capture 2 (HC2) (n = 9,405) or Linear Array HPV-Genotyping test (LA) (n = 1,533). Women with ≥1 subsequent examination in the register (n = 13,729) were followed for up to 9.5 years for high-grade cervical intraepithelial neoplasia (CIN) or cancer. After 3 years' follow-up, mRNA testing had higher specificity for CIN3 or worse (CIN3+) than HC2 testing (88.1% [95% confidence interval (CI): 86.8-89.6%] versus 59.3% [95% CI: 58.1-60.4%]) and higher positive predictive value (PPV) (38.2% [95% CI: 33.8%-43.1%] versus 19.5% [95% CI: 17.8-20.9%]). However, the sensitivity of mRNA testing was lower than that of HC2 testing (66.7% [95% CI: 59.3-74.5%] versus 97.0% [95% CI: 95.5-98.4%]), and women testing mRNA negative had higher 3-year risk for CIN3+ than those testing HC2 negative (3.2% [95% CI: 2.2-4.2%] versus 0.5% [95% CI: 0.3-0.7%]). Patterns were similar after 18 months and 5 years'; follow-up; for CIN2+ and cancer as outcomes; across all age groups; and when comparing mRNA testing to hrHPV DNA testing using LA. In conclusion, the HPV16/18/31/33/45 mRNA test is not optimal for ASC-US triage due to its low sensitivity and the substantial risk for precancer following a negative test. © 2016 UICC.

Non-Destructive Sampling of Ancient Insect DNA

PubMed Central

Thomsen, Philip Francis; Elias, Scott; Gilbert, M. Thomas P.; Haile, James; Munch, Kasper; Kuzmina, Svetlana; Froese, Duane G.; Holdaway, Richard N.; Willerslev, Eske

2009-01-01

Background A major challenge for ancient DNA (aDNA) studies on insect remains is that sampling procedures involve at least partial destruction of the specimens. A recent extraction protocol reveals the possibility of obtaining DNA from past insect remains without causing visual morphological damage. We test the applicability of this protocol on historic museum beetle specimens dating back to AD 1820 and on ancient beetle chitin remains from permafrost (permanently frozen soil) dating back more than 47,000 years. Finally, we test the possibility of obtaining ancient insect DNA directly from non-frozen sediments deposited 3280-1800 years ago - an alternative approach that also does not involve destruction of valuable material. Methodology/Principal Findings The success of the methodological approaches are tested by PCR and sequencing of COI and 16S mitochondrial DNA (mtDNA) fragments of 77–204 base pairs (-bp) in size using species-specific and general insect primers. Conclusion/Significance The applied non-destructive DNA extraction method shows promising potential on insect museum specimens of historical age as far back as AD 1820, but less so on the ancient permafrost-preserved insect fossil remains tested, where DNA was obtained from samples up to ca. 26,000 years old. The non-frozen sediment DNA approach appears to have great potential for recording the former presence of insect taxa not normally preserved as macrofossils and opens new frontiers in research on ancient biodiversity. PMID:19337382

Serological cross-reactions between four polyomaviruses of birds using virus-like particles expressed in yeast.

PubMed

Zielonka, Anja; Gedvilaite, Alma; Reetz, Jochen; Rösler, Uwe; Müller, Hermann; Johne, Reimar

2012-12-01

Polyomaviruses are aetiological agents of fatal acute diseases in various bird species. Genomic analysis revealed that avian polyomavirus (APyV), crow polyomavirus (CPyV), finch polyomavirus (FPyV) and goose hemorrhagic polyomavirus (GHPyV) are closely related to each other, but nevertheless form separate viral species; however, their serological relationship was previously unknown. As only APyV can be grown efficiently in tissue culture, virus-like particles (VLPs) were generated by expression of the genomic regions encoding the major structural protein VP1 of these viruses in yeast; these were used to elicit type-specific antibodies in rabbits and as antigens in serological reactions. For increased VLP assembly, a nuclear-localization signal was introduced into APyV-VP1. VLPs derived from the VP1 of the monkey polyomavirus simian virus 40 served as control. APyV-, GHPyV- and CPyV-VLPs showed haemagglutinating activity with chicken and human erythrocytes. CPyV- and GHPyV-specific sera showed slight cross-reactions in immunoblotting, haemagglutination-inhibition assay and indirect ELISA. The FPyV-specific serum inhibited the haemagglutination activity of APyV-VLPs slightly and showed a weak cross-neutralizing activity against APyV in cell-culture tests. Generally, these data indicate that the four polyomaviruses of birds are serologically distinct. However, in accordance with genetic data, a relationship between CPyV and GHPyV as well as between APyV and FPyV is evident, and grouping into two different serogroups may be suggested. The haemagglutinating activity of APyV, CPyV and GHPyV may indicate similar receptor-binding mechanisms for these viruses. Our data could be useful for the development of vaccines against the polyomavirus-induced diseases in birds and for interpretation of diagnostic test results.

Hypermethylated ERG as a cell-free fetal DNA biomarker for non-invasive prenatal testing of Down syndrome.

PubMed

Chen, Xi; Xiong, Likuan; Zeng, Ting; Xiao, Kelin; Huang, Yanping; Guo, Hui; Ren, Jinghui

2015-04-15

Previous reports have shown that the ERG gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. In this study, we explore the feasibility of hypermethylated ERG as a cell-free fetal (cff) DNA biomarker for non-invasive prenatal testing (NIPT) of Down syndrome. We randomly selected 90 healthy pregnant women, including 30 cases at each trimester of pregnancy. In addition, 15 pregnant women were recruited as the case group whose fetuses had been confirmed to have trisomy 21 by amniotic fluid analysis at 18th to 26th week gestation. Using HpaII, MspІ to digest cell-free maternal plasma DNA, we performed SYBR Green PCR to detect methylated sites of ERG sequences, and analyzed the concentrations of cff DNA in maternal plasma in different gestational trimesters and the case group. The ERG median concentrations of the maternal plasma after Hpa II digestion (LG copies/ml) in first, second and third-trimesters were 5.38, 6.10, and 7.04, respectively (Kruskal-Wallis, P<0.01); and that in the trisomy 21 case group was 6.85, which was higher than the second-trimester (Mann-Whitney, P<0.01). The study demonstrated that ERG gene is hypermethylated in cff DNA but hypomethylated in maternal DNA; and the median concentration of ERG gene in the trisomy 21 case group is higher than that in the gestational trimester matched normal group. ERG gene, as a fetal DNA biomarker, may be useful for NIPT of Down syndrome. Copyright © 2015 Elsevier B.V. All rights reserved.

“I think we’ve got too many tests!”: Prenatal providers’ reflections on ethical and clinical challenges in the practice integration of cell-free DNA screening

PubMed Central

Gammon, B.L.; Kraft, S.A.; Michie, M.; Allyse, M.

2016-01-01

Background The recent introduction of cell-free DNA-based non-invasive prenatal screening (cfDNA screening) into clinical practice was expected to revolutionize prenatal testing. cfDNA screening for fetal aneuploidy has demonstrated higher test sensitivity and specificity for some conditions than conventional serum screening and can be conducted early in the pregnancy. However, it is not clear whether and how clinical practices are assimilating this new type of testing into their informed consent and counselling processes. Since the introduction of cfDNA screening into practice in 2011, the uptake and scope have increased dramatically. Prenatal care providers are under pressure to stay up to date with rapidly changing cfDNA screening panels, manage increasing patient demands, and keep up with changing test costs, all while attempting to use the technology responsibly and ethically. While clinical literature on cfDNA screening has shown benefits for specific patient populations, it has also identified significant misunderstandings among providers and patients alike about the power of the technology. The unique features of cfDNA screening, in comparison to established prenatal testing technologies, have implications for informed decision-making and genetic counselling that must be addressed to ensure ethical practice. Objectives This study explored the experiences of prenatal care providers at the forefront of non-invasive genetic screening in the United States to understand how this testing changes the practice of prenatal medicine. We aimed to learn how the experience of providing and offering this testing differs from established prenatal testing methodologies. These differences may necessitate changes to patient education and consent procedures to maintain ethical practice. Methods We used the online American Congress of Obstetricians and Gynecologists Physician Directory to identify a systematic sample of five prenatal care providers in each U.S. state and the

A Method for Preparing DNA Sequencing Templates Using a DNA-Binding Microplate

PubMed Central

Yang, Yu; Hebron, Haroun R.; Hang, Jun

2009-01-01

A DNA-binding matrix was immobilized on the surface of a 96-well microplate and used for plasmid DNA preparation for DNA sequencing. The same DNA-binding plate was used for bacterial growth, cell lysis, DNA purification, and storage. In a single step using one buffer, bacterial cells were lysed by enzymes, and released DNA was captured on the plate simultaneously. After two wash steps, DNA was eluted and stored in the same plate. Inclusion of phosphates in the culture medium was found to enhance the yield of plasmid significantly. Purified DNA samples were used successfully in DNA sequencing with high consistency and reproducibility. Eleven vectors and nine libraries were tested using this method. In 10 μl sequencing reactions using 3 μl sample and 0.25 μl BigDye Terminator v3.1, the results from a 3730xl sequencer gave a success rate of 90–95% and read-lengths of 700 bases or more. The method is fully automatable and convenient for manual operation as well. It enables reproducible, high-throughput, rapid production of DNA with purity and yields sufficient for high-quality DNA sequencing at a substantially reduced cost. PMID:19568455

The future of forensic DNA analysis

PubMed Central

Butler, John M.

2015-01-01

The author's thoughts and opinions on where the field of forensic DNA testing is headed for the next decade are provided in the context of where the field has come over the past 30 years. Similar to the Olympic motto of ‘faster, higher, stronger’, forensic DNA protocols can be expected to become more rapid and sensitive and provide stronger investigative potential. New short tandem repeat (STR) loci have expanded the core set of genetic markers used for human identification in Europe and the USA. Rapid DNA testing is on the verge of enabling new applications. Next-generation sequencing has the potential to provide greater depth of coverage for information on STR alleles. Familial DNA searching has expanded capabilities of DNA databases in parts of the world where it is allowed. Challenges and opportunities that will impact the future of forensic DNA are explored including the need for education and training to improve interpretation of complex DNA profiles. PMID:26101278

Robust joint score tests in the application of DNA methylation data analysis.

PubMed

Li, Xuan; Fu, Yuejiao; Wang, Xiaogang; Qiu, Weiliang

2018-05-18

Recently differential variability has been showed to be valuable in evaluating the association of DNA methylation to the risks of complex human diseases. The statistical tests based on both differential methylation level and differential variability can be more powerful than those based only on differential methylation level. Anh and Wang (2013) proposed a joint score test (AW) to simultaneously detect for differential methylation and differential variability. However, AW's method seems to be quite conservative and has not been fully compared with existing joint tests. We proposed three improved joint score tests, namely iAW.Lev, iAW.BF, and iAW.TM, and have made extensive comparisons with the joint likelihood ratio test (jointLRT), the Kolmogorov-Smirnov (KS) test, and the AW test. Systematic simulation studies showed that: 1) the three improved tests performed better (i.e., having larger power, while keeping nominal Type I error rates) than the other three tests for data with outliers and having different variances between cases and controls; 2) for data from normal distributions, the three improved tests had slightly lower power than jointLRT and AW. The analyses of two Illumina HumanMethylation27 data sets GSE37020 and GSE20080 and one Illumina Infinium MethylationEPIC data set GSE107080 demonstrated that three improved tests had higher true validation rates than those from jointLRT, KS, and AW. The three proposed joint score tests are robust against the violation of normality assumption and presence of outlying observations in comparison with other three existing tests. Among the three proposed tests, iAW.BF seems to be the most robust and effective one for all simulated scenarios and also in real data analyses.

Method for assaying clustered DNA damages

DOEpatents

Sutherland, Betsy M.

2004-09-07

Disclosed is a method for detecting and quantifying clustered damages in DNA. In this method, a first aliquot of the DNA to be tested for clustered damages with one or more lesion-specific cleaving reagents under conditions appropriate for cleavage of the DNA to produce single-strand nicks in the DNA at sites of damage lesions. The number average molecular length (Ln) of double stranded DNA is then quantitatively determined for the treated DNA. The number average molecular length (Ln) of double stranded DNA is also quantitatively determined for a second, untreated aliquot of the DNA. The frequency of clustered damages (.PHI..sub.c) in the DNA is then calculated.

Mercaptoethanol-resistant human serum antibodies reacting with endotoxin from Neisseria gonorrhoeae.

PubMed Central

Maeland, J A; Larsen, B

1975-01-01

Sera from fifty patients with gonorrhoea, thirty with non-specific urethritis, and eighty blood donors were treated with mercaptoethanol (ME) and examined by the indirect haemagglutination test for antibodies against endotoxin from gonococci. Erythrocytes sensitized with determinant a of endotoxin from Strains 8551, V, and VII, or determinant b from Strain V were used. The percentage of sera active in the haemagglutination test was much higher in the gonorrhoea group than in the controls. The geometric mean titre was also significantly higher in the gonorrhoea group. This applied for all four antigens used. Results obtained in an anti-globulin test indicated that the titre of ME-treated serum was determined by IgG antibodies against the endotoxin. Many sera had titres which varied according to the strain origin of the antigen used in the test. The sensitivity of tests for antibodies was increased by using endotoxin from several different strains of gonococci for the examination of each serum. A simplified procedure for determination of antibodies against endotoxin from different strains of gonococci was elaborated. PMID:48404

DNA barcode goes two-dimensions: DNA QR code web server.

PubMed

Liu, Chang; Shi, Linchun; Xu, Xiaolan; Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

2012-01-01

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, "DNA barcode" actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications.

DNA Barcode Goes Two-Dimensions: DNA QR Code Web Server

PubMed Central

Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

2012-01-01

The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, “DNA barcode” actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications. PMID:22574113

Syphilis testing practices in the Americas.

PubMed

Trinh, Thuy T; Kamb, Mary L; Luu, Minh; Ham, D Cal; Perez, Freddy

2017-09-01

To present the findings of the Pan American Health Organization's 2014 survey on syphilis testing policies and practices in the Americas. Representatives of national/regional reference and large, lower-level laboratories from 35 member states were invited to participate. A semi-structured, electronically administered questionnaire collected data on syphilis tests, algorithms, equipment/commodities, challenges faced and basic quality assurance (QA) strategies employed (i.e. daily controls, standard operating procedures, technician training, participating in external QA programmes, on-site evaluations). The 69 participating laboratories from 30 (86%) member states included 41 (59%) national/regional reference and 28 (41%) lower-level laboratories. Common syphilis tests conducted were the rapid plasma reagin (RPR) (62% of surveyed laboratories), venereal disease research laboratory (VDRL) (54%), fluorescent treponemal antibody absorption (FTA-ABS) (41%) and Treponema pallidum haemagglutination assay (TPHA) (32%). Only three facilities reported using direct detection methods, and 28 (41% overall, 32% of lower-level facilities) used rapid tests. Most laboratories (62%) used only traditional testing algorithms (non-treponemal screening and treponemal confirmatory testing); however, 12% used only a reverse sequence algorithm (treponemal test first), and 14% employed both algorithms. Another nine (12%) laboratories conducted only one type of serologic test. Although most reference (97%) and lower-level (89%) laboratories used at least one QA strategy, only 16% reported using all five basic strategies. Commonly reported challenges were stock-outs of essential reagents or commodities (46%), limited staff training (73%) and insufficient equipment (39%). Many reference and clinical laboratories in the Americas face challenges in conducting appropriate syphilis testing and in ensuring quality of testing. © 2017 John Wiley & Sons Ltd The Pan-American Health Organization retains

Assessing DNA recovery from chewing gum.

PubMed

Eychner, Alison M; Schott, Kelly M; Elkins, Kelly M

2017-01-01

The purpose of this study was to evaluate which DNA extraction method yields the highest quantity of DNA from chewing gum. In this study, several popular extraction methods were tested, including Chelex-100, phenol-chloroform-isoamyl alcohol (PCIA), DNA IQ, PrepFiler, and QIAamp Investigator, and the quantity of DNA recovered from chewing gum was determined using real-time polymerase chain reaction with Quantifiler. Chewed gum control samples were submitted by anonymous healthy adult donors, and discarded environmental chewing gum samples simulating forensic evidence were collected from outside public areas (e.g., campus bus stops, streets, and sidewalks). As expected, results indicate that all methods tested yielded sufficient amplifiable human DNA from chewing gum using the wet-swab method. The QIAamp performed best when DNA was extracted from whole pieces of control gum (142.7 ng on average), and the DNA IQ method performed best on the environmental whole gum samples (29.0 ng on average). On average, the QIAamp kit also recovered the most DNA from saliva swabs. The PCIA method demonstrated the highest yield with wet swabs of the environmental gum (26.4 ng of DNA on average). However, this method should be avoided with whole gum samples (no DNA yield) due to the action of the organic reagents in dissolving and softening the gum and inhibiting DNA recovery during the extraction.

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells

PubMed Central

Jeon, Hye Lyun; Yi, Jung-Sun; Kim, Tae Sung; Oh, Youkyung; Lee, Hye Jeong; Lee, Minseong; Bang, Jin Seok; Ko, Kinarm; Ahn, Il Young; Ko, Kyungyuk; Kim, Joohwan; Park, Hye-Kyung; Lee, Jong Kwon; Sohn, Soo Jung

2017-01-01

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity. PMID:28443181

Development of a reference material of a single DNA molecule for the quality control of PCR testing.

PubMed

Mano, Junichi; Hatano, Shuko; Futo, Satoshi; Yoshii, Junji; Nakae, Hiroki; Naito, Shigehiro; Takabatake, Reona; Kitta, Kazumi

2014-09-02

We developed a reference material of a single DNA molecule with a specific nucleotide sequence. The double-strand linear DNA which has PCR target sequences at the both ends was prepared as a reference DNA molecule, and we named the PCR targets on each side as confirmation sequence and standard sequence. The highly diluted solution of the reference molecule was dispensed into 96 wells of a plastic PCR plate to make the average number of molecules in a well below one. Subsequently, the presence or absence of the reference molecule in each well was checked by real-time PCR targeting for the confirmation sequence. After an enzymatic treatment of the reaction mixture in the positive wells for the digestion of PCR products, the resultant solution was used as the reference material of a single DNA molecule with the standard sequence. PCR analyses revealed that the prepared samples included only one reference molecule with high probability. The single-molecule reference material developed in this study will be useful for the absolute evaluation of a detection limit of PCR-based testing methods, the quality control of PCR analyses, performance evaluations of PCR reagents and instruments, and the preparation of an accurate calibration curve for real-time PCR quantitation.

Patient acceptance of non-invasive testing for fetal aneuploidy via cell-free fetal DNA.

PubMed

Vahanian, Sevan A; Baraa Allaf, M; Yeh, Corinne; Chavez, Martin R; Kinzler, Wendy L; Vintzileos, Anthony M

2014-01-01

To evaluate factors associated with patient acceptance of noninvasive prenatal testing for trisomy 21, 18 and 13 via cell-free fetal DNA. This was a retrospective study of all patients who were offered noninvasive prenatal testing at a single institution from 1 March 2012 to 2 July 2012. Patients were identified through our perinatal ultrasound database; demographic information, testing indication and insurance coverage were compared between patients who accepted the test and those who declined. Parametric and nonparametric tests were used as appropriate. Significant variables were assessed using multivariate logistic regression. The value p < 0.05 was considered significant. Two hundred thirty-five patients were offered noninvasive prenatal testing. Ninety-three patients (40%) accepted testing and 142 (60%) declined. Women who accepted noninvasive prenatal testing were more commonly white, had private insurance and had more than one testing indication. There was no statistical difference in the number or the type of testing indications. Multivariable logistic regression analysis was then used to assess individual variables. After controlling for race, patients with public insurance were 83% less likely to accept noninvasive prenatal testing than those with private insurance (3% vs. 97%, adjusted RR 0.17, 95% CI 0.05-0.62). In our population, having public insurance was the factor most strongly associated with declining noninvasive prenatal testing.

The future of forensic DNA analysis.

PubMed

Butler, John M

2015-08-05

The author's thoughts and opinions on where the field of forensic DNA testing is headed for the next decade are provided in the context of where the field has come over the past 30 years. Similar to the Olympic motto of 'faster, higher, stronger', forensic DNA protocols can be expected to become more rapid and sensitive and provide stronger investigative potential. New short tandem repeat (STR) loci have expanded the core set of genetic markers used for human identification in Europe and the USA. Rapid DNA testing is on the verge of enabling new applications. Next-generation sequencing has the potential to provide greater depth of coverage for information on STR alleles. Familial DNA searching has expanded capabilities of DNA databases in parts of the world where it is allowed. Challenges and opportunities that will impact the future of forensic DNA are explored including the need for education and training to improve interpretation of complex DNA profiles. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Microsatellite DNA library for Caiman latirostris.

PubMed

Zucoloto, Rodrigo Barban; Verdade, Luciano Martins; Coutinho, Luiz Lehmann

2002-12-15

New genetic markers were characterized for the broad-snouted caiman (Caiman latirostris) by constructing libraries enriched for microsatellite DNA. Construction and characterization of these libraries are described in the present study. One microsatellite marker was developed from a (ACC-TGG)(n)enriched microsatellite DNA library, and 12 microsatellite markers were developed from a (AC-TG)(n)enriched microsatellite DNA library. These markers were tested in wild-caught animals, and these tests resulted in ten new polymorphic microsatellites for C. latirostris. Copyright 2002 Wiley-Liss, Inc.

Comparing triage algorithms using HPV DNA genotyping, HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women.

PubMed

Luttmer, Roosmarijn; Berkhof, Johannes; Dijkstra, Maaike G; van Kemenade, Folkert J; Snijders, Peter J F; Heideman, Daniëlle A M; Meijer, Chris J L M

2015-06-01

High-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2). Comparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women. hrHPV DNA-positive women aged 18-63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n=348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n=133). Sensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002-1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93-1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72-1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75-1.10). For detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology. Copyright © 2015 Elsevier B.V. All rights reserved.

Data from complete mtDNA sequencing of Tunisian centenarians: testing haplogroup association and the "golden mean" to longevity.

PubMed

Costa, Marta D; Cherni, Lotfi; Fernandes, Verónica; Freitas, Fernando; Ammar El Gaaied, Amel Ben; Pereira, Luísa

2009-04-01

Since the mitochondrial theory of ageing was proposed, mitochondrial DNA (mtDNA) diversity has been largely studied in old people, however complete genomes are still rare, being limited to Japanese and UK/US samples. In this work, we evaluated possible longevity associated polymorphisms/haplogroups in an African population, from Tunisia, by performing complete mtDNA sequencing. This population has a mixed Eurasian/sub-Saharan mtDNA gene pool, which could potentially facilitate the evaluation of association for sub-Saharan lineages. Sub-Saharan haplogroups were shown to be significantly less represented in centenarians (9.5%) than in controls (54.5%), but it is not possible to rule out an influence of population structure, which is high in these populations. No recurrent polymorphism were more frequent in centenarians than in controls, and although the Tunisian centenarians presented less synonymous and replacement polymorphisms than controls, this difference was not statistically significant. So far, it does not seem that centenarians have significantly less mildly deleterious substitutions, not only in Tunisia but also in Japanese and UK/US samples, as tested here, not favouring a "golden mean" to longevity.

Detection of Toxoplasma gondii DNA in horse meat from supermarkets in France and performance evaluation of two serological tests.

PubMed

Aroussi, Abdelkrim; Vignoles, Philippe; Dalmay, François; Wimel, Laurence; Dardé, Marie-Laure; Mercier, Aurélien; Ajzenberg, Daniel

2015-01-01

In France, some cases of severe toxoplasmosis have been linked to the consumption of horse meat that had been imported from the American continent where atypical strains of Toxoplasma gondii are more common than in Europe. Many seroprevalence studies are presented in the literature but risk assessment of T. gondii infection after horse meat consumption is not possible in the absence of validated serological tests and the unknown correlation between detection of antibodies against T. gondii and presence of tissue cysts. We performed magnetic-capture polymerase chain reaction (MC-PCR) to detect T. gondii DNA in 231 horse meat samples purchased in supermarkets in France and evaluated the performance and level of agreement of the modified agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA) in the meat juices. The serological tests lacked sensitivity, specificity, and agreement between them, and there was no correlation with the presence of T. gondii DNA in horse meat, raising concerns about the reliability of T. gondii seroprevalence data in horses from the literature. T. gondii DNA was detected in 43% of horse meat samples but the absence of strain isolation in mice following inoculation of more than 100 horse meat samples suggests a low distribution of cysts in skeletal muscles and a low risk of T. gondii infection associated with horse meat consumption. However, to avoid any risk of toxoplasmosis, thorough cooking of horse meat is recommended. © A. Aroussi et al., published by EDP Sciences, 2015.

Detection of Toxoplasma gondii DNA in horse meat from supermarkets in France and performance evaluation of two serological tests

PubMed Central

Aroussi, Abdelkrim; Vignoles, Philippe; Dalmay, François; Wimel, Laurence; Dardé, Marie-Laure; Mercier, Aurélien; Ajzenberg, Daniel

2015-01-01

In France, some cases of severe toxoplasmosis have been linked to the consumption of horse meat that had been imported from the American continent where atypical strains of Toxoplasma gondii are more common than in Europe. Many seroprevalence studies are presented in the literature but risk assessment of T. gondii infection after horse meat consumption is not possible in the absence of validated serological tests and the unknown correlation between detection of antibodies against T. gondii and presence of tissue cysts. We performed magnetic-capture polymerase chain reaction (MC-PCR) to detect T. gondii DNA in 231 horse meat samples purchased in supermarkets in France and evaluated the performance and level of agreement of the modified agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA) in the meat juices. The serological tests lacked sensitivity, specificity, and agreement between them, and there was no correlation with the presence of T. gondii DNA in horse meat, raising concerns about the reliability of T. gondii seroprevalence data in horses from the literature. T. gondii DNA was detected in 43% of horse meat samples but the absence of strain isolation in mice following inoculation of more than 100 horse meat samples suggests a low distribution of cysts in skeletal muscles and a low risk of T. gondii infection associated with horse meat consumption. However, to avoid any risk of toxoplasmosis, thorough cooking of horse meat is recommended. PMID:25809058

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

PubMed Central

Hashemipetroudi, Seyyed Hamidreza; Nematzadeh, Ghorbanali; Ahmadian, Gholamreza; Yamchi, Ahad; Kuhlmann, Markus

2018-01-01

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species. Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species PMID:29443017

A Novel Strategy to Predict Carcinogenicity of Antiparasitics Based on a Combination of DNA Lesions and Bacterial Mutagenicity Tests

PubMed Central

Liu, Qianying; Lei, Zhixin; Zhu, Feng; Ihsan, Awais; Wang, Xu; Yuan, Zonghui

2017-01-01

Genotoxicity and carcinogenicity testing of pharmaceuticals prior to commercialization is requested by regulatory agencies. The bacterial mutagenicity test was considered having the highest accuracy of carcinogenic prediction. However, some evidences suggest that it always results in false-positive responses when the bacterial mutagenicity test is used to predict carcinogenicity. Along with major changes made to the International Committee on Harmonization guidance on genotoxicity testing [S2 (R1)], the old data (especially the cytotgenetic data) may not meet current guidelines. This review provides a compendium of retrievable results of genotoxicity and animal carcinogenicity of 136 antiparasitics. Neither genotoxicity nor carcinogenicity data is available for 84 (61.8%), while 52 (38.2%) have been evaluated in at least one genotoxicity or carcinogenicity study, and only 20 (14.7%) in both genotoxicity and carcinogenicity studies. Among 33 antiparasitics with at least one old result in in vitro genotoxicity, 15 (45.5%) are in agreement with the current ICH S2 (R1) guidance for data acceptance. Compared with other genotoxicity assays, the DNA lesions can significantly increase the accuracy of prediction of carcinogenicity. Together, a combination of DNA lesion and bacterial tests is a more accurate way to predict carcinogenicity. PMID:29170735

DNA Carrier Testing and Newborn Screening for Maple Syrup Urine Disease in Old Order Mennonite Communities

PubMed Central

Carleton, Stephanie M.; Peck, Dawn S.; Grasela, Julie; Dietiker, Kristin L.

2010-01-01

Maple syrup urine disease (MSUD) is an inherited metabolic disorder caused by mutations in the branched chain α-keto acid dehydrogenase complex. Worldwide incidence of MSUD is 1:225,000 live births. However, within Old Order Mennonite communities, the incidence is 1:150 live births and results from a common tyrosine to asparagine substitution (Y438N) in the E1α subunit of branched chain α-keto acid dehydrogenase. We developed a new DNA diagnostic assay utilizing TaqMan® technology and compared its efficacy, sensitivity, and duration with an existing polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay. Carrier testing was performed by both TaqMan technology and PCR-RFLP on DNA isolated from buccal swabs of 160 individuals as well as from buccal swabs and blood spots of nine at-risk newborns; assay time, sensitivity, and reliability were also evaluated. The TaqMan assay, like the PCR-RFLP assay, accurately determined Y438N E1α allele status. However, the TaqMan assay appeared (1) more sensitive than the PCR-RFLP assay, requiring 10-fold less DNA (10 ng) to reliably determine genotype status and (2) faster, reducing the assay time required for diagnosis from ∼12 to 5 h. TaqMan technology allowed more rapid DNA diagnoses of MSUD in the neonate, thereby reducing the likelihood of neurological impairment while enhancing health and prognosis for affected infants. PMID:20136525

Test of synthetic DNA tracers in a periodic hydrodynamic system for time-variable transit time distribution assessment

NASA Astrophysics Data System (ADS)

Dahlke, H. E.; Wang, C.; McNew, C.; McLaughlin, S.; Lyon, S. W.

2016-12-01

Recent research on time-varying transport through hydrologic systems proposed using decomposed over-printed tracer breakthrough curves to directly observe transport through complex flow systems. This method, also known as the PERTH (Periodic Tracer Hierarchy) method requires periodic flow and multiple tracer injections to reveal changes in flow pathways and transport behavior. Time-variable transit time distributions (TTD) estimated from tracer breakthrough curves often vary with the storage state of the system, which in turn is influenced by internal and external variabilities, such as the arrangement of flow pathways and fluctuations in system inputs. Deciphering internal from external variabilities in TTDs might help to advance the use of TTDs for estimating the physical state of a system; however, thus far the finite number of unique conservative tracers available for tracing has limited deeper insights. Synthetic DNA tracers consisting of short strands of synthetic DNA encapsulated by polylactic acid (PLA) microspheres could potentially provide multiple unique tracers with identical transport properties needed to explore time varying transport through hydrologic systems in more detail. An experiment was conducted on the miniLeo hillslope, a 1 m3 sloping lysimeter, within the Biosphere 2 Landscape Evolution Observatory near Tucson, AZ to investigate transit time variability. The goal of the experiment was to 1) test the suitability of using synthetic DNA tracers for estimating TTDs in a hydrologic system and 2) to determine the TTDs of individual tracer pulses under periodic steady-state conditions. Five DNA tracers, consisting of four unique, encapsulated DNA sequences and one free/non-encapsulated DNA sequence, were applied as reference and probe tracers together with deuterium, using the PERTH method. The lysimeter received three 2-hour pulses of rainfall at a rate of 30 mm/hr for 10 days. Initial results show that both the encapsulated and free DNA tracers

A study of the transport and immobilisation mechanisms of human red blood cells in a paper-based blood typing device using confocal microscopy.

PubMed

Li, Lizi; Tian, Junfei; Ballerini, David; Li, Miaosi; Shen, Wei

2013-09-07

Recent research on the use of bioactive paper for human blood typing has led to the discovery of a new method for identifying the haemagglutination of red blood cells (RBCs). When a blood sample is introduced onto paper treated with the grouping antibodies, RBCs undergo haemagglutination with the corresponding grouping antibodies, forming agglutinated cell aggregates in the paper. A subsequent washing of the paper with saline buffer could not remove these aggregates from the paper; this phenomenon provides a new method for rapid, visual identification of the antibody-specific haemagglutination reactions and thus the determination of the blood type. This study aims to understand the mechanism of RBC immobilization inside the paper which follows haemagglutination reactions. Confocal microscopy is used to observe the morphology of the free and agglutinated RBCs that are labelled with FITC. Chromatographic elution patterns of both agglutinated and non-agglutinated RBCs are studied to gain insight into the transport behaviour of free RBCs and agglutinated aggregates. This work provides new information about RBC haemagglutination inside the fibre network of paper on a microscopic level, which is important for the future design of paper-based blood typing devices with high sensitivity and assaying speed.

The use of novel DNA nanotracers to determine groundwater flow paths - a test study at the Grimsel Deep Underground Geothermal (DUG) Laboratory in Switzerland

NASA Astrophysics Data System (ADS)

Kittilä, Anniina; Evans, Keith; Puddu, Michela; Mikutis, Gediminas; Grass, Robert N.; Deuber, Claudia; Saar, Martin O.

2016-04-01

Groundwater flow in fractured media is heterogeneous and takes place in structures with complex geometry and scale effects, which make the characterization and modeling of the groundwater flow technically challenging. Surface geophysical surveys have limited resolution of permeable structures, and often provide ambiguous results, whereas the interpretation of borehole flow logs to infer hydraulic flow paths within fractured reservoirs is usually non-unique. Nonetheless, knowledge of the hydraulic properties of individual fractures and the role they play in determining the larger-scale flow within the fracture network (i.e. the overall flow conditions) is required in many hydrogeological and geo-engineering situations, such as in geothermal reservoir studies. Tracer tests can overcome some of the aforementioned limitations by providing strong constraints on the geometry and characteristics of flow paths linking boreholes within both porous media and fracture-dominated types of reservoirs. In the case of geothermal reservoirs, tracer tests are often used to provide estimates of the pore/fracture volume swept by flow between injection and production wells. This in turn places constraints on the swept surface area, a parameter that is key for estimating the commercial longevity of the geothermal system. A problem with conventional tracer tests is that the solute species used as the tracer tend to persist in detectable quantities within the reservoir for a long time, thereby impeding repeat tracer tests. DNA nanotracers do not suffer from this problem as they can be designed with a unique signature for each test. DNA nanotracers are environmentally friendly, sub-micron sized silica particles encapsulating small fragments of synthetic DNA which can be fabricated to have a specified, uniquely detectable configuration. For this reason, repeat tracer tests conducted with a differently-encoded DNA fragment to that used in the original will not suffer interference from the

Assessment of the stability of DNA in specimens collected under conditions for drug testing-A pilot study.

PubMed

White, Robert M; Mitchell, John M; Hart, E Dale; Evans, Amy; Meaders, Meredith; Norsworthy, Sarah E; Hayes, Eugene D; Flegel, Ron; Maha, George C; Shaffer, Megan D; Hall, Erin M; Rogers, Kelley

2018-02-01

For forensic biological sample collections, the specimen donor is linked solidly to his or her specimen through a chain of custody (CoC) sometimes referenced as a chain of evidence. Rarely, a donor may deny that a urine or oral fluid (OF) specimen is his or her specimen even with a patent CoC. The goal of this pilot study was to determine the potential effects of short-term storage on the quality and quantity of DNA in both types of specimen under conditions that may be encountered with employment-related drug testing specimens. Fresh urine and freshly collected oral fluid all produced complete STR profiles. For the "pad" type OF collectors, acceptable DNA was extractable both from the buffer/preservative and the pad. Although fresh urine and OF produced complete STR profiles, partial profiles were obtained after storage for most samples. An exception was the DNA in the Quantisal OF collector, from which a complete profile was obtained for both freshly collected OF and stored OF. Copyright © 2017 Elsevier B.V. All rights reserved.

Faecal shedding of canine parvovirus after modified-live vaccination in healthy adult dogs.

PubMed

Freisl, M; Speck, S; Truyen, U; Reese, S; Proksch, A-L; Hartmann, K

2017-01-01

Since little is known about the persistence and faecal shedding of canine parvovirus (CPV) in dogs after modified-live vaccination, diagnostic tests for CPV can be difficult to interpret in the post-vaccination period. The primary aim of this study was to determine the incidence, duration and extent of CPV vaccine virus shedding in adult dogs and to investigate related factors, including the presence of protective antibodies, increase in anti-CPV antibody titres and development of any gastrointestinal side-effects. A secondary objective was to assess prevalence of CPV field virus shedding in clinically healthy dogs due to subclinical infections. One hundred adult, healthy privately owned dogs were vaccinated with a commercial CPV-2 modified-live vaccine (MLV). Faeces were tested for the presence of CPV DNA on days 0 (prior to vaccination), 3, 7, 14, 21 and 28 by quantitative real-time PCR. Pre- and post-vaccination serum titres were determined by haemagglutination inhibition on days 0, 7 and 28. Transient excretion of CPV DNA was detected in 2.0% of dogs before vaccination. About one quarter of dogs (23.0%) shed CPV DNA during the post-vaccination period, but field and vaccine virus differentiation by VP2 gene sequencing was only successful in few samples. Faecal CPV excretion occurred despite protective serum antibody titres. Post-vaccination CPV shedding was not related to adequate antibody response after vaccination or to the occurrence of gastrointestinal side-effects. Despite individual differences, CPV DNA was detectable for up to 28 days after vaccination, although the faecal CPV DNA load in these clinically healthy dogs was very low. Copyright © 2016 Elsevier Ltd. All rights reserved.

Loop-Mediated Isothermal Amplification on Crude DNA as a Point-of-Care Test for the Diagnosis of Mycoplasma-Related Vaginitis During Early Pregnancy.

PubMed

Wang, Yichao; Zhang, Bumei; Sun, Yan; Liu, Yunde; Gu, Yajun

2017-12-20

Mycoplasma-related vaginitis gradually has been growing as a threat in adults-genitourinary infection contributes to funisitis, spontaneous abortion, and low birth weight. Until now, use of loop-mediated isothermal amplification (LAMP) to detect Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), or Mycoplasma genitalium (MG) has been reported by some researchers. However, previous studies focused on purified DNA as the template for LAMP assay, which is usually extracted via commercial kit. We developed a LAMP assay for rapid detection of UU, MH, and MG genital mycoplasmas using a simple boiling method for DNA extraction, in a cohort of pregnant women with mycoplasma-related vaginitis. We monitored amplicons with the naked eye using SYBR Green I. The cohort in our study showed a prevalence of 22.6% in pregnant women, as detected by UU-LAMP assay. Compared to the polymerase chain reaction (PCR) test with purified DNA, the sensitivity of the UU-LAMP in clinical specimens with crude DNA was 87.5% (95% confidence interval [CI], 64.6%->99.9). For crude DNA specimens, UU-LAMP was more sensitive and reliable than PCR, with a higher agreement rate (96.8%) and Youden index value (0.88). As a point-of-care test, LAMP is a useful, specific, and efficient way to detect genital mycoplasmas in resource-limited settings, especially for crude DNA. © American Society for Clinical Pathology 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Vanadium accelerates polymerase chain reaction and expands the applicability of forensic DNA testing.

PubMed

Kaminiwa, Junko; Honda, Katsuya; Sugano, Yukiko; Yano, Shizue; Nishi, Takeki; Sekine, Yuko

2013-05-01

Polymerase chain reaction (PCR) has been rapidly established as one of the most widely used techniques in molecular biology. Because most DNA analysis is PCR-based, the analysis of unamplifiable DNA of poor quality or low quantity is nearly impossible. However, we observed that if an appropriate concentration of vanadium chloride is added to the standard reaction mixture, the enzymatic amplification of DNA could be enhanced. Using multiplex PCR with the addition of vanadium, DNA typing was possible from even trace amounts of DNA that we were unable to amplify using normal reaction conditions. This method might be an effective tool for not only criminal investigations and ancient DNA analysis, but also for nearly all fields using DNA technology. Copyright © 2012 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Maternal cfDNA screening for Down syndrome--a cost sensitivity analysis.

PubMed

Cuckle, Howard; Benn, Peter; Pergament, Eugene

2013-07-01

This study aimed to determine the principal factors contributing to the cost of avoiding a birth with Down syndrome by using cell-free DNA (cfDNA) to replace conventional screening. A range of unit costs were assigned to each item in the screening process. Detection rates were estimated by meta-analysis and modeling. The marginal cost associated with the detection of additional cases using cfDNA was estimated from the difference in average costs divided by the difference in detection. The main factor was the unit cost of cfDNA testing. For example, replacing a combined test costing $150 with 3% false-positive rate and invasive testing at $1000, by cfDNA tests at $2000, $1500, $1000, and $500, the marginal cost is $8.0, $5.8, $3.6, and $1.4m, respectively. Costs were lower when replacing a quadruple test and higher for a 5% false-positive rate, but the relative importance of cfDNA unit cost was unchanged. A contingent policy whereby 10% to 20% women were selected for cfDNA testing by conventional screening was considerably more cost-efficient. Costs were sensitive to cfDNA uptake. Universal cfDNA screening for Down syndrome will only become affordable by public health purchasers if costs fall substantially. Until this happens, the contingent use of cfDNA is recommended. © 2013 John Wiley & Sons, Ltd.

Exploring mechanisms of transport and persistence of environmental DNA (eDNA)

NASA Astrophysics Data System (ADS)

Shogren, A.; Tank, J. L.; Riis, T.; Rosi, E. J.; Bolster, D.

2017-12-01

Sampling for eDNA is a non-intrusive method to detect species presence without direct observation, which allows for earlier detection and more rapid response than conventional sampling methods. However, our current understanding of how eDNA is transported and persists in flowing waters (e.g., streams and rivers) remains imprecise; in flowing waters, the target organism may be some distance away from where the eDNA in water is collected. It is uncertain how the unique transport properties of suspended eDNA or the inherent heterogeneity of natural flowing systems may impact the probability of downstream eDNA detection. To improve understanding of eDNA fate, we first conducted experimental releases and modeled the impact of benthic substrate heterogeneity and size on eDNA transport and retention in streams. We also used recirculating artificial streams to constrain estimates of eDNA degradation in systems with varying flow and microbial biofilm coverage. We found that eDNA retention in streams is substrate-specific, and that streambed hydraulics have significant influence on how far eDNA is transported downstream. Through the degradation experiments, we found that eDNA degradation is strongly context dependent, but even in systems with low velocity, eDNA can remain detectable in the water column >24hrs after introduction. This differential persistence of eDNA particles confirms that eDNA dynamics in flowing waters are not constant along a spatial continuum, which complicates interpretation of a positive detection in flowing waters, which presents a scaling problem for future modeling efforts to support transport predictions. To test our experimental results in a natural system, we compared our previous estimates for eDNA transport, retention, and degradation to field data collected during a longitudinal field survey for zebra mussel eDNA on the Gudena River in Silkeborg, Denmark. We found that though heterogeneity indeed complicates scaling efforts to extrapolate

Direct-to-consumer DNA testing: the fallout for individuals and their families unexpectedly learning of their donor conception origins.

PubMed

Crawshaw, Marilyn

2017-07-11

Increasing numbers of donor-conceived individuals (and/or parents) are seeking individuals genetically related through donor conception. One route is through 'direct-to-consumer' (DTC) DNA testing, prompting calls for fertility services to alert donors and prospective parents to the increasing unsustainability of anonymity and secrecy. The complexity of interpreting DNA results in this context has also been discussed, including their lack of absolute certainty, as has the need for professional and peer support. This commentary highlights a different 'threat', from individuals learning of their donor-conception origins through the use of such tests by themselves or relatives for such purposes as genealogy or health checks. It illustrates the personal complexities faced by three older women and their families on learning not only of their genetic relationship to each other but also to 15 more donor-related siblings. DTC DNA services are a growing feature of modern life. This commentary raises ethical questions about their responsibilities towards those inadvertently learning of donor conception origins and the responsibilities of fertility services to inform prospective parents and donors of this new phenomenon. Considerations of how and when parents should tell their children of their donor-conception origins here instead become how and when children should inform their parents.

Development and Validation of Environmental DNA (eDNA) Markers for Detection of Freshwater Turtles.

PubMed

Davy, Christina M; Kidd, Anne G; Wilson, Chris C

2015-01-01

Environmental DNA (eDNA) is a potentially powerful tool for detection and monitoring of rare species, including threatened native species and recently arrived invasive species. Here, we develop DNA primers for a suite of nine sympatric freshwater turtles, and use it to test whether turtle eDNA can be successfully detected in samples from aquaria and an outdoor pond. We also conduct a cost comparison between eDNA detection and detection through traditional survey methods, using data from field surveys at two sites in our target area. We find that eDNA from turtles can be detected using both conventional polymerase chain reaction (PCR) and quantitative PCR (qPCR), and that the cost of detection through traditional survey methods is 2-10X higher than eDNA detection for the species in our study range. We summarize necessary future steps for application of eDNA surveys to turtle monitoring and conservation and propose specific cases in which the application of eDNA could further the conservation of threatened turtle species.

Development and Validation of Environmental DNA (eDNA) Markers for Detection of Freshwater Turtles

PubMed Central

Davy, Christina M.; Kidd, Anne G.; Wilson, Chris C.

2015-01-01

Environmental DNA (eDNA) is a potentially powerful tool for detection and monitoring of rare species, including threatened native species and recently arrived invasive species. Here, we develop DNA primers for a suite of nine sympatric freshwater turtles, and use it to test whether turtle eDNA can be successfully detected in samples from aquaria and an outdoor pond. We also conduct a cost comparison between eDNA detection and detection through traditional survey methods, using data from field surveys at two sites in our target area. We find that eDNA from turtles can be detected using both conventional polymerase chain reaction (PCR) and quantitative PCR (qPCR), and that the cost of detection through traditional survey methods is 2–10X higher than eDNA detection for the species in our study range. We summarize necessary future steps for application of eDNA surveys to turtle monitoring and conservation and propose specific cases in which the application of eDNA could further the conservation of threatened turtle species. PMID:26200348

Detection of parvovirus B19 DNA in blood: Viruses or DNA remnants?

PubMed

Molenaar-de Backer, M W A; Russcher, A; Kroes, A C M; Koppelman, M H G M; Lanfermeijer, M; Zaaijer, H L

2016-11-01

Parvovirus B19 (B19V) DNA can be detected in blood over a long period after acute infection. Several reports associate the presence of B19V DNA with disease, irrespective of timing of the initial B19V infection. This study aims to analyze the properties of B19V DNA in blood, differentiating between bare, non-infectious strands of DNA and B19V DNA in viable virions. Ten blood donors with asymptomatic acute B19V infection were followed and sampled up to 22 months after infection. The samples were treated with and without an endonuclease and tested for B19V DNA, to distinguish between DNA in virions and naked DNA. In the acute phase of infection, high levels of B19V DNA were detected, concurrent with B19V IgM antibodies. B19V DNA apparently was encapsidated, as indicated by resistance to endonuclease degradation. Subsequently, B19V DNA remained detectable for more than one year in all donors at low levels (<10 5 IU/mL). Approximately 150days after infection B19V DNA became degradable by an endonuclease, indicating that this concerned naked DNA. In some donors a second endonuclease-resistant peak occurred. Detection of B19V DNA in blood by PCR does not necessarily imply that B19V replication takes place and that infectious B19V virions are present. We propose that remnant B19V DNA strands can be released from tissues without active replication. This finding urges to reconsider an assumed role of B19V infection mainly based on B19V DNA detection in blood, a much debated subject in clinical syndromes such as myocarditis and arthritis. Copyright © 2016 Elsevier B.V. All rights reserved.

A ranking index for quality assessment of forensic DNA profiles forensic DNA profiles

PubMed Central

2010-01-01

Background Assessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms. Results We recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI) (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009) 951-958). FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles. Conclusions The developed tool provides unbiased quality assessment of forensic DNA profiles. It can be applied for any DNA profiling system based on Short Tandem Repeat markers. Apart from crime related DNA analysis, FI can therefore be used as a quality tool in paternal or familial testing as well as in disaster victim identification. PMID:21062433

Immunomodulatory activity of methanolic extract of Morus alba Linn. (mulberry) leaves.

PubMed

Bharani, Shendige Eswara Rao; Asad, Mohammed; Dhamanigi, Sunil Samson; Chandrakala, Gowda Kallenahalli

2010-01-01

The leaves of Morus alba Linn. (Family: Moraceae) commonly known as mulberry are mainly used as food for the silkworms and they are sometimes eaten as vegetable or used as cattle fodder in different parts of the world. The effect of Morus alba on the immune system was evaluated by using different experimental models such as carbon clearance test, cyclophosphamide induced neutropenia, neutrophil adhesion test, effect on serum immunoglobulins, mice lethality test and indirect haemagglutination test. Methanolic extract of Morus alba was administered orally at low dose and high dose of 100 mg/kg and 1 g/kg respectively and Ocimum sanctum (100 mg/kg, po) was used as standard drug. Morus alba extract in both doses increased the levels of serum immunoglobulins and prevented the mortality induced by bovine Pasteurella multocida in mice. It also increased the circulating antibody titre in indirect haemagglutination test. On the other hand, it showed significant increase in the phagocytic index in carbon clearance assay, a significant protection against cyclophosphamide induced neutropenia and increased the adhesion of neutrophils in the neutrophil adhesion test. Hence, it was concluded that Morus alba increases both humoral immunity and cell mediated immunity.

Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification.

PubMed

Poon, Leo L M; Wong, Bonnie W Y; Ma, Edmund H T; Chan, Kwok H; Chow, Larry M C; Abeyewickreme, Wimal; Tangpukdee, Noppadon; Yuen, Kwok Y; Guan, Yi; Looareesuwan, Sornchai; Peiris, J S Malik

2006-02-01

Malaria is one of the most important parasitic infections in humans. A sensitive diagnostic test for malaria that could be applied at the community level could be useful in programs to control the disease. The aim of the present work was to develop a simple, inexpensive molecular test for Plasmodium falciparum. Blood was collected from controls (n = 100) and from patients diagnosed with falciparum malaria infection (n = 102), who were recruited to the study. Heat-treated blood samples were tested by a loop-mediated isothermal amplification (LAMP) assay for P. falciparum. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. To evaluate the assay, DNA from these samples was purified and tested by PCR. Results from the LAMP and PCR assays were compared. The LAMP assay detected P. falciparum directly from heat-treated blood. The quantitative data from the assay correlated to the parasite counts obtained by blood-film microscopic analyses. When we used the PCR assay as the comparison method, the sensitivity and specificity of the LAMP assay were 95% and 99%, respectively. Unlike PCR, the LAMP assay does not require purified DNA for efficient DNA amplification, thereby reducing the cost and turnaround time for P. falciparum diagnosis. The assay requires only basic instruments, and assay positivity can be verified by visual inspection.

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

PubMed Central

Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

2014-01-01

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA.

PubMed

Ludgate, Jackie L; Wright, James; Stockwell, Peter A; Morison, Ian M; Eccles, Michael R; Chatterjee, Aniruddha

2017-08-31

Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis. Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing. The main features and advantages of this protocol are: An optimized method for extracting good quality DNA from FFPE tissues. An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue. Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing. We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.

Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay

PubMed Central

Krajden, Mel; Comanor, Lorraine; Rifkin, Oretta; Grigoriew, Anna; Minor, James M.; Kapke, Gordon F.

1998-01-01

Quantification of hepatitis B virus (HBV) DNA in serum is used to establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the conditions that preserve sample integrity is of paramount importance. We therefore evaluated the stability of HBV DNA in 26 previously frozen (PF) and 5 fresh, never previously frozen serum specimens. PF specimens, covering a 3-log10 HBV DNA dynamic range, were thawed and stored at −70, 4, 23, 37, and 45°C (±1.5°C) for 0, 24, 72, and 120 h (±2 h) and were refrozen at −70°C prior to testing. Five fresh specimens were split into two groups. Both group FG1 and group FG2 specimens were handled as described above; however, group FG1 specimens were subsequently maintained at 4°C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at ≤4°C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45°C, respectively. Three independent statistical methods confirmed that the probability of specimen failure, defined as a loss of 20% or more of HBV DNA and/or coagulation of serum, was lowest at ≤4°C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of ≥20% at 23 or 37°C, sufficient numbers of serum specimens must be evaluated to determine overall statistical trends. In conclusion, HBV DNA integrity in separated serum specimens is preserved for at least 5 days when the specimens are stored at −70 or 4°C. PMID:9466745

Diagnosing systemic lupus erythematosus: new-generation immunoassays for measurement of anti-dsDNA antibodies are an effective alternative to the Farr technique and the Crithidia luciliae immunofluorescence test.

PubMed

Antico, A; Platzgummer, S; Bassetti, D; Bizzaro, N; Tozzoli, R; Villalta, D

2010-07-01

The aim of this study was to evaluate the diagnostic performance of four new enzyme immunoassays (EIAs) for anti-double-stranded-DNA (anti-dsDNA) antibodies, in comparison with the Farr assay and the Crithidia luciliae immunofluorescence test (CLIFT). To this purpose, sera from four patient groups were collected: 52 sera from patients with systemic lupus erythematosus (SLE); 28 from patients with other connective tissue diseases (CTD); 36 from patients with hepatitis C virus (HCV) infection; and 24 from those with acute viral infection. All sera were tested for anti-dsDNA antibodies by four EIA methods using a different antigenic DNA source [synthetic oligonucleotide (Method A), circular plasmid (Method B), recombinant (Method C), and purified extracted (Method D)], and by CLIFT and Farr assays. The diagnostic sensitivity of the assays was as follows: 84.6% (Method A), 73% (B), 82.7% (C), 84.6% (D), 55.8% (CLIFT), and 78.8% (Farr). Specificity was 82.9% (A), 97.7% (B), 96.5% (C), 94.3% (D), 96.5% (CLIFT), and 90.9% (Farr). From these data, we can conclude that the new-generation EIA methods evaluated in this study have higher sensitivity than the CLIFT and Farr assays and, with the exception of Method A, have specificity similar to the CLIFT and slightly higher than the Farr assay. These findings suggest that EIA tests may replace CLIFT as a screening test and the Farr assay as a specific test, for anti-dsDNA antibody detection.

Factors affecting levels of circulating cell-free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing.

PubMed

Kinnings, Sarah L; Geis, Jennifer A; Almasri, Eyad; Wang, Huiquan; Guan, Xiaojun; McCullough, Ron M; Bombard, Allan T; Saldivar, Juan-Sebastian; Oeth, Paul; Deciu, Cosmin

2015-08-01

Sufficient fetal DNA in a maternal plasma sample is required for accurate aneuploidy detection via noninvasive prenatal testing, thus highlighting a need to understand the factors affecting fetal fraction. The MaterniT21™ PLUS test uses massively parallel sequencing to analyze cell-free fetal DNA in maternal plasma and detect chromosomal abnormalities. We assess the impact of a variety of factors, both maternal and fetal, on the fetal fraction across a large number of samples processed by Sequenom Laboratories. The rate of increase in fetal fraction with increasing gestational age varies across the duration of the testing period and is also influenced by fetal aneuploidy status. Maternal weight trends inversely with fetal fraction, and we find no added benefit from analyzing body mass index or blood volume instead of weight. Strong correlations exist between fetal fractions from aliquots taken from the same patient at the same blood draw and also at different blood draws. While a number of factors trend with fetal fraction across the cohort as a whole, they are not the sole determinants of fetal fraction. In this study, the variability for any one patient does not appear large enough to justify postponing testing to a later gestational age. © 2015 John Wiley & Sons, Ltd.

Clinical experience of laboratory follow-up with noninvasive prenatal testing using cell-free DNA and positive microdeletion results in 349 cases.

PubMed

Schwartz, S; Kohan, M; Pasion, R; Papenhausen, P R; Platt, L D

2018-02-01

Screening via noninvasive prenatal testing (NIPT) involving the analysis of cell-free DNA (cfDNA) from plasma has become readily available to screen for chromosomal and DNA aberrations through maternal blood. This report reviews a laboratory's experience with follow-up of positive NIPT screens for microdeletions. Patients that were screened positive by NIPT for a microdeletion involving 1p, 4p, 5p, 15q, or 22q who underwent diagnostic studies by either chorionic villus sampling or amniocentesis were evaluated. The overall positive predictive value for 349 patients was 9.2%. When a microdeletion was confirmed, 39.3% of the cases had additional abnormal microarray findings. Unrelated abnormal microarray findings were detected in 11.8% of the patients in whom the screen positive microdeletion was not confirmed. Stretches of homozygosity in the microdeletion were frequently associated with a false positive cfDNA microdeletion result. Overall, this report reveals that while cfDNA analysis will screen for microdeletions, the positive predictive value is low; in our series it is 9.2%. Therefore, the patient should be counseled accordingly. Confirmatory diagnostic microarray studies are imperative because of the high percentage of false positives and the frequent additional abnormalities not delineated by cfDNA analysis. © 2018 John Wiley & Sons, Ltd.

Construction and Evaluation of Internal Control DNA for PCR Amplification of Chlamydia trachomatis DNA from Urine Samples

PubMed Central

Betsou, Fotini; Beaumont, Katy; Sueur, Jean Marie; Orfila, Jeanne

2003-01-01

An internal control DNA (ICD) with the same primer binding sequences as the target Chlamydia trachomatis DNA was constructed and evaluated in a PCR assay with immunoenzymatic detection. One hundred urine specimens were tested, and 23 were found to contain inhibitors of the PCR, if not subjected to DNA extraction prior to amplification. Coamplification and detection of the ICD appeared to be a useful method for estimating the effects of inhibitors on C. trachomatis DNA amplification. PMID:12624066

DNA in the Criminal Justice System: The DNA Success Story in Perspective.

PubMed

Mapes, Anna A; Kloosterman, Ate D; de Poot, Christianne J

2015-07-01

Current figures on the efficiency of DNA as an investigative tool in criminal investigations only tell part of the story. To get the DNA success story in the right perspective, we examined all forensic reports from serious (N = 116) and high-volume crime cases (N = 2791) over the year 2011 from one police region in the Netherlands. These data show that 38% of analyzed serious crime traces (N = 384) and 17% of analyzed high-volume crime traces (N = 386) did not result in a DNA profile. Turnaround times (from crime scene to DNA report) were 66 days for traces from serious crimes and 44 days for traces from high-volume crimes. Suspects were truly identified through a match with the Offender DNA database of the Netherlands in 3% of the serious crime cases and in 1% of the high-volume crime cases. These data are important for both the forensic laboratory and the professionals in the criminal justice system to further optimize forensic DNA testing as an investigative tool. © 2015 American Academy of Forensic Sciences.

Attempted DNA extraction from a Rancho La Brea Columbian mammoth (Mammuthus columbi): prospects for ancient DNA from asphalt deposits.

PubMed

Gold, David A; Robinson, Jacqueline; Farrell, Aisling B; Harris, John M; Thalmann, Olaf; Jacobs, David K

2014-02-01

Fossil-bearing asphalt deposits are an understudied and potentially significant source of ancient DNA. Previous attempts to extract DNA from skeletons preserved at the Rancho La Brea tar pits in Los Angeles, California, have proven unsuccessful, but it is unclear whether this is due to a lack of endogenous DNA, or if the problem is caused by asphalt-mediated inhibition. In an attempt to test these hypotheses, a recently recovered Columbian mammoth (Mammuthus columbi) skeleton with an unusual pattern of asphalt impregnation was studied. Ultimately, none of the bone samples tested successfully amplified M. columbi DNA. Our work suggests that reagents typically used to remove asphalt from ancient samples also inhibit DNA extraction. Ultimately, we conclude that the probability of recovering ancient DNA from fossils in asphalt deposits is strongly (perhaps fatally) hindered by the organic compounds that permeate the bones and that at the Rancho La Brea tar pits, environmental conditions might not have been ideal for the general preservation of genetic material.

STRBase: a short tandem repeat DNA database for the human identity testing community

PubMed Central

Ruitberg, Christian M.; Reeder, Dennis J.; Butler, John M.

2001-01-01

The National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database (http://www.cstl.nist.gov/biotech/strbase/) since 1997 commonly referred to as STRBase. This database is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. STRBase consolidates and organizes the abundant literature on this subject to facilitate on-going efforts in DNA typing. Observed alleles and annotated sequence for each STR locus are described along with a review of STR analysis technologies. Additionally, commercially available STR multiplex kits are described, published polymerase chain reaction (PCR) primer sequences are reported, and validation studies conducted by a number of forensic laboratories are listed. To supplement the technical information, addresses for scientists and hyperlinks to organizations working in this area are available, along with the comprehensive reference list of over 1300 publications on STRs used for DNA typing purposes. PMID:11125125

Reflex high-risk human papilloma virus DNA test is useful in the triage of women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion.

PubMed

Wu, Howard Her-Juing; Allen, Susan L; Kirkpatrick, Joseph L; Elsheikh, Tarik M

2006-10-01

This study is aimed to investigate the role of reflex high-risk human papilloma virus (HPV) DNA testing as an alternative triage method to colposcopy for women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H) on Papanicolaou (Pap) tests. Reflex HPV DNA testing using Hybrid Capture II method was carried out on 88 women with ASC-H diagnosed by Thin Prep Pap test. Correlation with follow-up biopsies was available on 42 of these patients. The reflex HPV DNA test showed an overall positive rate of 67% and negative rate of 33% in 88 patients with ASC-H. Using age 30 as the cut off point, the positive rate had increased to 83.3% (35/42) in patients 30 yr or younger, while the positive rate for patients older than 30 yr had decreased to 52.2% (24/46). Follow-up colposcopic biopsy results were available in 35 of 59 HPV-positive women, which revealed 15 (43%) high-grade squamous intraepithelial lesions (HSIL), 12 low-grade squamous intraepithelial lesions (LSIL), and 8 negative for dysplasia. In 7 HPV-negative patients, the follow-up biopsies showed no evidence of HSIL or LSIL. Correlation between clinical risk factors and the HPV results demonstrated no significant differences in HPV positivity between the high-risk and low-risk patients. The high sensitivity (100%) and negative predictive rate (100%) in detecting HSIL in our study provide strong evidence that, instead of automatic referral to colposcopy, reflex HPV DNA testing may be used as an alternative triage method for women diagnosed with ASC-H on Thin Prep Pap test, especially for women older than 30 yr of age.

A simple quantitative diagnostic alternative for MGMT DNA-methylation testing on RCL2 fixed paraffin embedded tumors using restriction coupled qPCR.

PubMed

Pulverer, Walter; Hofner, Manuela; Preusser, Matthias; Dirnberger, Elisabeth; Hainfellner, Johannes A; Weinhaeusel, Andreas

2014-01-01

MGMT promoter methylation is associated with favorable prognosis and chemosensitivity in glioblastoma multiforme (GBM), especially in elderly patients. We aimed to develop a simple methylation-sensitive restriction enzyme (MSRE)-based quantitative PCR (qPCR) assay, allowing the quantification of MGMT promoter methylation. DNA was extracted from non-neoplastic brain (n = 24) and GBM samples (n = 20) upon 3 different sample conservation conditions (-80 °C, formalin-fixed and paraffin-embedded (FFPE); RCL2-fixed). We evaluated the suitability of each fixation method with respect to the MSRE-coupled qPCR methylation analyses. Methylation data were validated by MALDITOF. qPCR was used for evaluation of alternative tissue conservation procedures. DNA from FFPE tissue failed reliable testing; DNA from both RCL2-fixed and fresh frozen tissues performed equally well and was further used for validation of the quantitative MGMT methylation assay (limit of detection (LOD): 19.58 pg), using individual's undigested sample DNA for calibration. MGMT methylation analysis in non-neoplastic brain identified a background methylation of 0.10 ± 11% which we used for defining a cut-off of 0.32% for patient stratification. Of GBM patients 9 were MGMT methylationpositive (range: 0.56 - 91.95%), and 11 tested negative. MALDI-TOF measurements resulted in a concordant classification of 94% of GBM samples in comparison to qPCR. The presented methodology allows quantitative MGMT promoter methylation analyses. An amount of 200 ng DNA is sufficient for triplicate analyses including control reactions and individual calibration curves, thus excluding any DNA qualityderived bias. The combination of RCL2-fixation and quantitative methylation analyses improves pathological routine examination when histological and molecular analyses on limited amounts of tumor samples are necessary for patient stratification.

Differential impact of ionic and coordinate covalent chromium (Cr)-DNA binding on DNA replication.

PubMed

Fornsaglio, Jamie L; O'Brien, Travis J; Patierno, Steven R

2005-11-01

The reactive species produced by the reduction of Cr(VI), particularly Cr(III), can form both ionic and coordinate covalent complexes with DNA. These Cr(III)-DNA interactions consist of Cr-DNA monoadducts, Cr-DNA ternary adducts, and Cr-DNA interstrand cross-links (Cr-ICLs), the latter of which are DNA polymerase arresting lesions (PALs). We sought to determine the impact of Cr-DNA interactions on the formation of replication blocking lesions in S. cerevisiae using a PCR-based method. We found that target sequence (TS) amplification using DNA isolated from Cr(VI)-treated yeast actually increased as a function of Cr(VI) concentration. Moreover, the enhanced TS amplification was reproduced in vitro using Cr(III)-treated DNA. In contrast, PCR amplification of TS from DNA isolated from yeast exposed to equitoxic doses of the inorganic DNA cross-linking agent cisplatin (CDDP), was decreased in a concentration-dependent manner. This paradox suggested that a specific Cr-DNA interaction, such as an ionic Cr-DNA complex, was responsible for the enhanced TS amplification, thereby masking the replication-blocking effect of certain ternary Cr-DNA adducts (i.e. interstrand cross-links). To test this possibility, we removed ionically associated Cr from the DNA using salt extraction prior to PCR analysis. This procedure obviated the increased amplification and revealed a dose-dependent decrease in TS amplification and an increase in Cr-PALs. These data from DNA analyzed ex vivo after treatment of intact cells indicate that ionic interactions of Cr with DNA result in increased DNA amplification whereas coordinate-covalent Cr-DNA complexes lead to formation of Cr-PALs. Thus, these results suggest that treatment of living cells with Cr(VI) leads to two modes of Cr-binding, which may have conflicting effects on DNA replication.

Development and validation of a multiplex reaction analyzing eight miniSTRs of the X chromosome for identity and kinship testing with degraded DNA.

PubMed

Castañeda, María; Odriozola, Adrián; Gómez, Javier; Zarrabeitia, María T

2013-07-01

We report the development of an effective system for analyzing X chromosome-linked mini short tandem repeat loci with reduced-size amplicons (less than 220 bp), useful for analyzing highly degraded DNA samples. To generate smaller amplicons, we redesigned primers for eight X-linked microsatellites (DXS7132, DXS10079, DXS10074, DXS10075, DXS6801, DXS6809, DXS6789, and DXS6799) and established efficient conditions for a multiplex PCR system (miniX). The validation tests confirmed that it has good sensitivity, requiring as little as 20 pg of DNA, and performs well with DNA from paraffin-embedded tissues, thus showing potential for improved analysis and identification of highly degraded and/or very limited DNA samples. Consequently, this system may help to solve complex forensic cases, particularly when autosomal markers convey insufficient information.

DNA evidence: current perspective and future challenges in India.

PubMed

Verma, Sunil K; Goswami, Gajendra K

2014-08-01

Since the discovery of DNA fingerprinting technology in 1985 it has been used extensively as evidence in the court of law world-wide to establish the individual identity both in civil and criminal matters. In India, the first case of parentage dispute solved by the use of DNA fingerprinting technology was in 1989. Since then till date, the DNA technology has been used not only to resolve the cases of paternity and maternity disputes, but also for the establishment of individual identity in various criminal cases and for wildlife forensic identification. Since last half a decade, India is exercising to enact legislation on the use of DNA in the judicial realm and the draft 'Human DNA Bill-2012' is pending in the parliament. Largely, the promoters of forensic DNA testing have anticipated that DNA tests are nearly infallible and DNA technology could be the greatest single advance step in search for truth, conviction of the perpetrator, and acquittal of the innocent. The current article provides a comprehensive review on the status of DNA testing in India and elucidates the consequences of the admissibility of DNA as 'evidence' in the judicial dominion. In this backdrop of civil and criminal laws and changing ethical and societal attitudes, it is concluded that the DNA legislation in India and world-wide needs to be designed with utmost care. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Development of biometric DNA ink for authentication security.

PubMed

Hashiyada, Masaki

2004-10-01

Among the various types of biometric personal identification systems, DNA provides the most reliable personal identification. It is intrinsically digital and unchangeable while the person is alive, and even after his/her death. Increasing the number of DNA loci examined can enhance the power of discrimination. This report describes the development of DNA ink, which contains synthetic DNA mixed with printing inks. Single-stranded DNA fragments encoding a personalized set of short tandem repeats (STR) were synthesized. The sequence was defined as follows. First, a decimal DNA personal identification (DNA-ID) was established based on the number of STRs in the locus. Next, this DNA-ID was encrypted using a binary, 160-bit algorithm, using a hashing function to protect privacy. Since this function is irreversible, no one can recover the original information from the encrypted code. Finally, the bit series generated above is transformed into base sequences, and double-stranded DNA fragments are amplified by the polymerase chain reaction (PCR) to protect against physical attacks. Synthesized DNA was detected successfully after samples printed in DNA ink were subjected to several resistance tests used to assess the stability of printing inks. Endurance test results showed that this DNA ink would be suitable for practical use as a printing ink and was resistant to 40 hours of ultraviolet exposure, performance commensurate with that of photogravure ink. Copyright 2004 Tohoku University Medical Press

DNA Probe for Lactobacillus delbrueckii

PubMed Central

Delley, Michèle; Mollet, Beat; Hottinger, Herbert

1990-01-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-labeled DNA probe. Images PMID:16348233

Mitochondrial DNA alteration in obstructive sleep apnea.

PubMed

Lacedonia, Donato; Carpagnano, Giovanna E; Crisetti, Elisabetta; Cotugno, Grazia; Palladino, Grazia P; Patricelli, Giulia; Sabato, Roberto; Foschino Barbaro, Maria P

2015-04-07

Obstructive Sleep Apnea (OSAS) is a disease associated with the increase of cardiovascular risk and it is characterized by repeated episodes of Intermittent Hypoxia (IH) which inducing oxidative stress and systemic inflammation. Mitochondria are cell organelles involved in the respiratory that have their own DNA (MtDNA). The aim of this study was to investigate if the increase of oxidative stress in OSAS patients can induce also MtDNA alterations. 46 OSAS patients (age 59.27 ± 11.38; BMI 30.84 ± 3.64; AHI 36.63 ± 24.18) were compared with 36 control subjects (age 54.42 ± 6.63; BMI 29.06 ± 4.7; AHI 3.8 ± 1.10). In blood cells Content of MtDNA and nuclear DNA (nDNA) was measured in OSAS patients by Real Time PCR. The ratio between MtDNA/nDNA was then calculated. Presence of oxidative stress was evaluated by levels of Reactive Oxygen Metabolites (ROMs), measured by diacron reactive oxygen metabolite test (d-ROM test). MtDNA/nDNA was higher in patients with OSAS than in the control group (150.94 ± 49.14 vs 128.96 ± 45.8; p = 0.04), the levels of ROMs were also higher in OSAS subjects (329.71 ± 70.17 vs 226 ± 36.76; p = 0.04) and they were positively correlated with MtDNA/nDNA (R = 0.5, p < 0.01). In OSAS patients there is a Mitochondrial DNA damage induced by the increase of oxidative stress. Intermittent hypoxia seems to be the main mechanism which leads to this process.

Simulated radioactive decontamination of biological samples using a portable DNA extraction instrument for rapid DNA profiling.

PubMed

Frégeau, Chantal J; Dalpé, Claude

2016-02-01

A portable DNA extraction instrument was evaluated for its ability to decontaminate blood and saliva samples deposited on different surfaces (metal, plastic and glass) contaminated with stable isotopes of cobalt (Co), cesium (Cs), and strontium (Sr) as equivalents to their radiogenic (60)Co, (137)Cs, and (90)Sr isotopes, respectively, that could be released during a nuclear weapon accident or a radiological dispersal device (RDD) detonation. Despite the very high contamination levels tested in this study, successful removal of greater than 99.996% of the Co, Cs, Sr contaminants was achieved based on inductively coupled plasma-mass spectrometry (ICP-MS) and neutron activation analyses carried out on all liquids (including DNA eluates) and solid waste produced during automated DNA extraction. The remaining amounts of Co, Cs and Sr in the DNA eluates, when converted to dose rates (corresponding to (60)Co, (137)Cs and (90)Sr), were determined to be below the recommended dose limits for the general public in most of the scenarios tested. The presence of Co, Cs and Sr contaminants in the cell lysates had no adverse impact on the binding of DNA onto the magnetic DNA IQ™ beads. DNA yields were similar to uncontaminated controls. The remaining Co, Cs and Sr in the DNA eluates did not interfere with real-time PCR DNA quantification. In addition, the quality of the AmpFlSTR(®) Identifiler(®) profiles derived in 26min using an accelerated protocol was very good and comparable to controls. This study emphasizes the use of an accelerated process involving a portable DNA extraction instrument to significantly reduce radioactive dose rates to allow contaminated samples to be processed safely in a forensic mobile laboratory to expedite the identification of individuals potentially involved in the dispersal of nuclear or other radioactive materials. Crown Copyright © 2016. Published by Elsevier Ireland Ltd. All rights reserved.

DNA recovery from soils of diverse composition.

PubMed

Zhou, J; Bruns, M A; Tiedje, J M

1996-02-01

A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.

Effectiveness of VIA, Pap, and HPV DNA Testing in a Cervical Cancer Screening Program in a Peri-Urban Community in Andhra Pradesh, India

PubMed Central

Gravitt, Patti E.; Paul, Proma; Katki, Hormuzd A.; Vendantham, Haripriya; Ramakrishna, Gayatri; Sudula, Mrudula; Kalpana, Basany; Ronnett, Brigitte M.; Vijayaraghavan, K.; Shah, Keerti V.

2010-01-01

Background While many studies have compared the efficacy of Pap cytology, visual inspection with acetic acid (VIA) and human papillomavirus (HPV) DNA assays for the detection cervical intraepithelial neoplasia and cancer, few have evaluated the program effectiveness. Methods and Findings A population-based sample of 5603 women from Medchal Mandal in Andhra Pradesh, India were invited to participate in a study comparing Pap cytology, VIA, and HPV DNA screening for the detection of CIN3+. Participation in primary screening and all subsequent follow-up visits was rigorously tracked. A 20% random sample of all women screened, in addition to all women with a positive screening test result underwent colposcopy with directed biopsy for final diagnosis. Sensitivity, specificity, positive and negative predictive values were adjusted for verification bias. HPV testing had a higher sensitivity (100%) and specificity (90.6%) compared to Pap cytology (sensitivity  =  78.2%; specificity = 86.0%) and VIA (sensitivity = 31.6%; specificity = 87.5%). Since 58% of the sample refused involvement and another 28% refused colposcopy or biopsy, we estimated that potentially 87.6% of the total underlying cases of CIN3 and cancer may have been missed due to program failures. Conclusions We conclude that despite our use of available resources, infrastructure, and guidelines for cervical cancer screening implementation in resource limited areas, community participation and non-compliance remain the major obstacles to successful reduction in cervical cancer mortality in this Indian population. HPV DNA testing was both more sensitive and specific than Pap cytology and VIA. The use of a less invasive and more user-friendly primary screening strategy (such as self-collected swabs for HPV DNA testing) may be required to achieve the coverage necessary for effective reduction in cervical cancer mortality. PMID:21060889

Short Tandem Repeat DNA Internet Database

National Institute of Standards and Technology Data Gateway

SRD 130 Short Tandem Repeat DNA Internet Database (Web, free access)   Short Tandem Repeat DNA Internet Database is intended to benefit research and application of short tandem repeat DNA markers for human identity testing. Facts and sequence information on each STR system, population data, commonly used multiplex STR systems, PCR primers and conditions, and a review of various technologies for analysis of STR alleles have been included.

A cargo-sorting DNA robot.

PubMed

Thubagere, Anupama J; Li, Wei; Johnson, Robert F; Chen, Zibo; Doroudi, Shayan; Lee, Yae Lim; Izatt, Gregory; Wittman, Sarah; Srinivas, Niranjan; Woods, Damien; Winfree, Erik; Qian, Lulu

2017-09-15

Two critical challenges in the design and synthesis of molecular robots are modularity and algorithm simplicity. We demonstrate three modular building blocks for a DNA robot that performs cargo sorting at the molecular level. A simple algorithm encoding recognition between cargos and their destinations allows for a simple robot design: a single-stranded DNA with one leg and two foot domains for walking, and one arm and one hand domain for picking up and dropping off cargos. The robot explores a two-dimensional testing ground on the surface of DNA origami, picks up multiple cargos of two types that are initially at unordered locations, and delivers them to specified destinations until all molecules are sorted into two distinct piles. The robot is designed to perform a random walk without any energy supply. Exploiting this feature, a single robot can repeatedly sort multiple cargos. Localization on DNA origami allows for distinct cargo-sorting tasks to take place simultaneously in one test tube or for multiple robots to collectively perform the same task. Copyright © 2017, American Association for the Advancement of Science.

Environmental DNA (eDNA) Detection Probability Is Influenced by Seasonal Activity of Organisms.

PubMed

de Souza, Lesley S; Godwin, James C; Renshaw, Mark A; Larson, Eric

2016-01-01

Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with replicated eDNA water samples, which were analyzed in the laboratory using species-specific quantitative PCR (qPCR) assays. We then used occupancy estimation with detection probability modeling to evaluate both the effects of landscape attributes on organism presence and season of sampling on detection probability of eDNA. Importantly, we found that season strongly affected eDNA detection probability for both species, with N. alabamensis having higher eDNA detection probabilities during the cool season and S. depressus have higher eDNA detection probabilities during the warm season. These results illustrate the influence of organismal behavior or activity on eDNA detection in the environment and identify an important role for basic natural history in designing eDNA monitoring programs.

Comparative analysis between saliva and buccal swabs as source of DNA: lesson from HLA-B*57:01 testing.

PubMed

Cascella, Raffaella; Stocchi, Laura; Strafella, Claudia; Mezzaroma, Ivano; Mannazzu, Marco; Vullo, Vincenzo; Montella, Francesco; Parruti, Giustino; Borgiani, Paola; Sangiuolo, Federica; Novelli, Giuseppe; Pirazzoli, Antonella; Zampatti, Stefania; Giardina, Emiliano

2015-01-01

Our work aimed to designate the optimal DNA source for pharmacogenetic assays, such as the screening for HLA-B*57:01 allele. A saliva and four buccal swab samples were taken from 104 patients. All the samples were stored at different time and temperature conditions and then genotyped for the HLA-B*57:01 allele by SSP-PCR and classical/capillary electrophoresis. The genotyping analysis reported different performance rates depending on the storage conditions of the samples. Given our results, the buccal swab demonstrated to be more resistant and stable in time with respect to the saliva. Our investigation designates the buccal swab as the optimal DNA source for pharmacogenetic assays in terms of resistance, low infectivity, low-invasiveness and easy sampling, and safe transport in centralized medical centers providing specialized pharmacogenetic tests.

Allostery through protein-induced DNA bubbles

DOE PAGES

Traverso, Joseph J.; Manoranjan, Valipuram S.; Bishop, A. R.; ...

2015-03-12

Allostery through DNA is increasingly recognized as an important modulator of DNA functions. Here, we show that the coalescence of protein-induced DNA bubbles can mediate allosteric interactions that drive protein aggregation. We propose that such allostery may regulate DNA's flexibility and the assembly of the transcription machinery. Mitochondrial transcription factor A (TFAM), a dual-function protein involved in mitochondrial DNA (mtDNA) packaging and transcription initiation, is an ideal candidate to test such a hypothesis owing to its ability to locally unwind the double helix. Numerical simulations demonstrate that the coalescence of TFAM-induced bubbles can explain experimentally observed TFAM oligomerization. The resultingmore » melted DNA segment, approximately 10 base pairs long, around the joints of the oligomers act as flexible hinges, which explains the efficiency of TFAM in compacting DNA. Since mitochondrial polymerase (mitoRNAP) is involved in melting the transcription bubble, TFAM may use the same allosteric interaction to both recruit mitoRNAP and initiate transcription.« less

Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

Testing a structural model for viral DNA packaging motor function by optical tweezers measurements, site directed mutagenesis, and molecular dynamics calculations

NASA Astrophysics Data System (ADS)

Keller, Nicholas A.; Migliori, Amy D.; Arya, Gaurav; Rao, Venigalla B.; Smith, Douglas E.

2013-09-01

Many double-stranded DNA viruses employ a molecular motor to package DNA into preformed capsid shells. Based on structures of phage T4 motor proteins determined by X-ray crystallography and cryo-electron microscopy, Rao, Rossmann and coworkers recently proposed a structural model for motor function. They proposed that DNA is ratcheted by a large conformational change driven by electrostatic interactions between charged residues at an interface between two globular domains of the motor protein. We have conducted experiments to test this model by studying the effect on packaging under applied load of site-directed changes altering these residues. We observe significant impairment of packaging activity including reductions in packaging rate, percent time packaging, and time active under high load. We show that these measured impairments correlate well with alterations in free energies associated with the conformational change predicted by molecular dynamics simulations.

Fluorescent carbon nanoparticle-based lateral flow biosensor for ultrasensitive detection of DNA.

PubMed

Takalkar, Sunitha; Baryeh, Kwaku; Liu, Guodong

2017-12-15

We report a fluorescent carbon nanoparticle (FCN)-based lateral flow biosensor for ultrasensitive detection of DNA. Fluorescent carbon nanoparticle with a diameter of around 15nm was used as a tag to label a detection DNA probe, which was complementary with the part of target DNA. A capture DNA probe was immobilized on the test zone of the lateral flow biosensor. Sandwich-type hybridization reactions among the FCN-labeled DNA probe, target DNA and capture DNA probe were performed on the lateral flow biosensor. In the presence of target DNA, FCNs were captured on the test zone of the biosensor and the fluorescent intensity of the captured FCNs was measured with a portable fluorescent reader. After systematic optimizations of experimental parameters (the components of running buffers, the concentration of detection DNA probe used in the preparation of FCN-DNA conjugates, the amount of FCN-DNA dispensed on the conjugate pad and the dispensing cycles of the capture DNA probes on the test-zone), the biosensor could detect a minimum concentration of 0.4 fM DNA. This study provides a rapid and low-cost approach for DNA detection with high sensitivity, showing great promise for clinical application and biomedical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of the diagnostic accuracy of a rapid immunochromatographic test and the rapid plasma reagin test for antenatal syphilis screening in Mozambique.

PubMed Central

Montoya, Pablo J.; Lukehart, Sheila A.; Brentlinger, Paula E.; Blanco, Ana J.; Floriano, Florencia; Sairosse, Josefa; Gloyd, Stephen

2006-01-01

OBJECTIVE: Programmes to control syphilis in developing countries are hampered by a lack of laboratory services, delayed diagnosis, and doubts about current screening methods. We aimed to compare the diagnostic accuracy of an immunochromatographic strip (ICS) test and the rapid plasma reagin (RPR) test with the combined gold standard (RPR, Treponema pallidum haemagglutination assay and direct immunofluorescence stain done at a reference laboratory) for the detection of syphilis in pregnancy. METHODS: We included test results from 4789 women attending their first antenatal visit at one of six health facilities in Sofala Province, central Mozambique. We compared diagnostic accuracy (sensitivity, specificity, and positive and negative predictive values) of ICS and RPR done at the health facilities and ICS performed at the reference laboratory. We also made subgroup comparisons by human immunodeficiency virus (HIV) and malaria status. FINDINGS: For active syphilis, the sensitivity of the ICS was 95.3% at the reference laboratory, and 84.1% at the health facility. The sensitivity of the RPR at the health facility was 70.7%. Specificity and positive and negative predictive values showed a similar pattern. The ICS outperformed RPR in all comparisons (P<0.001). CONCLUSION: The diagnostic accuracy of the ICS compared favourably with that of the gold standard. The use of the ICS in Mozambique and similar settings may improve the diagnosis of syphilis in health facilities, both with and without laboratories. PMID:16501726

Flavonoids and DNA Repair in Prostate Cancer

DTIC Science & Technology

2005-12-01

1-0114 TITLE: Flavonoids and DNA Repair in Prostate Cancer...SUBTITLE Flavonoids and DNA Repair in Prostate Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-04-1-0114 5c. PROGRAM ELEMENT NUMBER...test the hypothesis that natural products such as flavonoids are able to stimulate the repair of oxidative DNA damage. For this purpose LNCaP

Evaluation of the efficacy of the four tests (p16 immunochemistry, PCR, DNA and RNA In situ Hybridization) to evaluate a Human Papillomavirus infection in head and neck cancers: a cohort of 348 French squamous cell carcinomas.

PubMed

Augustin, Jérémy; Outh-Gauer, Sophie; Mandavit, Marion; Gasne, Cassandre; Grard, Ophélie; Denize, Thomas; Nervo, Marine; Mirghani, Haïtham; Laccourreye, Ollivier; Bonfils, Pierre; Bruneval, Patrick; Veyer, David; Péré, Hélène; Tartour, Eric; Badoual, Cécile

2018-04-20

It is now established that HPV plays a role in the development of a subset of head and neck squamous cell carcinomas (HNSCCs), notably oropharyngeal squamous cell carcinomas (SCCs). However, it is not clear which test one should use to detect HPV in oropharyngeal (OP) and non-OP SCCs. In this study, using 348 HNSCCs (126 OP SCCs and 222 non-OP SCCs), we evaluated diagnostic performances of different HPV tests in OP and non-OP SCCs: PCR, p16 immunostaining, in situ hybridization targeting DNA (DNA-CISH) and RNA (RNA-CISH), combined p16 + DNA-CISH, and combined p16 + RNA-CISH. HPV DNA (PCR) was detected in 26% of all tumors (44% of OP SCCs and 17% of non-OP SCCs). For OP SCCs, RNA-CISH was the most sensitive standalone test (88%), but p16 + RNA-CISH was even more sensitive (95%). Specificities were the same for RNA-CISH and DNA-CISH (97%) but it was better for p16 + RNA-CISH (100%). For non-OP SCCs, all tests had sensitivities below 50%, and RNA-CISH, DNA-CISH and p16 + DNA-CISH had respectively 100%, 97% and 99% specificities. As a standalone test, RNA-CISH is the most performant assay to detect HPV in OP SCCs, and combined p16 + RNA-CISH test slightly improves its performances. However, RNA-CISH has the advantage of being one single test. Like p16 and DNA-CISH, RNA-CISH performances are poor in non-OP SCCs to detect HPV, and combining tests does not improve performances. Copyright © 2018. Published by Elsevier Inc.

FBI's DNA analysis program

NASA Astrophysics Data System (ADS)

Brown, John R.

1994-03-01

Forensic DNA profiling technology is a significant law enforcement tool due to its superior discriminating power. Applying the principles of population genetics to the DNA profile obtained in violent crime investigations results in low frequency of occurrence estimates for the DNA profile. These estimates often range from a frequency of occurrence of 1 in 50 unrelated individuals to 1 in a million unrelated individuals or even smaller. It is this power to discriminate among individuals in the population that has propelled forensic DNA technology to the forefront of forensic testing in violent crime cases. Not only is the technology extremely powerful in including or excluding a criminal suspect as the perpetrator, but it also gives rise to the potential of identifying criminal suspects in cases where the investigators of unknown suspect cases have exhausted all other available leads.

Platinum nanoparticles induce damage to DNA and inhibit DNA replication.

PubMed

Nejdl, Lukas; Kudr, Jiri; Moulick, Amitava; Hegerova, Dagmar; Ruttkay-Nedecky, Branislav; Gumulec, Jaromir; Cihalova, Kristyna; Smerkova, Kristyna; Dostalova, Simona; Krizkova, Sona; Novotna, Marie; Kopel, Pavel; Adam, Vojtech

2017-01-01

Sparsely tested group of platinum nanoparticles (PtNPs) may have a comparable effect as complex platinum compounds. The aim of this study was to observe the effect of PtNPs in in vitro amplification of DNA fragment of phage λ, on the bacterial cultures (Staphylococcus aureus), human foreskin fibroblasts and erythrocytes. In vitro synthesized PtNPs were characterized by dynamic light scattering (PtNPs size range 4.8-11.7 nm), zeta potential measurements (-15 mV at pH 7.4), X-ray fluorescence, UV/vis spectrophotometry and atomic absorption spectrometry. The PtNPs inhibited the DNA replication and affected the secondary structure of DNA at higher concentrations, which was confirmed by polymerase chain reaction, DNA sequencing and DNA denaturation experiments. Further, cisplatin (CisPt), as traditional chemotherapy agent, was used in all parallel experiments. Moreover, the encapsulation of PtNPs in liposomes (LipoPtNPs) caused an approximately 2.4x higher of DNA damage in comparison with CisPt, LipoCisPt and PtNPs. The encapsulation of PtNPs in liposomes also increased their antibacterial, cytostatic and cytotoxic effect, which was determined by the method of growth curves on S. aureus and HFF cells. In addition, both the bare and encapsulated PtNPs caused lower oxidative stress (determined by GSH/GSSG ratio) in the human erythrocytes compared to the bare and encapsulated CisPt. CisPt was used in all parallel experiments as traditional chemotherapy agent.

Platinum nanoparticles induce damage to DNA and inhibit DNA replication

PubMed Central

Nejdl, Lukas; Kudr, Jiri; Moulick, Amitava; Hegerova, Dagmar; Ruttkay-Nedecky, Branislav; Gumulec, Jaromir; Cihalova, Kristyna; Smerkova, Kristyna; Dostalova, Simona; Krizkova, Sona; Novotna, Marie; Kopel, Pavel

2017-01-01

Sparsely tested group of platinum nanoparticles (PtNPs) may have a comparable effect as complex platinum compounds. The aim of this study was to observe the effect of PtNPs in in vitro amplification of DNA fragment of phage λ, on the bacterial cultures (Staphylococcus aureus), human foreskin fibroblasts and erythrocytes. In vitro synthesized PtNPs were characterized by dynamic light scattering (PtNPs size range 4.8–11.7 nm), zeta potential measurements (-15 mV at pH 7.4), X-ray fluorescence, UV/vis spectrophotometry and atomic absorption spectrometry. The PtNPs inhibited the DNA replication and affected the secondary structure of DNA at higher concentrations, which was confirmed by polymerase chain reaction, DNA sequencing and DNA denaturation experiments. Further, cisplatin (CisPt), as traditional chemotherapy agent, was used in all parallel experiments. Moreover, the encapsulation of PtNPs in liposomes (LipoPtNPs) caused an approximately 2.4x higher of DNA damage in comparison with CisPt, LipoCisPt and PtNPs. The encapsulation of PtNPs in liposomes also increased their antibacterial, cytostatic and cytotoxic effect, which was determined by the method of growth curves on S. aureus and HFF cells. In addition, both the bare and encapsulated PtNPs caused lower oxidative stress (determined by GSH/GSSG ratio) in the human erythrocytes compared to the bare and encapsulated CisPt. CisPt was used in all parallel experiments as traditional chemotherapy agent. PMID:28704436

Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

PubMed Central

Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Iêda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

2011-01-01

Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults. PMID:21731912

Application and comparison of large-scale solution-based DNA capture-enrichment methods on ancient DNA

PubMed Central

Ávila-Arcos, María C.; Cappellini, Enrico; Romero-Navarro, J. Alberto; Wales, Nathan; Moreno-Mayar, J. Víctor; Rasmussen, Morten; Fordyce, Sarah L.; Montiel, Rafael; Vielle-Calzada, Jean-Philippe; Willerslev, Eske; Gilbert, M. Thomas P.

2011-01-01

The development of second-generation sequencing technologies has greatly benefitted the field of ancient DNA (aDNA). Its application can be further exploited by the use of targeted capture-enrichment methods to overcome restrictions posed by low endogenous and contaminating DNA in ancient samples. We tested the performance of Agilent's SureSelect and Mycroarray's MySelect in-solution capture systems on Illumina sequencing libraries built from ancient maize to identify key factors influencing aDNA capture experiments. High levels of clonality as well as the presence of multiple-copy sequences in the capture targets led to biases in the data regardless of the capture method. Neither method consistently outperformed the other in terms of average target enrichment, and no obvious difference was observed either when two tiling designs were compared. In addition to demonstrating the plausibility of capturing aDNA from ancient plant material, our results also enable us to provide useful recommendations for those planning targeted-sequencing on aDNA. PMID:22355593

Environmental DNA (eDNA) Detection Probability Is Influenced by Seasonal Activity of Organisms

PubMed Central

de Souza, Lesley S.; Godwin, James C.; Renshaw, Mark A.; Larson, Eric

2016-01-01

Environmental DNA (eDNA) holds great promise for conservation applications like the monitoring of invasive or imperiled species, yet this emerging technique requires ongoing testing in order to determine the contexts over which it is effective. For example, little research to date has evaluated how seasonality of organism behavior or activity may influence detection probability of eDNA. We applied eDNA to survey for two highly imperiled species endemic to the upper Black Warrior River basin in Alabama, US: the Black Warrior Waterdog (Necturus alabamensis) and the Flattened Musk Turtle (Sternotherus depressus). Importantly, these species have contrasting patterns of seasonal activity, with N. alabamensis more active in the cool season (October-April) and S. depressus more active in the warm season (May-September). We surveyed sites historically occupied by these species across cool and warm seasons over two years with replicated eDNA water samples, which were analyzed in the laboratory using species-specific quantitative PCR (qPCR) assays. We then used occupancy estimation with detection probability modeling to evaluate both the effects of landscape attributes on organism presence and season of sampling on detection probability of eDNA. Importantly, we found that season strongly affected eDNA detection probability for both species, with N. alabamensis having higher eDNA detection probabilities during the cool season and S. depressus have higher eDNA detection probabilities during the warm season. These results illustrate the influence of organismal behavior or activity on eDNA detection in the environment and identify an important role for basic natural history in designing eDNA monitoring programs. PMID:27776150

Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparison with Other Methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Liyou; Yi, T. Y.; Van Nostrand, Joy

Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site [Hanford Reach of the Columbia River (HRCR), 11 strains], Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the averagemore » nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.« less

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes.

PubMed

Klymus, Katy E; Marshall, Nathaniel T; Stepien, Carol A

2017-01-01

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

DNA Origami Reorganizes upon Interaction with Graphite: Implications for High-Resolution DNA Directed Protein Patterning

PubMed Central

Rahman, Masudur; Neff, David; Green, Nathaniel; Norton, Michael L.

2016-01-01

Although there is a long history of the study of the interaction of DNA with carbon surfaces, limited information exists regarding the interaction of complex DNA-based nanostructures with the important material graphite, which is closely related to graphene. In view of the capacity of DNA to direct the assembly of proteins and optical and electronic nanoparticles, the potential for combining DNA-based materials with graphite, which is an ultra-flat, conductive carbon substrate, requires evaluation. A series of imaging studies utilizing Atomic Force Microscopy has been applied in order to provide a unified picture of this important interaction of structured DNA and graphite. For the test structure examined, we observe a rapid destabilization of the complex DNA origami structure, consistent with a strong interaction of single-stranded DNA with the carbon surface. This destabilizing interaction can be obscured by an intentional or unintentional primary intervening layer of single-stranded DNA. Because the interaction of origami with graphite is not completely dissociative, and because the frustrated, expanded structure is relatively stable over time in solution, it is demonstrated that organized structures of pairs of the model protein streptavidin can be produced on carbon surfaces using DNA origami as the directing material. PMID:28335324

DNA cards: determinants of DNA yield and quality in collecting genetic samples for pharmacogenetic studies.

PubMed

Mas, Sergi; Crescenti, Anna; Gassó, Patricia; Vidal-Taboada, Jose M; Lafuente, Amalia

2007-08-01

As pharmacogenetic studies frequently require establishment of DNA banks containing large cohorts with multi-centric designs, inexpensive methods for collecting and storing high-quality DNA are needed. The aims of this study were two-fold: to compare the amount and quality of DNA obtained from two different DNA cards (IsoCode Cards or FTA Classic Cards, Whatman plc, Brentford, Middlesex, UK); and to evaluate the effects of time and storage temperature, as well as the influence of anticoagulant ethylenediaminetetraacetic acid on the DNA elution procedure. The samples were genotyped by several methods typically used in pharmacogenetic studies: multiplex PCR, PCR-restriction fragment length polymorphism, single nucleotide primer extension, and allelic discrimination assay. In addition, they were amplified by whole genome amplification to increase genomic DNA mass. Time, storage temperature and ethylenediaminetetraacetic acid had no significant effects on either DNA card. This study reveals the importance of drying blood spots prior to isolation to avoid haemoglobin interference. Moreover, our results demonstrate that re-isolation protocols could be applied to increase the amount of DNA recovered. The samples analysed were accurately genotyped with all the methods examined herein. In conclusion, our study shows that both DNA cards, IsoCode Cards and FTA Classic Cards, facilitate genetic and pharmacogenetic testing for routine clinical practice.

DNA Profiles from Fingerprint Lifts-Enhancing the Evidential Value of Fingermarks Through Successful DNA Typing.

PubMed

Subhani, Zuhaib; Daniel, Barbara; Frascione, Nunzianda

2018-05-25

This study evaluated the compatibility of the most common enhancement methods and lifting techniques with DNA profiling. Emphasis is placed on modern lifting techniques (i.e., gelatin lifters and Isomark™) and historical fingerprint lifts for which limited research has been previously conducted. A total of 180 fingerprints were deposited on a glass surface, enhanced, lifted, and processed for DNA typing. DNA could be extracted and profiled for all the powders and lifts tested and from both groomed fingerprints and natural prints with no significant difference in the percentage of profile recovered. DNA profiles could also be obtained from historical fingerprint lifts (79.2% of 72 lifts) with one or more alleles detected. These results demonstrate the compatibility between different powder/lift combinations and DNA profiling therefore augmenting the evidential value of fingerprints in forensic casework. © 2018 American Academy of Forensic Sciences.

"It's our DNA, we deserve the right to test!" A content analysis of a petition for the right to access direct-to-consumer genetic testing.

PubMed

Su, Yeyang; Borry, Pascal; Otte, Ina C; Howard, Heidi C

2013-09-01

Various companies are currently advertising or selling genetic tests over the internet using a model of provision referred to as 'direct-to-consumer' (DTC). This commercial offer of DTC genetic testing (GT) has fueled a number of scientific, ethical and policy debates. To date there have been few studies published regarding the users' perspective. This study aimed to obtain information regarding the issues raised by individuals who signed a petition in support of DTC GT and the 'unrestricted' access to their genetic information. We conducted qualitative content analysis of comments written by individuals who signed a public online petition initiated by DIYgenomics (CA, USA) to support "personal access to genetic information". Of the 523 individuals who signed the petition sponsored by DIYgenomics, 247 individuals also wrote individual comments. A content analysis of these comments reveals that petitioners raised six main issues in support of unrestricted access to DTC GT: that their ownership of their DNA should allow them to have unrestricted access to their genomic information; that they should have the right to their genomic information; that the government has no place in (further) regulating DTC GT; that healthcare professionals should not be placed as intermediaries when purchasing DTC GT services; that many petioners who had already obtained DTC GT had positive experiences with this model of provision; and that genealogy or ancestry DNA testing is one of the main activities petitioners wish to have 'unrestricted' or 'direct' access. These results give insight into why individuals may support unrestricted access to their genomic information and confirm some of the motivations of users for purchasing DTC GT. Our analysis also brings to the forefront themes that have been raised less often in empirical studies involving motivations to purchase DTC GT services; these include the strongly held beliefs of some petitioners that, since they own their DNA, they

Comparing Facial 3D Analysis With DNA Testing to Determine Zygosities of Twins.

PubMed

Vuollo, Ville; Sidlauskas, Mantas; Sidlauskas, Antanas; Harila, Virpi; Salomskiene, Loreta; Zhurov, Alexei; Holmström, Lasse; Pirttiniemi, Pertti; Heikkinen, Tuomo

2015-06-01

The aim of this study was to compare facial 3D analysis to DNA testing in twin zygosity determinations. Facial 3D images of 106 pairs of young adult Lithuanian twins were taken with a stereophotogrammetric device (3dMD, Atlanta, Georgia) and zygosity was determined according to similarity of facial form. Statistical pattern recognition methodology was used for classification. The results showed that in 75% to 90% of the cases, zygosity determinations were similar to DNA-based results. There were 81 different classification scenarios, including 3 groups, 3 features, 3 different scaling methods, and 3 threshold levels. It appeared that coincidence with 0.5 mm tolerance is the most suitable feature for classification. Also, leaving out scaling improves results in most cases. Scaling was expected to equalize the magnitude of differences and therefore lead to better recognition performance. Still, better classification features and a more effective scaling method or classification in different facial areas could further improve the results. In most of the cases, male pair zygosity recognition was at a higher level compared with females. Erroneously classified twin pairs appear to be obvious outliers in the sample. In particular, faces of young dizygotic (DZ) twins may be so similar that it is very hard to define a feature that would help classify the pair as DZ. Correspondingly, monozygotic (MZ) twins may have faces with quite different shapes. Such anomalous twin pairs are interesting exceptions, but they form a considerable portion in both zygosity groups.

Hepatitis B virus DNA-positive, hepatitis B surface antigen-negative blood donations intercepted by anti-hepatitis B core antigen testing: the Canadian Blood Services experience.

PubMed

O'Brien, Sheila F; Fearon, Margaret A; Yi, Qi-Long; Fan, Wenli; Scalia, Vito; Muntz, Irene R; Vamvakas, Eleftherios C

2007-10-01

The benefit of introducing anti-hepatitis B core antigen (HBc) screening for intercepting potentially infectious donations missed by hepatitis B surface antigen (HBsAg) screening in Canada was studied. Anti-HBc testing of all donations was implemented in April 2005, along with antibody to hepatitis B surface antigen (anti-HBs) and hepatitis B virus (HBV) DNA supplemental testing of anti-HBc repeat-reactive, HBsAg-negative donations. The proportion of potentially infectious donations intercepted by anti-HBc over the initial 18 months of testing was calculated based on three assumptions relating infectivity of HBV DNA-positive units to anti-HBs levels. Lookback was conducted for all DNA-positive donations. Of 493,344 donors, 5,585 (1.13%) were repeat-reactive for the presence of anti-HBc, with 29 (0.52%) being HBV DNA-positive and HBsAg-negative. The proportion of potentially infectious donations intercepted by anti-HBc screening was 1 in 17,800 if all HBV DNA-positive donations were infectious, 1 in 26,900 if infectivity was limited to donations with an anti-HBs level of not more than 100 mIU per mL, and 1 in 69,300 if only donations with undetectable anti-HBs were infectious. For 279 components in the lookback study, no traced recipients were HBsAg-positive and 7 recipients were anti-HBc-reactive in association with 4 donors, 3 of whom had an anti-HBs level of more than 100 mIU per mL and 1 of whom had a level of 61 mIU per mL. Implementation of anti-HBc screening reduced the risk of transfusing potentially infectious units by at least as much as had been expected based on the literature. The lookback did not provide proof of transfusion transmission of HBV from HBV DNA-positive, anti-HBc-reactive, HBsAg-negative donors but it did not establish lack of transmission either.

Comparison of DNA testing strategies in monitoring human papillomavirus infection prevalence through simulation.

PubMed

Lin, Carol Y; Li, Ling

2016-11-07

HPV DNA diagnostic tests for epidemiology monitoring (research purpose) or cervical cancer screening (clinical purpose) have often been considered separately. Women with positive Linear Array (LA) polymerase chain reaction (PCR) research test results typically are neither informed nor referred for colposcopy. Recently, a sequential testing by using Hybrid Capture 2 (HC2) HPV clinical test as a triage before genotype by LA has been adopted for monitoring HPV infections. Also, HC2 has been reported as a more feasible screening approach for cervical cancer in low-resource countries. Thus, knowing the performance of testing strategies incorporating HPV clinical test (i.e., HC2-only or using HC2 as a triage before genotype by LA) compared with LA-only testing in measuring HPV prevalence will be informative for public health practice. We conducted a Monte Carlo simulation study. Data were generated using mathematical algorithms. We designated the reported HPV infection prevalence in the U.S. and Latin America as the "true" underlying type-specific HPV prevalence. Analytical sensitivity of HC2 for detecting 14 high-risk (oncogenic) types was considered to be less than LA. Estimated-to-true prevalence ratios and percentage reductions were calculated. When the "true" HPV prevalence was designated as the reported prevalence in the U.S., with LA genotyping sensitivity and specificity of (0.95, 0.95), estimated-to-true prevalence ratios of 14 high-risk types were 2.132, 1.056, 0.958 for LA-only, HC2-only, and sequential testing, respectively. Estimated-to-true prevalence ratios of two vaccine-associated high-risk types were 2.359 and 1.063 for LA-only and sequential testing, respectively. When designated type-specific prevalence of HPV16 and 18 were reduced by 50 %, using either LA-only or sequential testing, prevalence estimates were reduced by 18 %. Estimated-to-true HPV infection prevalence ratios using LA-only testing strategy are generally higher than using HC2-only or

iDNA-Prot: Identification of DNA Binding Proteins Using Random Forest with Grey Model

PubMed Central

Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

2011-01-01

DNA-binding proteins play crucial roles in various cellular processes. Developing high throughput tools for rapidly and effectively identifying DNA-binding proteins is one of the major challenges in the field of genome annotation. Although many efforts have been made in this regard, further effort is needed to enhance the prediction power. By incorporating the features into the general form of pseudo amino acid composition that were extracted from protein sequences via the “grey model” and by adopting the random forest operation engine, we proposed a new predictor, called iDNA-Prot, for identifying uncharacterized proteins as DNA-binding proteins or non-DNA binding proteins based on their amino acid sequences information alone. The overall success rate by iDNA-Prot was 83.96% that was obtained via jackknife tests on a newly constructed stringent benchmark dataset in which none of the proteins included has pairwise sequence identity to any other in a same subset. In addition to achieving high success rate, the computational time for iDNA-Prot is remarkably shorter in comparison with the relevant existing predictors. Hence it is anticipated that iDNA-Prot may become a useful high throughput tool for large-scale analysis of DNA-binding proteins. As a user-friendly web-server, iDNA-Prot is freely accessible to the public at the web-site on http://icpr.jci.edu.cn/bioinfo/iDNA-Prot or http://www.jci-bioinfo.cn/iDNA-Prot. Moreover, for the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results. PMID:21935457

Screening of Caatinga plants as sources of lectins and trypsin inhibitors.

PubMed

Arcoverde, José Hélton Vasconcelos; Carvalho, Aline de Souza; Neves, Fernanda Pacífico de Almeida; Dionízio, Bianca Paiva; Pontual, Emmanuel Viana; Paiva, Patrícia Maria Guedes; Napoleão, Thiago Henrique; Correia, Maria Tereza dos Santos; da Silva, Márcia Vanusa; Carneiro-da-Cunha, Maria das Graças

2014-01-01

Although it is one of the most threatened areas in the Earth, there are few studies on the biotechnological potential of the Caatinga. This work evaluated 36 extracts from 27 Caatinga plants for lectin and trypsin inhibitor activities. The presence of lectin was detected in 77.7% of samples by haemagglutinating assay. The highest values of specific haemagglutinating activity were found in extracts of leaves from Mimosa lewesii, Bauhinia acuruana and Manilkara rufula and in branches from Myracrodruon urundeuva. Trypsin inhibitor activity was detected in 63.9% of the tested extracts, strong inhibitory effect (>70%) being found in 11 samples. This work demonstrates that Caatinga is a potential source of bioactive plant proteins that can be isolated and studied for several applications. The biochemical prospecting of Caatinga is essential for collection of bioactive principles so as to add conservation value to the region.

DNA Yield From Tissue Samples in Surgical Pathology and Minimum Tissue Requirements for Molecular Testing.

PubMed

Austin, Melissa C; Smith, Christina; Pritchard, Colin C; Tait, Jonathan F

2016-02-01

Complex molecular assays are increasingly used to direct therapy and provide diagnostic and prognostic information but can require relatively large amounts of DNA. To provide data to pathologists to help them assess tissue adequacy and provide prospective guidance on the amount of tissue that should be procured. We used slide-based measurements to establish a relationship between processed tissue volume and DNA yield by A260 from 366 formalin-fixed, paraffin-embedded tissue samples submitted for the 3 most common molecular assays performed in our laboratory (EGFR, KRAS, and BRAF). We determined the average DNA yield per unit of tissue volume, and we used the distribution of DNA yields to calculate the minimum volume of tissue that should yield sufficient DNA 99% of the time. All samples with a volume greater than 8 mm(3) yielded at least 1 μg of DNA, and more than 80% of samples producing less than 1 μg were extracted from less than 4 mm(3) of tissue. Nine square millimeters of tissue should produce more than 1 μg of DNA 99% of the time. We conclude that 2 tissue cores, each 1 cm long and obtained with an 18-gauge needle, will almost always provide enough DNA for complex multigene assays, and our methodology may be readily extrapolated to individual institutional practice.

DNA microarrays for identifying fishes.

PubMed

Kochzius, M; Nölte, M; Weber, H; Silkenbeumer, N; Hjörleifsdottir, S; Hreggvidsson, G O; Marteinsson, V; Kappel, K; Planes, S; Tinti, F; Magoulas, A; Garcia Vazquez, E; Turan, C; Hervet, C; Campo Falgueras, D; Antoniou, A; Landi, M; Blohm, D

2008-01-01

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a "Fish Chip" for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.

A rapid low-cost high-density DNA-based multi-detection test for routine inspection of meat species.

PubMed

Lin, Chun Chi; Fung, Lai Ling; Chan, Po Kwok; Lee, Cheuk Man; Chow, Kwok Fai; Cheng, Shuk Han

2014-02-01

The increasing occurrence of food frauds suggests that species identification should be part of food authentication. Current molecular-based species identification methods have their own limitations or drawbacks, such as relatively time-consuming experimental steps, expensive equipment and, in particular, these methods cannot identify mixed species in a single experiment. This project proposes an improved method involving PCR amplification of the COI gene and detection of species-specific sequences by hybridisation. Major innovative breakthrough lies in the detection of multiple species, including pork, beef, lamb, horse, cat, dog and mouse, from a mixed sample within a single experiment. The probes used are species-specific either in sole or mixed species samples. As little as 5 pg of DNA template in the PCR is detectable in the proposed method. By designing species-specific probes and adopting reverse dot blot hybridisation and flow-through hybridisation, a low-cost high-density DNA-based multi-detection test suitable for routine inspection of meat species was developed. © 2013.

Detection of Invasive Mosquito Vectors Using Environmental DNA (eDNA) from Water Samples

PubMed Central

Schneider, Judith; Valentini, Alice; Dejean, Tony; Montarsi, Fabrizio; Taberlet, Pierre

2016-01-01

Repeated introductions and spread of invasive mosquito species (IMS) have been recorded on a large scale these last decades worldwide. In this context, members of the mosquito genus Aedes can present serious risks to public health as they have or may develop vector competence for various viral diseases. While the Tiger mosquito (Aedes albopictus) is a well-known vector for e.g. dengue and chikungunya viruses, the Asian bush mosquito (Ae. j. japonicus) and Ae. koreicus have shown vector competence in the field and the laboratory for a number of viruses including dengue, West Nile fever and Japanese encephalitis. Early detection and identification is therefore crucial for successful eradication or control strategies. Traditional specific identification and monitoring of different and/or cryptic life stages of the invasive Aedes species based on morphological grounds may lead to misidentifications, and are problematic when extensive surveillance is needed. In this study, we developed, tested and applied an environmental DNA (eDNA) approach for the detection of three IMS, based on water samples collected in the field in several European countries. We compared real-time quantitative PCR (qPCR) assays specific for these three species and an eDNA metabarcoding approach with traditional sampling, and discussed the advantages and limitations of these methods. Detection probabilities for eDNA-based approaches were in most of the specific comparisons higher than for traditional survey and the results were congruent between both molecular methods, confirming the reliability and efficiency of alternative eDNA-based techniques for the early and unambiguous detection and surveillance of invasive mosquito vectors. The ease of water sampling procedures in the eDNA approach tested here allows the development of large-scale monitoring and surveillance programs of IMS, especially using citizen science projects. PMID:27626642

Detection of Invasive Mosquito Vectors Using Environmental DNA (eDNA) from Water Samples.

PubMed

Schneider, Judith; Valentini, Alice; Dejean, Tony; Montarsi, Fabrizio; Taberlet, Pierre; Glaizot, Olivier; Fumagalli, Luca

2016-01-01

Repeated introductions and spread of invasive mosquito species (IMS) have been recorded on a large scale these last decades worldwide. In this context, members of the mosquito genus Aedes can present serious risks to public health as they have or may develop vector competence for various viral diseases. While the Tiger mosquito (Aedes albopictus) is a well-known vector for e.g. dengue and chikungunya viruses, the Asian bush mosquito (Ae. j. japonicus) and Ae. koreicus have shown vector competence in the field and the laboratory for a number of viruses including dengue, West Nile fever and Japanese encephalitis. Early detection and identification is therefore crucial for successful eradication or control strategies. Traditional specific identification and monitoring of different and/or cryptic life stages of the invasive Aedes species based on morphological grounds may lead to misidentifications, and are problematic when extensive surveillance is needed. In this study, we developed, tested and applied an environmental DNA (eDNA) approach for the detection of three IMS, based on water samples collected in the field in several European countries. We compared real-time quantitative PCR (qPCR) assays specific for these three species and an eDNA metabarcoding approach with traditional sampling, and discussed the advantages and limitations of these methods. Detection probabilities for eDNA-based approaches were in most of the specific comparisons higher than for traditional survey and the results were congruent between both molecular methods, confirming the reliability and efficiency of alternative eDNA-based techniques for the early and unambiguous detection and surveillance of invasive mosquito vectors. The ease of water sampling procedures in the eDNA approach tested here allows the development of large-scale monitoring and surveillance programs of IMS, especially using citizen science projects.

Myeloperoxidase-induced Genomic DNA-centered Radicals*

PubMed Central

Gomez-Mejiba, Sandra E.; Zhai, Zili; Gimenez, Maria S.; Ashby, Michael T.; Chilakapati, Jaya; Kitchin, Kirk; Mason, Ronald P.; Ramirez, Dario C.

2010-01-01

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis. PMID:20406811

Studying DNA Looping by Single-Molecule FRET

PubMed Central

Le, Tung T.; Kim, Harold D.

2014-01-01

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA. PMID:24998459

Studying DNA looping by single-molecule FRET.

PubMed

Le, Tung T; Kim, Harold D

2014-06-28

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

Immunological aspects of circulating DNA.

PubMed

Anker, Philippe; Stroun, Maurice

2006-09-01

Nude mice were injected with DNA released by T lymphocytes previously exposed to inactivated herpes symplex type 1 or polio viruses. The serum of these mice was tested for its neutralizing activity. Injected nude mice synthesized antiherpetic or antipolio antibodies, depending on the antigen used to sensitize the T lymphocytes. Mice injected with DNA released by human T cells produced antibodies carrying human allotypes as they could be neutralized by antiallotype sera. However, mice that were injected with DNA released by antigen-stimulated murine T lymphocytes produced antiviral antibodies, which were not neutralized by anti-human allotype sera.

Reliable noninvasive prenatal testing by massively parallel sequencing of circulating cell-free DNA from maternal plasma processed up to 24h after venipuncture.

PubMed

Buysse, Karen; Beulen, Lean; Gomes, Ingrid; Gilissen, Christian; Keesmaat, Chantal; Janssen, Irene M; Derks-Willemen, Judith J H T; de Ligt, Joep; Feenstra, Ilse; Bekker, Mireille N; van Vugt, John M G; Geurts van Kessel, Ad; Vissers, Lisenka E L M; Faas, Brigitte H W

2013-12-01

Circulating cell-free fetal DNA (ccffDNA) in maternal plasma is an attractive source for noninvasive prenatal testing (NIPT). The amount of total cell-free DNA significantly increases 24h after venipuncture, leading to a relative decrease of the ccffDNA fraction in the blood sample. In this study, we evaluated the downstream effects of extended processing times on the reliability of aneuploidy detection by massively parallel sequencing (MPS). Whole blood from pregnant women carrying normal and trisomy 21 (T21) fetuses was collected in regular EDTA anti-coagulated tubes and processed within 6h, 24 and 48h after venipuncture. Samples of all three different time points were further analyzed by MPS using Z-score calculation and the percentage of ccffDNA based on X-chromosome reads. Both T21 samples were correctly identified as such at all time-points. However, after 48h, a higher deviation in Z-scores was noticed. Even though the percentage of ccffDNA in a plasma sample has been shown previously to significantly decrease 24h after venipuncture, the percentages based on MPS results did not show a significant decrease after 6, 24 or 48h. The quality and quantity of ccffDNA extracted from plasma samples processed up to 24h after venipuncture are sufficiently high for reliable downstream NIPT analysis by MPS. Furthermore, we show that it is important to determine the percentage of ccffDNA in the fraction of the sample that is actually used for NIPT, as downstream procedures might influence the fetal or maternal fraction. © 2013.

Checking of individuality by DNA profiling.

PubMed

Brdicka, R; Nürnberg, P

1993-08-25

A review of methods of DNA analysis used in forensic medicine for identification, paternity testing, etc. is provided. Among other techniques, DNA fingerprinting using different probes and polymerase chain reaction-based techniques such as amplified sequence polymorphisms and minisatellite variant repeat mapping are thoroughly described and both theoretical and practical aspects are discussed.

Value of DNA tests: a decision perspective.

PubMed

Taroni, Franco; Bozza, Silvia; Bernard, Magali; Champod, Christophe

2007-01-01

Before a Court of Law testifying in DNA-evidence cases, scientists are often challenged with the idea that the more markers (loci) the better, i.e., why does the scientist not use 16 or more markers? This paper introduces a new perspective, decision analysis, to deal with the problem of the number of markers to type in a criminal context. The decision-making process, which plays a key role in the routine work of a forensic scientist, consists of the rational choice, given personal objectives, between two or more possible outcomes when the consequences of the choice are uncertain. Simulated results support the hypothesis that analytical added value does not increase with the number of markers.

Selling biotechnology in the dental medicine marketplace: the OmniGene Diagnostics DNA probe tests for periodontal pathogens.

PubMed

Van Arsdell, S W; DiFronzo, F; Backman, K C; Mahler, P H

1996-09-01

OmniGene Diagnostics, Inc. has applied the principles of genetic engineering to develop species-specific DNA probe tests for eight periodontal pathogens (Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetem-comitans, Fusobacterium nucleatum, Eikenella corrodens, Campylobacter rectus, Bacteroides forsythus, and Treponema denticola). The test requires minimal effort on the part of the clinician: subgingival plaque samples are collected from the patient and sent through the mail for analysis by OmniGene Diagnostics' fully licensed clinical reference laboratory. Results are transmitted to the practitioner by phone, fax, or mail. The use of diagnostic tests for periodontal pathogens is a relatively new concept in dentistry and acceptance of the OmniGene Diagnostics tests by the dental marketplace has been slower than anticipated. OmniGene Diagnostics' challenge for the future is to persuade the dental community that monitoring periodontal pathogen levels, as well as other clinical indicators of disease, is essential to providing optimal care to the periodontitis patient.

The Trypanosoma cruzi Satellite DNA OligoC-TesT and Trypanosoma cruzi Kinetoplast DNA OligoC-TesT for Diagnosis of Chagas Disease: A Multi-cohort Comparative Evaluation Study

PubMed Central

De Winne, Koen; Büscher, Philippe; Luquetti, Alejandro O.; Tavares, Suelene B. N.; Oliveira, Rodrigo A.; Solari, Aldo; Zulantay, Ines; Apt, Werner; Diosque, Patricio; Monje Rumi, Mercedes; Gironès, Nuria; Fresno, Manuel; Lopez-Velez, Rogelio; Perez-Molina, José A.; Monge-Maillo, Begoña; Garcia, Lineth; Deborggraeve, Stijn

2014-01-01

Background The Trypanosoma cruzi satellite DNA (satDNA) OligoC-TesT is a standardised PCR format for diagnosis of Chagas disease. The sensitivity of the test is lower for discrete typing unit (DTU) TcI than for TcII-VI and the test has not been evaluated in chronic Chagas disease patients. Methodology/Principal Findings We developed a new prototype of the OligoC-TesT based on kinetoplast DNA (kDNA) detection. We evaluated the satDNA and kDNA OligoC-TesTs in a multi-cohort study with 187 chronic Chagas patients and 88 healthy endemic controls recruited in Argentina, Chile and Spain and 26 diseased non-endemic controls from D.R. Congo and Sudan. All specimens were tested in duplicate. The overall specificity in the controls was 99.1% (95% CI 95.2%–99.8%) for the satDNA OligoC-TesT and 97.4% (95% CI 92.6%–99.1%) for the kDNA OligoC-TesT. The overall sensitivity in the patients was 67.9% (95% CI 60.9%–74.2%) for the satDNA OligoC-TesT and 79.1% (95% CI 72.8%–84.4%) for the kDNA OligoC-Test. Conclusions/Significance Specificities of the two T. cruzi OligoC-TesT prototypes are high on non-endemic and endemic controls. Sensitivities are moderate but significantly (p = 0.0004) higher for the kDNA OligoC-TesT compared to the satDNA OligoC-TesT. PMID:24392177

Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand

PubMed Central

Patel, Maha P.; Baum, Dana A.; Silverman, Scott K.

2008-01-01

DNA with a 5′-adenylpyrophosphoryl cap (5′-adenylated DNA; AppDNA) is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5′-Adenylated DNA is also useful for in vitro selection experiments. Efficient preparation of 5′-adenylated DNA is therefore desirable for several biochemical applications. Here we have developed a DNA adenylation procedure that uses T4 DNA ligase and is more reliable than a previously reported approach that used the 5′-phosphorylated donor DNA substrate to be adenylated, a DNA template, and ATP but no acceptor strand. Our improved DNA adenylation procedure uses the above components as well as an acceptor strand that has a strategically chosen C-T acceptor-template mismatch directly adjacent to the adenylation site. This mismatch permits adenylation of the donor DNA substrate but largely suppresses subsequent ligation of the donor with the acceptor, as assayed on nine different DNA substrates that collectively have all four DNA nucleotides represented at each of the first two positions. The new DNA adenylation procedure is successful using either laboratory-prepared or commercial T4 DNA ligase and works well on the preparative (2 nmol) scale for all nine of the test DNA substrates. PMID:18022669

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

PubMed

Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations.

PubMed Central

Fynan, E F; Webster, R G; Fuller, D H; Haynes, J R; Santoro, J C; Robinson, H L

1993-01-01

Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 micrograms of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 micrograms of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines. Images Fig. 1 PMID:8265577

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes

PubMed Central

Klymus, Katy E.; Marshall, Nathaniel T.

2017-01-01

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity. PMID:28542313

Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification.

PubMed

Jorgez, Carolina J; Bischoff, Farideh Z

2009-01-01

Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured. Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template. Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA. Copyright 2009 S. Karger AG, Basel.

Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

Nanogravimetric and voltammetric DNA-hybridization biosensors for studies of DNA damage by common toxicants and pollutants.

PubMed

Nowicka, Anna M; Kowalczyk, Agata; Stojek, Zbigniew; Hepel, Maria

2010-01-01

Electrochemical and nanogravimetric DNA-hybridization biosensors have been developed for sensing single mismatches in the probe-target ssDNA sequences. The voltammetric transduction was achieved by coupling ferrocene moiety to streptavidin linked to biotinylated tDNA. The mass-related frequency transduction was implemented by immobilizing the sensory pDNA on a gold-coated quartz crystal piezoresonators oscillating in the 10MHz band. The high sensitivity of these sensors enabled us to study DNA damage caused by representative toxicants and environmental pollutants, including Cr(VI) species, common pesticides and herbicides. We have found that the sensor responds rapidly to any damage caused by Cr(VI) species, with more severe DNA damage observed for Cr(2)O(7)(2-) and for CrO(4)(2-) in the presence of H(2)O(2) as compared to CrO(4)(2-) alone. All herbicides and pesticides examined caused DNA damage or structural alterations leading to the double-helix unwinding. Among these compounds, paraoxon-ethyl and atrazine caused the fastest and most severe damage to DNA. The physico-chemical mechanism of damaging interactions between toxicants and DNA has been proposed. The methodology of testing voltammetric and nanogravimetric DNA-hybridization biosensors developed in this work can be employed as a simple protocol to obtain rapid comparative data concerning DNA damage caused by herbicide, pesticides and other toxic pollutants. The DNA-hybridization biosensor can, therefore, be utilized as a rapid screening device for classifying environmental pollutants and to evaluate DNA damage induced by these compounds.

Widespread recombination in published animal mtDNA sequences.

PubMed

Tsaousis, A D; Martin, D P; Ladoukakis, E D; Posada, D; Zouros, E

2005-04-01

Mitochondrial DNA (mtDNA) recombination has been observed in several animal species, but there are doubts as to whether it is common or only occurs under special circumstances. Animal mtDNA sequences retrieved from public databases were unambiguously aligned and rigorously tested for evidence of recombination. At least 30 recombination events were detected among 186 alignments examined. Recombinant sequences were found in invertebrates and vertebrates, including primates. It appears that mtDNA recombination may occur regularly in the animal cell but rarely produces new haplotypes because of homoplasmy. Common animal mtDNA recombination would necessitate a reexamination of phylogenetic and biohistorical inference based on the assumption of clonal mtDNA transmission. Recombination may also have an important role in producing and purging mtDNA mutations and thus in mtDNA-based diseases and senescence.

Searching target sites on DNA by proteins: Role of DNA dynamics under confinement

PubMed Central

Mondal, Anupam; Bhattacherjee, Arnab

2015-01-01

DNA-binding proteins (DBPs) rapidly search and specifically bind to their target sites on genomic DNA in order to trigger many cellular regulatory processes. It has been suggested that the facilitation of search dynamics is achieved by combining 3D diffusion with one-dimensional sliding and hopping dynamics of interacting proteins. Although, recent studies have advanced the knowledge of molecular determinants that affect one-dimensional search efficiency, the role of DNA molecule is poorly understood. In this study, by using coarse-grained simulations, we propose that dynamics of DNA molecule and its degree of confinement due to cellular crowding concertedly regulate its groove geometry and modulate the inter-communication with DBPs. Under weak confinement, DNA dynamics promotes many short, rotation-decoupled sliding events interspersed by hopping dynamics. While this results in faster 1D diffusion, associated probability of missing targets by jumping over them increases. In contrast, strong confinement favours rotation-coupled sliding to locate targets but lacks structural flexibility to achieve desired specificity. By testing under physiological crowding, our study provides a plausible mechanism on how DNA molecule may help in maintaining an optimal balance between fast hopping and rotation-coupled sliding dynamics, to locate target sites rapidly and form specific complexes precisely. PMID:26400158

Brief report on the United States Food and Drug Administration Blood Products Advisory Committee recommendations for management of donors and units testing positive for hepatitis B virus DNA.

PubMed

Lucey, C

2006-11-01

This article briefly recounts the 21st July 2005, Blood Products Advisory Committee (BPAC) meeting concerning recommendations for management of donors and units testing positive for hepatitis B virus (HBV) DNA. The author attended the meeting. The United States Food and Drug Administration (FDA) web site was used for meeting materials, and handouts were collected at the meeting to provide narrative information. Two European experts assisted with HBV subject matter. The proceedings of the advisory committee, the issue briefing materials, and testing algorithms are presented. BPAC voted concurrence with the FDA algorithm for Management of Donors and Units Testing Positive for Hepatitis B Virus DNA.

Profile of Roche's Ariosa Harmony prenatal test.

PubMed

Bevilacqua, Elisa; Resta, Serena; Carlin, Andrew; Kang, Xin; Cos Sanchez, Teresa; de Marchin, Jérôme; Jani, Jacques C

2018-06-18

Roche's Ariosa Harmony TM Prenatal Test, a noninvasive cfDNA (cell-free DNA) method for major trisomies has been available since January-2013 at the authors unit and tests were sent to California. From July-2017 onwards, prenatal cfDNA has been reimbursed in Belgium for all pregnancies, however since then samples are sent to a local technology transfer center. Little data are available on patient's profile and choices towards cfDNA and on the performance of local technology transfer centers. Areas covered: The profiles and choices of women regarding this test were evaluated. Further, the performance of cfDNA at the local center was compared to the one in California. The results showed that women from the Netherlands, as compared to Belgium, were more likely to undergo cfDNA testing for maternal request and would be less likely to undergo karyotyping if cfDNA were unavailable, and therefore are better candidates for cfDNA testing, when this is used as first-line screening. The local test failure rate was nearly twice that of the main laboratory in California, however when repeated, the success rate was quite high. Expert commentary: The findings highlight the importance of conducting these types of studies, before decisions about clinical implementation are made by national governments and ministries of health.

Ames and random amplified polymorphic DNA tests for the validation of the mutagenic and/or genotoxic potential of the drinking water disinfection by-products chloroform and bromoform.

PubMed

Khallef, Messaouda; Cenkci, Süleyman; Akyil, Dilek; Özkara, Arzu; Konuk, Muhsin; Benouareth, Djamel Eddine

2018-01-28

Chloroform and Bromoform are two abundant trihalomethanes found in Algerian drinking water. The investigation of the mutagenic hazard of these disinfection by-products was studied by Ames test as prokaryotic bioassay to show their mutagenic effects. For this, Salmonella typhimurium TA98 and TA100 strains were employed. Both chloroform and bromoform showed a direct mutagenic effect since the number of revertant colonies gradually increase in dose-dependent manner with all concentrations tested with the two bacterial strains and these were both in the absence and presence of S9 metabolic activation. The genotoxic hazard was also studied by random amplified polymorphic DNA test on the root cells of Allium cepa as eukaryotic bioassay. DNA extracted from the roots of the onion were incubated at different concentrations of chloroform and bromoform and then amplified by polymerase chain reaction. This was based on demonstrating a major effect of disappearance of bands compared to roots incubated in the negative control (distilled water). The results showed that these two compounds affected genomic DNA by breaks although by mutations.

Assessment of MagNA pure LC extraction system for detection of human papillomavirus (HPV) DNA in PreservCyt samples by the Roche AMPLICOR and LINEAR ARRAY HPV tests.

PubMed

Stevens, Matthew P; Rudland, Elice; Garland, Suzanne M; Tabrizi, Sepehr N

2006-07-01

Roche Molecular Systems recently released two PCR-based assays, AMPLICOR and LINEAR ARRAY (LA), for the detection and genotyping, respectively, of human papillomaviruses (HPVs). The manual specimen processing method recommended for use with both assays, AmpliLute, can be time-consuming and labor-intensive and is open to potential specimen cross-contamination. We evaluated the Roche MagNA Pure LC (MP) as an alternative for specimen processing prior to use with either assay. DNA was extracted from cervical brushings, collected in PreservCyt media, by AmpliLute and MP using DNA-I and Total Nucleic Acid (TNA) kits, from 150 patients with histologically confirmed cervical abnormalities. DNA was amplified and detected by AMPLICOR and the LA HPV test. Concordances of 96.5% (139 of 144) (kappa=0.93) and 95.1% (135 of 142) (kappa=0.90) were generated by AMPLICOR when we compared DNA extracts from AmpliLute to MP DNA-I and TNA, respectively. The HPV genotype profiles were identical in 78.7 and 74.7% of samples between AmpliLute and DNA-I or TNA, respectively. To improve LA concordance, all 150 specimens were extracted by MP DNA-I protocol after the centrifugation of 1-ml PreservCyt samples. This modified approach improved HPV genotype concordance levels between AmpliLute and MP DNA-I to 88.0% (P=0.043) without affecting AMPLICOR sensitivity. Laboratories that have an automated MP extraction system would find this procedure more feasible and easier to handle than the recommended manual extraction method and could substitute such extractions for AMPLICOR and LA HPV tests once internally validated.

Recombinant DNA Paper Model Simulation: The Genetic Engineer.

ERIC Educational Resources Information Center

Wagner, Joan

1998-01-01

Describes a course for talented high school students that focuses on DNA science and technology. Employs Cold Spring Harbor's DNA Science laboratory manual. Engages students in performing sickle-cell anemia and thalassemia tests in rabbits. (DDR)

Evolution of DNA Methylation across Insects

PubMed Central

Vogel, Kevin J.; Moore, Allen J.; Schmitz, Robert J.

2017-01-01

DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects. PMID:28025279

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

PubMed

Jordan, Jeanne A; Ibe, Christine O; Moore, Miranda S; Host, Christel; Simon, Gary L

2012-05-01

In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay. Copyright © 2012 Elsevier B.V. All rights reserved.

Twisting, supercoiling and stretching in protein bound DNA

NASA Astrophysics Data System (ADS)

Lam, Pui-Man; Zhen, Yi

2018-04-01

We have calculated theoretical results for the torque and slope of the twisted DNA, with various proteins bound on it, using the Neukirch-Marko model, in the regime where plectonemes exist. We found that the torque in the protein bound DNA decreases compared to that in the bare DNA. This is caused by the decrease in the free energy g(f) , and hence the smaller persistence lengths, in the case of protein bound DNA. We hope our results will encourage experimental investigations of supercoiling in protein bound DNA, which can provide further tests of the Neukirch-Marko model.