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Sample records for dna haemagglutination test

  1. Simplified method for the rubella haemagglutination inhibition screening test.

    PubMed Central

    Mortimer, P P; Jordan, S M; Smith, I V

    1977-01-01

    A modification of the rubella haemagglutination inhibition (HAI) test in which both pretreatment and serum titration are carried out in wells of the same microtitre plate saves time, labour, and materials and gives results comparable to a conventional HAI procedure. PMID:886015

  2. Treponema pallidum specific IgM haemagglutination test for serodiagnosis of syphilis.

    PubMed Central

    Sato, T; Kubo, E; Yokota, M; Kayashima, T; Tomizawa, T

    1984-01-01

    The Treponema pallidum specific IgM haemagglutination (TP-IgM-HA) test uses erythrocytes sensitised with antiserum to human IgM to separate IgM from IgG in serum. Specific antitreponemal IgM captured in this way is detected by adding a second reagent comprising erythrocytes sensitised with T pallidum antigen. Eighty two serum samples from 82 patients with untreated syphilis, 521 samples from 73 patients with treated syphilis, and 1872 samples from people who did not have syphilis were examined by the 19S(IgM)-TPHA (T pallidum haemagglutination), IgM-FTA-ABS (fluorescent treponemal antibody absorbed), TP-IgM-ELISA (enzyme linked immunosorbent assay), and TP-IgM-HA tests for the presence of 19S(IgM) antibodies specific to treponemes. The sensitivity of the TP-IgM-HA test was 97.6% and the specificity was 99.7%. We also traced IgM specific to treponemes in untreated patients with primary syphilis by four different tests. The TP-IgM-HA test results clearly reflected the effect of the treatment. PMID:6394097

  3. Indirect Haemagglutination Test in Comparison with ELISA for Detection of Antibodies against Invasive Amoebiasis

    PubMed Central

    Dhanalakshmi, Sankaramoorthy; Meenachi, Chidambaram

    2016-01-01

    Introduction Diagnosis of amoebiasis is based on combination of tests like microscopy, imaging, serology and molecular methods. In absence of molecular techniques, serology can be used as an alternative aid. Various serological techniques were reported with different sensitivity and specificity. The diagnostic efficiency of these assays mainly depends on the characteristics of antigen that is being used and various conditions of performance. Aim To evaluate the efficiency of recombinant calcium binding domain containing protein by Indirect Haemagglutination Assay (IHA) against a commercial ELISA among amoebic liver abscess cases and control group. Materials and Methods The study was carried out during the period of 2011-2015 and blood samples were collected from suspected amoebiasis cases who were attending the clinics of Medicine and Paediatrics department, JIPMER. A total of 200 sera samples which included 100 Amoebic Liver Abscess (ALA), 50 cases of other parasitic infections and liver diseases and 50 presumed healthy controls were examined by IHA and commercial ELISA. In brief, chick cells were stabilized by Double Aldehyde Sensitization (DAS) method. Optimum Sensitizing Dose (OSD) of the antigen was determined. The test was performed in a U-bottomed microtiter plate with recombinant amoebic antigen (12.5μg/ml), incubated at Room Temperature (RT) for 2 hours. RIDASCREEN Entamoeba IgG ELISA kit which is commercially available was used to evaluate the samples as per manufacturer’s instruction. Results The overall sensitivity and specificity of the IHA was 62% and 96%, respectively when compared to ELISA having sensitivity and specificity of 69% and 90%, respectively. The positive predictive value of the IHA was 91% while negative predictive value was 79%. Similarly, the positive predictive value of the ELISA was 87% while negative predictive value was 74%. Conclusion As serology heavily suffers due to lack of a standardised test system employing the native

  4. Indirect Haemagglutination Test in Comparison with ELISA for Detection of Antibodies against Invasive Amoebiasis.

    PubMed

    Dhanalakshmi, Sankaramoorthy; Meenachi, Chidambaram; Parija, Subhash Chandra

    2016-08-01

    Diagnosis of amoebiasis is based on combination of tests like microscopy, imaging, serology and molecular methods. In absence of molecular techniques, serology can be used as an alternative aid. Various serological techniques were reported with different sensitivity and specificity. The diagnostic efficiency of these assays mainly depends on the characteristics of antigen that is being used and various conditions of performance. To evaluate the efficiency of recombinant calcium binding domain containing protein by Indirect Haemagglutination Assay (IHA) against a commercial ELISA among amoebic liver abscess cases and control group. The study was carried out during the period of 2011-2015 and blood samples were collected from suspected amoebiasis cases who were attending the clinics of Medicine and Paediatrics department, JIPMER. A total of 200 sera samples which included 100 Amoebic Liver Abscess (ALA), 50 cases of other parasitic infections and liver diseases and 50 presumed healthy controls were examined by IHA and commercial ELISA. In brief, chick cells were stabilized by Double Aldehyde Sensitization (DAS) method. Optimum Sensitizing Dose (OSD) of the antigen was determined. The test was performed in a U-bottomed microtiter plate with recombinant amoebic antigen (12.5μg/ml), incubated at Room Temperature (RT) for 2 hours. RIDASCREEN Entamoeba IgG ELISA kit which is commercially available was used to evaluate the samples as per manufacturer's instruction. The overall sensitivity and specificity of the IHA was 62% and 96%, respectively when compared to ELISA having sensitivity and specificity of 69% and 90%, respectively. The positive predictive value of the IHA was 91% while negative predictive value was 79%. Similarly, the positive predictive value of the ELISA was 87% while negative predictive value was 74%. As serology heavily suffers due to lack of a standardised test system employing the native antigen, there arises need to identify alternative source of

  5. The application of the haemagglutination test to a study of the immunity to malaria in protected and unprotected population groups in Australian New Guinea*

    PubMed Central

    Desowitz, R. S.; Saave, J. J.

    1965-01-01

    The formolized tanned sheep erythrocyte haemagglutination test has been applied to an immuno-malariometric study in Australian New Guinea to determine whether the haemagglutination titre reflects a subject's immune state and to measure the effect of malaria control operations on a population's immunity. Two population groups were studied—one (unprotected) living in holoendemic malaria conditions, the other (protected) living in an area subject to malaria control measures for four years. An increase in both serological positivity rates and geometric mean titres among the unprotected group with increasing age suggests that the test does serve to assess the state of immunity; the corresponding rates were much lower in the protected population, particularly among the children. The authors foresee the possible use of the haemagglutination test as a supplement to other procedures in assessing the progress of a malaria campaign. They, note, however, that more immuno-malariometric studies on populations subject to different degrees of malaria endemicity will need to be carried out before the relationship between the immune state and serological results can be clearly established. PMID:14310901

  6. International collaborative study to establish reference preparations to standardise haemagglutination testing for anti-A and anti-B in normal intravenous immunoglobulins by the direct method.

    PubMed

    Thorpe, S J; Fox, B; Sharp, G; Heath, A B; Behr-Gross, M-E; Terao, E; Virata-Theimer, M L; Yu, M W

    2010-04-01

    A joint project (coded BSP089) was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM) of the Council of Europe, the National Institute for Biological Standards and Control (NIBSC) on behalf of the World Health Organization (WHO) and the Center for Biologics Evaluation and Research (CBER) of the U.S. Food and Drug Administration (FDA) to evaluate, in an international collaborative study, 3 lyophilised intravenous immunoglobulin (IVIG) preparations for their suitability to serve as Reference Preparations to standardise and control the highly variable haemagglutination testing for anti-A and anti-B in IVIG products. 23 laboratories tested candidate IVIG reference preparations consisting of a Positive control, a Negative control and a specifically formulated Limit test reference preparation to define the maximum (e.g., pharmacopoeial) limits of anti-A and anti-B haemagglutinins in IVIG products, where limits are applicable. Laboratories performed direct haemagglutination using papain-treated erythrocytes and/or indirect anti-globulin tests. For both methods, there was up to 16-fold variation in anti-A and anti-B titres, although there was good agreement over a 2-fold titre range for anti-A and anti-B between laboratories using the direct method for both the Positive control and Limit reference preparations. Comparative titration data for the Positive control and Limit reference preparations indicated that the use of a 'Limit' test reference preparation would facilitate identification of higher titre batches when the direct haemagglutination method is used. The Positive control, Negative control and Limit test preparations were adopted in November 2008 by the Commission of the European Pharmacopoeia (Ph. Eur.) as Biological Reference Preparations. The same preparations have been established as reference reagents by the WHO and the U.S FDA, including the maximal specifications defined by the Limit test preparation. This will facilitate

  7. An alternative method for inactivating heteroagglutinins in human sera applicable to rubella haemagglutination inhibition testing at low dilutions.

    PubMed Central

    Mortimer, P P

    1976-01-01

    Serum agglutinins of chick and pigeon cells are predominantly immunoglobulin M and can be inactivated by 2 mercaptoethanol. 2 Mercaptoethanol is more effective than strong suspensions of red cells in removing the agglutinins of indicator cells from sera being prepared for HAI tests. In rubella HAI tests from a dilution of 1 to 10 dilute 2 mercaptoethanol offer a convenient method of removing agglutinins, and, at higher concentration, allow dilutions of sera from 1 in 2-5 to be tested without significant interference by heteroagglutinins. HAI titres are comparable when red cell absorbed and 2 mercaptoethanol treated sera are tested in parallel. Images PMID:946972

  8. Evaluation of enzyme-linked immunosorbent assays and a haemagglutination inhibition tests for the detection of antibodies to Newcastle disease virus in village chickens using a Bayesian approach.

    PubMed

    Chaka, H; Thompson, P N; Goutard, F; Grosbois, V

    2015-04-01

    Newcastle disease (ND) is an endemic disease in village chickens in Ethiopia with substantial economic importance. The sensitivity (Se) and specificity (Sp) of a blocking enzyme-linked immunosorbent assay (bELISA, Svanova Biotech), indirect ELISA (iELISA, Laboratoire Service International) and haemagglutination inhibition (HI) test for ND virus (NDV) antibody detection were evaluated in a Bayesian framework in the absence of a gold standard test, on sera collected from unvaccinated chickens kept under the village production system in household flocks and at markets in two woredas (i.e. districts) of the Eastern Shewa zone, Ethiopia. The outcomes of the iELISA test differed dramatically from those of the two other tests with 92% of the samples testing positive as compared with less than 15% for bELISA and HI. iELISA results were also inconsistent with previous estimations of Newcastle serological prevalence. The information provided by the iELISA test was thus considered as highly unreliable, probably due to an extremely low specificity, and thus not considered in the Bayesian models aiming at estimating serological prevalence and test performance parameters. Bayesian modelling of HI and bELISA test results suggested that bELISA had both the highest Se (86.6%; 95% posterior credible interval (PCI): 61.8%; 98.5%), and the highest Sp (98.3%; 95% PCI: 97.2%; 99.5%), while HI had a Se of 80.2% (95% PCI: 59.1%; 94.3%), and a Sp of 96.1% (95% PCI: 95.1%; 97.4%). Model selection and the range of the posterior distribution of the correlation between bELISA and HI test outcomes for truly seropositive animals (median at 0.461; PCI: -0.055; 0.894) suggested a tendency for bELISA and HI to detect the same truly positive animals and to fail to detect the same truly positive animals. The use of bELISA in screening and surveillance for NDV antibodies is indicated given its high Se and Sp, in addition to its ease of automation to handle large numbers of samples compared to HI. The

  9. A WHO reference reagent to standardize haemagglutination testing for anti-A and anti-B in serum and plasma: international collaborative study to evaluate a candidate preparation.

    PubMed

    Thorpe, S J; Fox, B; Sharp, G; White, J; Milkins, C

    2016-08-01

    The aim of the study was to evaluate a lyophilized serum preparation, 14/300, for its suitability to serve as a World Health Organization (WHO) Reference Reagent to standardize and control haemagglutination titrations for anti-A and anti-B in serum and plasma, in an international collaborative study. Serum preparation 14/300 and two plasma-based reserve preparations, 14/304 (high titre anti-A) and 14/208 (high titre anti-B), were titrated by 24 laboratories in 13 countries using direct (DRT) and indirect (IAT) haemagglutination techniques. There was eightfold to 64-fold variation in reported titres per preparation and method across laboratories, that is, titres extended over 4-7 dilutions, although intralaboratory variability was generally good, with over 90% of replicate titres within a twofold range. There was a reduction in interlaboratory variability when titres of the reserve preparations were adjusted relative to those of the candidate Reference Reagent. The establishment of 14/300 as a WHO Reference Reagent for high titre anti-A and anti-B in serum, with nominal anti-A and anti-B titres of 128 for DRT, and nominal anti-A and anti-B titres of 256 for IAT, will facilitate global standardization of haemagglutination titrations for anti-A and anti-B in patient samples and blood components. © 2016 Crown copyright. Vox Sanguinis © 2016 International Society of Blood Transfusion.

  10. Estimation of Tetanus Toxoid by Different Methods, including Haemagglutination Inhibition

    PubMed Central

    Fulthorpe, A. J.

    1958-01-01

    Comparative quantitative estimations of tetanus toxoid by different methods have been made. The in vivo total combining power test is considered likely to give a true assessment of the combination of toxoid with antitoxin since it is based on the biological action of toxin. The haemagglutination inhibition test has been found to give good agreement with the in vivo test and it is cheap, sensitive and readily repeatable. Since the haemagglutination inhibition test can be performed with toxin or toxoid by the same method, it is possible to relate the quantitative estimation of toxoid to the L+ dose of a routine test toxin, and thus to a standard antitoxin, which is convenient. Considerable discrepancies have been found between results in the flocculation test and the in vivo total combining power test, particularly with toxoids denatured by heat and phenol, or modified by formalin. Discrepanicies between the flocculation and in vivo tests with many preparations have been related to the concentration of residual free formalin. PMID:13610419

  11. Forensic DNA testing.

    PubMed

    Butler, John M

    2011-12-01

    Forensic DNA testing has a number of applications, including parentage testing, identifying human remains from natural or man-made disasters or terrorist attacks, and solving crimes. This article provides background information followed by an overview of the process of forensic DNA testing, including sample collection, DNA extraction, PCR amplification, short tandem repeat (STR) allele separation and sizing, typing and profile interpretation, statistical analysis, and quality assurance. The article concludes with discussions of possible problems with the data and other forensic DNA testing techniques.

  12. Laboratory diagnosis of psittacine beak and feather disease by haemagglutination and haemagglutination inhibition.

    PubMed

    Raidal, S R; Sabine, M; Cross, G M

    1993-04-01

    Simple and sensitive haemagglutination and haemagglutination inhibition assays were developed for psittacine beak and feather disease (PBFD) virus and serum antibody, respectively. The assays were used in the examination of samples from 73 birds clinically affected with PBFD. High antigen titres (log2 9 to log2 12) were detected in feathers, faeces and cloacal contents of PBFD-affected birds. Antigen was not detected in either faecal or feather samples from 20 normal galahs (Eolophus roseicapillus) and 9 normal sulphur crested cockatoos (Cacatua galerita). After kaolin treatment and haemadsorption of serum, haemagglutination inhibition (HI) antibody titres could not be detected in serum from 42 PBFD-affected birds, whereas serum HI titres from 64 normal psittacine birds ranged from less than log2 1 to log2 8. Serum and yolk HI antibody responses of 6 PBFD virus-inoculated layer hens were measured. Pre-inoculation chicken sera contained high concentrations of non-specific haemagglutination inhibitors (not detected in chloroform-extracted yolk), which were removed by kaolin treatment and haemadsorption.

  13. Determination of serum antibodies against swine-origin influenza A virus H1N1/09 by immunofluorescence, haemagglutination inhibition, and by neutralization tests: how is the prevalence rate of protecting antibodies in humans?

    PubMed

    Allwinn, Regina; Geiler, Janina; Berger, Annemarie; Cinatl, J; Doerr, H W

    2010-05-01

    In April 2009, a new variant of influenza A virus, subtype H1N1v emerged in Mexico and spread all over the world producing the H1N1 pandemic in mankind after 1918-1920 and 1978/1979. Obviously there was no herd immunity against this new virus variant. Mainly young people, but less elderly were affected and presented severe and even lethal courses of disease. Since virus-specific antibodies are commonly regarded as markers of partial or complete immunoprotection, we performed antibody determinations in serum samples obtained from people before and after the pandemic has arrived in our region (Frankfurt/M., Germany). The assays were done by indirect immunofluorescence, by neutralization test, and by a haemagglutination inhibition test (HI), which was established in a practical modification for general and easy use. Among 145 individuals, of whom serum specimens had been drawn before the onset of pandemic, 19 revealed humoral immunity, i.e. titres of H1N1v neutralizing antibodies (at least 1:64). Eleven were older than 60 years, one belonged to the age group 40-59 years, three to the age group 20-39 years, and two to the age group 15-19 years. After the onset of pandemic in Frankfurt, serum specimens drawn from n = 225 randomly selected patients of our local university hospital were investigated for antibodies against H1N1v by HI, which is generally recommended for routine check of immunity. Twenty-eight individuals revealed the protecting antibody titre of at least 1:40. The age distribution had moved to mean age groups. The results fit to the incidence of influenza A/H1N1(09) disease, as confirmed by RT-PCR in patients admitted to our hospital, peaking in the younger age groups up to 30 years (second affected group: 30-40 years). While commonly used solid-phase antibody tests (like immunofluorescence) are not suitable to diagnose passed H1N1(09) infection and acquired immunity, this can be easily done by HI. Expecting the next waves of influenza A/H1N1v infections

  14. 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...

  15. Stool DNA Test

    MedlinePlus

    ... result. A test is considered negative if DNA markers common to colon cancer or precancerous polyps and signs of blood are ... result. A test is considered positive if DNA markers common to colon cancer or precancerous polyps or signs of blood are ...

  16. Studies on marine algae for haemagglutinic activity.

    PubMed

    Alam, M T; Usmanghani, K

    1994-07-01

    Lectins (agglutinins) are important in medical and immunological applications. Phytohaemagglutinins have been found useful in blood banking. Keeping in view of these facts, the marine algae found at Karachi coastal region have been screened for agglutinic activity by using human erythrocytes of A, B, AB and 0 group. Altogether 53 algal samples were collected and subjected to extraction, fractionation serial dilution and titre determinations. The total marine algae screened for haemagglutinic activity were 44 out of these 14, 13 and 17 belonged to Chlorophyta, Phaeophyta, and Rhodophyta respectively. Among these three groups the Rhodophyta showed the highest number of lytic activity. The green marine alga Valoniopsis pachynema showed a titre value between 2(2) and 2(3), which is statistically significant. In case of brown marine algae Colpomenia sinuosa was found to be active (titre 2(3)), while Dictyota dichotoma, D. indica and Iyengaria stellata, furnished week titre value as 2(2). The red marine algae screened were 17, out of these 4 spp. showed significant activity (titre 2(3)), and these are Gelidium usmanghani, Gracilaria foliifera Hypnea pannosa and Hynea valentiae. While Scinaia fascicularis, Scinaia indica and Champia parvula were found to be weak in their onset on human erythrocytes. The results obtained were quite in agreement with those reported in the literature.

  17. Interferon and antibody titrations using haemagglutinating Togaviridae and trypsinized human erythrocytes.

    PubMed

    Sedmak, J J; Dixon, M; Schoenherr, C; Sabran, J L; Grossberg, S E

    1983-02-01

    Several Togaviridae of the alphavirus and flavivirus genera agglutinate trypsinized human group O erythrocytes (THOE) (Shortridge and Hu, 1976). Haemagglutinin titers of Semliki Forest virus (SFV) and Japanese encephalitis virus (JEV) measured with THOE were equivalent to, if not higher than, those obtained with Embden gander erythrocytes, even with unextracted haemagglutinin. Results obtained with THOE in JEV haemagglutination-inhibition tests on sera taken from a previously infected individual over a 20-yr period were similar to those measured during the initial JEV infection. The inhibition of SFV haemagglutinin production as measured with THOE was a very sensitive bioassay for chicken interferon: interferon titers were 6- to 10-fold higher than those obtained with the vesicular stomatitis virus plaque-reduction method. The generally greater availability of human erythrocytes (including those stabilized with glutaraldehyde), the simplicity of the trypsin treatment, and the possibility of using unextracted haemagglutinin recommend this technique for use with haemagglutinating Togaviridae.

  18. Rubella virus serology:detection of residual lipoprotein inhibitors of haemagglutination using sensitive indicator arboviruses.

    PubMed Central

    Shortridge, K F

    1977-01-01

    Immune and non-immune rubella sera were examined in the haemagglutination inhibition (HAI) test to evaluate the removal of lipoprotein non-specific inhibitors (NSI) of haemagglutination after kaolin and manganous chloride/heparin treatments. The sera were titrated fresh or after lipoprotein deterioration brought about by ageing the samples at 4 degrees C for various periods of time and by freezing at -20 degrees C with subsequent thawing. Deterioration was seen as altered electrophoretic mobility while the lipoproteins in treated sera were detected by indicator arboviruses whose haemagglutination is known to be strongly inhibited by the native macromolecules. After both treatments, notably manganous chloride/heparin, residual NSI activity was detected in deteriorated samples, particularly with group B arboviruses such as Japanese encephalitis and west Nile viruses but generally less so or not at all with the group A arboviruses employed. Absolutely fresh sera are considered highly desirable for rubella virus HAI assay, and it is suggested that the efficiency of lipoprotein NSI removal regardless of treatment protocol could be monitored in parallel HAI tests using carefully chosen indicator arboviruses. This could be done in conjunction with density gradient centrifugation of doubtful sera should ultracentrifuging facilities be available. The suitability of the monitoring procedure would be dependent to some extent on whether certain arboviruses are known to be endemic in a particular area. Images PMID:864010

  19. Comparison of radioassay and haemagglutination methods for anti-thyroid microsomal antibodies.

    PubMed Central

    Mariotti, S; Pinchera, A; Vitti, P; Chiovato, L; Marcocci, C; Urbano, C; Tosi, M; Baschieri, L

    1978-01-01

    Parallel measurements of circulating anti-thyroid microsomal (anti-M) antibodies by radioassay and haemagglutination were performed on subjects with or without thyroid disorders. Three-quarters (75.4%) of control subjects had undetectable antibody levels (less than 10 u/ml) by radioassay and only 3.1% had concentrations of greater than or equal to 75 u/ml. Abnormally elevated levels (greater than or equal to 75 u/ml) were found in most of the patients with Hashimoto's thyroiditis (94.1%) or idiopathic myxoedema (86.7%), in the majority (75.0%) of those with Graves' disease and only in a minority of those with other thyroid disorders. The percentage of positive sera by haemagglutination was very similar in all groups to that of abnormal values observed in the radioassay. Direct comparison of parallel tests on a total of 631 sera revealed a highly significant correlation (r = 0.91, P less than 0.001) between the two methods, but elevated antibody titres by haemagglutination were found in some sera with negative radioassays. All these sera were from a single patient with thyroid carcinoma associated with Hashimoto's thyroiditis and had elevated levels of anti-thyroglobulin (anti-Tg) antibodies. Evidence that such discrepancies were due to anti-Tg antibodies reacting with microsomal-bound Tg was provided by the demonstration that the haemagglutination produced by these sera could be completely inhibited by the addition of Tg. A similar inhibition was observed with two rabbit antisera to human Tg, but not with sera from patients with thyroid autoimmune disorders containing high levels of anti-microsomal anti-bodies. PMID:582027

  20. The effect of haemagglutinating factor from boar seminal vesicle fluid on leucocytes.

    PubMed

    Veselský, L; Sedláková, E; Dostál, J; Hruban, V; Pazdera, J

    1981-01-01

    Complete agglutination of porcine, bovine, ovine, and rabbit leucocytes and of porcine and bovine thrombocytes was observed after their exposure to a 1% solution of the haemagglutinating protein isolated from boar seminal fluid. Bull seminal vesicle fluid had the same agglutinating effect on leucocytes, but did not agglutinate thrombocytes. Ram seminal plasma and other fluids from the reproductive tract of boar and bull did not agglutinate either leucocytes or thrombocytes. A viability test showed that the agglutinin of boar seminal vesicle fluid, bull seminal vesicle fluid, and boar prostate fluid were all lethal for leucocytes.

  1. 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

    2017-01-01

    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.

  2. Attitudes on DNA ancestry tests.

    PubMed

    Wagner, Jennifer K; Weiss, Kenneth M

    2012-01-01

    The DNA ancestry testing industry is more than a decade old, yet details about it remain a mystery: there remain no reliable, empirical data on the number, motivations, and attitudes of customers to date, the number of products available and their characteristics, or the industry customs and standard practices that have emerged in the absence of specific governmental regulations. Here, we provide preliminary data collected in 2009 through indirect and direct participant observation, namely blog post analysis, generalized survey analysis, and targeted survey analysis. The attitudes include the first available data on attitudes of those of individuals who have and have not had their own DNA ancestry tested as well as individuals who are members of DNA ancestry-related social networking groups. In a new and fluid landscape, the results highlight the need for empirical data to guide policy discussions and should be interpreted collectively as an invitation for additional investigation of (1) the opinions of individuals purchasing these tests, individuals obtaining these tests through research participation, and individuals not obtaining these tests; (2) the psychosocial and behavioral reactions of individuals obtaining their DNA ancestry information with attention given both to expectations prior to testing and the sociotechnical architecture of the test used; and (3) the applications of DNA ancestry information in varying contexts.

  3. The derivation of a minimum immune titre of rubella haemagglutination-inhibition (HI) antibody*

    PubMed Central

    Bradstreet, C. M. Patricia; Kirkwood, B.; Pattison, J. R.; Tobin, J. O'H.

    1978-01-01

    Ten laboratories collaborated in a study of minimum immune titre (MIT) of rubella haemagglutination-inhibiting (HI) antibody with one laboratory acting as a reference laboratory to provide a uniform basis for comparison of the HI results. The international unitage equivalent to the MIT used by the ten laboratories was found to vary from 24 to 98 units. Testing of the sera by immunofluorescence and by HI after flotation centrifugation indicated that residual non-specific inhibitors may interfere with HI antibody testing to an extent equivalent to 12-15 units. An acceptable MIT would therefore be equivalent to 24-48 units of rubella HI antibody. The single radial haemolysis (SRH) results on the sera indicate that this is a sensitive and specific test for rubella antibody. PMID:366017

  4. DNA testing in hereditary neuropathies.

    PubMed

    Murphy, Sinéad M; Laurá, Matilde; Reilly, Mary M

    2013-01-01

    The inherited neuropathies are a clinically and genetically heterogeneous group of disorders in which there have been rapid advances in the last two decades. Molecular genetic testing is now an integral part of the evaluation of patients with inherited neuropathies. In this chapter we describe the genes responsible for the primary inherited neuropathies. We briefly discuss the clinical phenotype of each of the known inherited neuropathy subgroups, describe algorithms for molecular genetic testing of affected patients and discuss genetic counseling. The basic principles of careful phenotyping, documenting an accurate family history, and testing the available genes in an appropriate manner should identify the vast majority of individuals with CMT1 and many of those with CMT2. In this chapter we also describe the current methods of genetic testing. As advances are made in molecular genetic technologies and improvements are made in bioinformatics, it is likely that the current time-consuming methods of DNA sequencing will give way to quicker and more efficient high-throughput methods, which are briefly discussed here. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Testing the Efficacy of a Multi-Component DNA-Prime/DNA-Boost Vaccine against Trypanosoma cruzi Infection in Dogs

    PubMed Central

    Aparicio-Burgos, José E.; Ochoa-García, Laucel; Zepeda-Escobar, José Antonio; Gupta, Shivali; Dhiman, Monisha; Martínez, José Simón; de Oca-Jiménez, Roberto Montes; Arreola, Margarita Val; Barbabosa-Pliego, Alberto; Vázquez-Chagoyán, Juan C.; Garg, Nisha Jain

    2011-01-01

    Background Trypanosoma cruzi, the etiologic agent of Chagas Disease, is a major vector borne health problem in Latin America and an emerging infectious disease in the United States. Methods We tested the efficacy of a multi-component DNA-prime/DNA-boost vaccine (TcVac1) against experimental T. cruzi infection in a canine model. Dogs were immunized with antigen-encoding plasmids and cytokine adjuvants, and two weeks after the last immunization, challenged with T. cruzi trypomastigotes. We measured antibody responses by ELISA and haemagglutination assay, parasitemia and infectivity to triatomines by xenodiagnosis, and performed electrocardiography and histology to assess myocardial damage and tissue pathology. Results Vaccination with TcVac1 elicited parasite-and antigen-specific IgM and IgG (IgG2>IgG1) responses. Upon challenge infection, TcVac1-vaccinated dogs, as compared to non-vaccinated controls dogs, responded to T. cruzi with a rapid expansion of antibody response, moderately enhanced CD8+ T cell proliferation and IFN-γ production, and suppression of phagocytes’ activity evidenced by decreased myeloperoxidase and nitrite levels. Subsequently, vaccinated dogs controlled the acute parasitemia by day 37 pi (44 dpi in non-vaccinated dogs), and exhibited a moderate decline in infectivity to triatomines. TcVac1-immunized dogs did not control the myocardial parasite burden and electrocardiographic and histopatholgic cardiac alterations that are the hallmarks of acute Chagas disease. During the chronic stage, TcVac1-vaccinated dogs exhibited a moderate decline in cardiac alterations determined by EKG and anatomo-/histo-pathological analysis while chronically-infected/non-vaccinated dogs continued to exhibit severe EKG alterations. Conclusions Overall, these results demonstrated that TcVac1 provided a partial resistance to T. cruzi infection and Chagas disease, and provide an impetus to improve the vaccination strategy against Chagas disease. PMID:21625470

  6. Colistin inhibition of mannose-resistant haemagglutination by K88-positive and K99-positive escherichia coli strains. A preliminary report.

    PubMed

    Søgaard, H; Larsen, J L; Christensen, S

    1983-03-01

    Two enteropathogenic E. coli strains isolated from a calf and a piglet succumbed to diarrhoea were studied. The bovine strain carried K99-antigen and the porcine strain was K88-positive. Both strains agglutinated pig erythrocytes in the presence of D-mannose. In the test a bacterial cell density of 3 x 10(9) per ml and doubling dilutions hereof were used. The haemagglutination titres were 16 and 128, respectively. When the bacteria were exposed to colistin before mixing with the red cells, haemagglutination was inhibited completely with 1.0 and 0.5 microgram/mg of colistin. At a colistin concentration of 0.25 microgram/ml (1/4-1/2 of the MIC's) the titres were lowered by a factor of 16-32.

  7. DNA test--a forensic boon.

    PubMed

    Mohanty, Nayan Kishore; Haldar, Swaraj

    2006-02-01

    DNA fingerprinting (DNAFP) profiles can be applied to identify an individual in criminal as well as in civil cases. The main advantage of the technique is its ability to analyse small and environmentally challenged samples and to accurately establish their origins with a high degree of certainty: DNAFP can identify an individual in criminal and civil cases eg, rape, kidnapping, assassination and so on. The forensic DNA analysis can be stored in a data bank. It can help in crime prevention by giving the information of potential criminals. Reliability of DNA technique is very important. In paternity and maternity identification, when called for, DNA testing plays very much positive role.

  8. A DNA test to sex ratite birds.

    PubMed

    Huynen, Leon; Millar, Craig D; Lambert, David M

    2002-04-01

    DNA-based sex tests now exist for many avian species. However, none of these tests are widely applicable to ratites. We present DNA sequence data for a locus that is W chromosome-linked in the kiwi, ostrich, cassowary, rhea, and emu. At the amino acid level, this sequence has significant homology to X-linked genes in platyfish and Caenorhabditis elegans. Polymerase chain reaction (PCR) primers designed to this locus allow the assignment of sex in all species of living ratites.

  9. Haemagglutination-inhibiting antibodies against arboviruses of the families Togaviridae and Bunyaviridae in birds caught in southern Moravia, Czechoslovakia.

    PubMed

    Juricová, Z; Hubálek, Z; Halouzka, J; Pellantová, J; Chytil, J

    1987-01-01

    A total of 295 birds belonging to 19 species of 7 families of wild Passeriformes were examined by haemagglutination-inhibition test. The birds were caught for an international research program "Balt" at the time of autumn migration (August-September 1984). Their blood sera were examined for antibodies against 6 arbovirus antigens of the genera Alphavirus (Sindbis-SIN) and Flavivirus (tick-borne encephalitis-TBE, West Nile-WN) and family Bunyaviridae (Tahyna-TAH, Calovo-CVO and Bhanja-BHA). Antibodies against all studied viruses were detected at different frequencies: SIN 6.4%, TBE 7.1%, WN 9.7%, TAH 16.3%, CVO 12.1%, and BHA 1.0%.

  10. Apoptosis of granulocytes and lymphocytes in peripheral blood in rabbits infected with haemagglutinating and non-haemagglutinating antigenic variants of the RHD (rabbit haemorrhagic disease) virus.

    PubMed

    Niedźwiedzka-Rystwej, P; Tokarz-Deptuła, B; Deptuła, W

    2013-01-01

    This paper attempts to study the dynamics of apoptosis of granulocytes and lymphocytes in peripheral blood in rabbits infected with haemagglutinating (Vt97, Triptis, Hartmannsdorf) and non-haemagglutinating (Pv97, 9905 RHDVa) antigenic variants of the RHD virus. The pathogenicity of those antigenic variants was also assessed by recording the mortality of the infected animals. The animals were infected with antigenic variants and blood was sampled at hour 0, 4, 8, 12, 24, 36 p.i. and the percentage of apoptotic granulocytes and lymphocytes was measured with the use of flow cytometry. The results of the study showed that apoptosis is included during RHDV infection, as the number of apoptotic granulocytes and lymphocytes increases throughout the experiment; depending on the antigenic variant, apoptosis joins in at 4-8-12 h p.i. and lasts until 24-36 h p.i. Furthermore, the mortality of rabbits infected with the examined strains of RHD virus varied from 30% to 100%. This study performed for the first time in this manner, indicates the importance of apoptosis during infection with the RHD virus.

  11. [Microsatellite DNA analysis as a tool for forensic paternity testing (DNA paternity testing)].

    PubMed

    Veselinović, Igor

    2006-01-01

    MICROSATELLITE ANALYSIS: By using serological or HLA-testing, the alleged father can be excluded as the biological father, but, regardless of the degree of probability, positive paternity results cannot be obtained without DNA testing. According to the results of the National Human Genome Project, human genome consists of approximately 30.000 genes. The vast majority of human DNA is not organized in genes and has no genetic expression or visible function. Non-coding DNA contains genetic markers important for human identification. Short tandem repeats, or STRs, are a class of microsatellites consisting of tandemly repeated sequences of 2 to 6 base pair length monomers. Most of the microsatellites show a high degree of polymorphism, which can be evaluated by PCR technique, and used in criminalistics, forensic identification and parentage testing. A source of DNA in parentage testing are blood samples or buccal swabs which are routinelly used. Amplification of isolated DNA can be performed in 25-30 cycles by PCR, and fragments are separated by capillary electrophoresis. The probability of paternity of 99.99% or higher corresponds to the paternity "practically proven", indicating that the alleged father is the biological father. Such results can be obtained only by DNA testing. DNA-testing laboratories are required to conduct validation of laboratory facilities, equipment and staff and are subject to permanent control by the society.

  12. Comparative evaluation of haemagglutination potential of haemolymph from two species of giant African land snails (Archachatina marginata and Achatina achatina).

    PubMed

    Abiona, John Adesanya; Akinduti, Paul Akinniyi; Oyekunle, Mufutao Atanda; Osinowo, Olusegun Ayodeji; Onagbesan, A Okanlawon Mohammed

    2014-05-01

    A comparative study was conducted to evaluate haemagglutination potential in the haemolymph of two species of giant African land snails (Archachatina marginata and Achatina achatina). Three liveweight groups of snails (<100 g, 101-150 g and >150 g) were used with 4 replicates per liveweight per species for haemagglutination assay (HA). The effect of aestivation on haemagglutination potential was also evaluated. Erythrocytes (2%) from cattle, sheep, goat and chicken were used for HA assay. Results showed that agglutinin-like substances that agglutinate erythrocytes of sheep, goat, cattle and chicken were present in the haemolymph of the two species of giant African land snails. Effect of species was found to be significant (P < 0.001) on haemagglutination titre. Haemolymph of A. marginata, had higher haemagglutination titre than that of A. achatina across the three liveweight groups used in this study. Snail liveweight had no significant effect (P > 0.05) on agglutinin content of the haemolymph in both species. Agglutination level depended on the source of erythrocyte used. Sheep erythrocyte recorded the highest haemagglutination titre, followed by goat, cattle, and chicken in that order. To our knowledge, this is the first evidence that Giant African land snails (GALS) haemolymph contain agglutinins as previously reported for Helix species. This evidence may be the basis for its survivability in the wild and thus establish the use of GALS for African herbal medicinal applications.

  13. Haemagglutination, haemolysin production and serum resistance of proteus and related species isolated from clinical sources.

    PubMed

    Mishra, M; Thakar, Y S; Pathak, A A

    2001-01-01

    A total of 148 strains of Proteus and related species comprising of Proteus mirabilis (116), Proteus vulgaris (24), Providentia rettgeri (4), Providentia alcalifaciens (2), Providentia stuarti (1) and Morganella morganii (1), isolated from various sources, were examined for haemagglutination (HA), haemolysin production (HL) and serum resistance (SR). Maximum isolates were obtained from urine (47.30%) and pus (40.54%) and they were multidrug resistant. The sensitivity to Ciprofloxacin was 78.38%, Gentamicin: 62.84%, Cefotaxime: 29.73%, Norfloxacin: 22.97%, Tetracycline: 20.95% and Ampicillin: 6.76%. There were four commonest resistance patterns shown by 58.62% of Proteus mirabilis and 66.67% of Proteus vulgaris strains. Haemagglutination was shown by 91 (61.49%) strains, HL production in 126 (85.14%) strains and SR by 124 (83.78%) isolates. All the three i.e. HA, HL and SR were simultaneously present in 77 (52.27%) strains, any two were present in 40 (27.03%) strains and any one was positive in 30 (20.03%) strains. Thus in as many as 147 (98.32%) isolates, any one or more virulence factors were present. The virulence in commensal pathogen like Proteus is basically a multifactorial phenomenon. The presence of more virulence factors in one strain may increase its pathogenic ability. The evaluation of multiple virulence factors instead of one single parameter will be of greater help in assessing its pathogenic potential.

  14. A novel multitarget stool DNA test for colorectal cancer screening.

    PubMed

    Malik, Pramod

    2016-01-01

    Review of: Imperiale TF, Ransohoff DF, Itzkowitz SH, Levin TR, Lavin P, Lidgard GP, Ahlquist DA, Berger BM. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med 2014;370(14):1287-97. This Practice Pearl reviews the results of a prospective, multicenter, cross-sectional clinical study that evaluated the performance of a new multitarget stool DNA (or mt-sDNA) screening test for colorectal cancer (CRC) and compared it with a fecal immunochemical test (FIT) in individuals at average risk for CRC. The potential impact of this test on the future of CRC screening is also discussed in a brief commentary. mt-sDNA testing is a noninvasive screening test designed to detect DNA biomarkers associated with colorectal neoplasia and occult hemoglobin in the stool. The sensitivity of mt-sDNA testing for detection of CRC was 92.3%, compared with 73.8% for FIT (p = 0.002). Sensitivity for detecting advanced precancerous lesions was 42.4% for mt-sDNA testing and 23.8% for FIT (p < 0.001). The specificities of mt-sDNA testing and FIT were 86.6% and 94.9%, respectively (p < 0.001). mt-sDNA testing thus may be a first-line screening option for asymptomatic individuals at average risk for CRC who do not want to have a colonoscopy.

  15. Performance of 14 rubella IgG immunoassays on samples with low positive or negative haemagglutination inhibition results.

    PubMed

    Huzly, Daniela; Hanselmann, Ingeborg; Neumann-Haefelin, Dieter; Panning, Marcus

    2016-01-01

    Rubella IgG testing is routinely done in prenatal care and seroepidemiological studies. Recently concern was raised that seropositivity rates were decreasing questioning vaccination policies. Manufacturers of rubella IgG assays and authors of seroepidemiological studies use different cut-offs for the definition of seropositivity. As rubella virus circulation is reduced since many years, seronegativity rates might be overestimated using an inappropriate cut-off. Using different cut-off definitions we compared fourteen current rubella IgG immunoassays for sensitivity and qualitative result concordance in samples with low positive or negative haemagglutination inhibition (HI) titre. 150 clinical samples from patients and health care workers were included in the study. All samples were measured in 14 different rubella IgG immunoassays. Seropositivity was defined using recombinant rubella IgG immunoblot as reference standard. The concordance of qualitative results using the manufacturers cut-off definitions was 56.4% if grey-zone results were analysed separately and 69.8% if grey-zone results were defined as positive. Using universal cut-offs of 10 IU/ml or 15 IU/ml the concordance was 70% and 61.4% respectively. Using the different cut-off definitions up to 71 out of the 124 immunoblot-positive samples tested negative in the immunoassays. The mean coefficient of variation (CV) of quantitative results in positive samples was 51% (range 19-113%). Determination of rubella immunity by measurement of rubella-IgG in a population with high vaccination coverage with current assays leads to a high number of false negative results. The value of routine rubella antibody testing in countries with high vaccination coverage should be discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Haemagglutination-inhibiting activity to type A influenzaviruses in the sera of wild birds from the far east of the USSR

    PubMed Central

    Slepuškin, A. N.; Pysina, T. V.; Gonsovsky, F. K.; Sazonov, A. A.; Isačenko, V. A.; Sokolova, N. N.; Polivanov, V. M.; Lvov, D. K.; Zakstel'skaja, L. Ja.

    1972-01-01

    The discovery in migrating birds of influenzaviruses and the demonstration of relationships between strains found in man and in birds show the importance of these investigations for influenzavirus ecology. Investigation of birds in the far-east regions of the USSR is particularly important as many of them migrate to South-East Asia and China, the regions from which human influenza pandemics seem to originate. A total of 262 bird sera from these regions were titrated by the HI method with 20 different influenzavirus antigens (5 human, 2 equine, 1 swine, and 12 avian influenzaviruses). Haemagglutination-inhibiting activity was found in 16.8% of the tested sera, most frequently in sera of Anas formosa, Gallinago gallinago, Anas falcata, and Larus crassirostris. HI activity to A/turkey/Wisconsin/66 (Hav6N2) was found in 7.3% of the sera. PMID:4541006

  17. DNA testing of sexual assault evidence: the laboratory perspective.

    PubMed

    Burg, Abby; Kahn, Roger; Welch, Katherine

    2011-09-01

    The availability of DNA testing has dramatically changed the way that crimes are investigated. DNA results can link offenders to their crimes, exonerate wrongfully accused individuals, identify mass fatality victims and more. In the case of sexual assault, DNA evidence alone cannot prove that a sexual assault has occurred. DNA analysis can only reveal whether a person's DNA is, or is not, present. In this paper, the authors provide readers with an overview of the advantages and limitations of DNA analysis, the importance of proper evidence collection, the technologies available, and the amount of sample needed for testing. Through proper evidence collection and quality laboratory services, the full value of DNA will be realized.

  18. 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.

  19. DNA nanotechnology from the test tube to the cell

    NASA Astrophysics Data System (ADS)

    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.

  20. 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.

  1. A blind testing design for authenticating ancient DNA sequences.

    PubMed

    Yang, H; Golenberg, E M; Shoshani, J

    1997-04-01

    Reproducibility is a serious concern among researchers of ancient DNA. We designed a blind testing procedure to evaluate laboratory accuracy and authenticity of ancient DNA obtained from closely related extant and extinct species. Soft tissue and bones of fossil and contemporary museum proboscideans were collected and identified based on morphology by one researcher, and other researchers carried out DNA testing on the samples, which were assigned anonymous numbers. DNA extracted using three principal isolation methods served as template in PCR amplifications of a segment of the cytochrome b gene (mitochondrial genome), and the PCR product was directly sequenced and analyzed. The results show that such a blind testing design performed in one laboratory, when coupled with phylogenetic analysis, can nonarbitrarily test the consistency and reliability of ancient DNA results. Such reproducible results obtained from the blind testing can increase confidence in the authenticity of ancient sequences obtained from postmortem specimens and avoid bias in phylogenetic analysis. A blind testing design may be applicable as an alternative to confirm ancient DNA results in one laboratory when independent testing by two laboratories is not available.

  2. 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.

  3. Post-conviction DNA testing: the UK's first 'exoneration' case?

    PubMed

    Johnson, P; Williams, R

    2004-01-01

    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.

  4. 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.

  5. 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.

  6. Highly efficient and minimally invasive in-vivo gene transfer to the mouse uterus using haemagglutinating virus of Japan (HVJ) envelope vector.

    PubMed

    Nakamura, Hitomi; Kimura, Tadashi; Ikegami, Hiroyuki; Ogita, Kazuhide; Koyama, Shinsuke; Shimoya, Koichiro; Tsujie, Tomoko; Koyama, Masayasu; Kaneda, Yasufumi; Murata, Yuji

    2003-10-01

    The uterus is obviously critical in implantation, development of the fetus and parturition. Endometrial cancer derived from endometrial epithelium is one of the common malignancies in the female reproductive tract. In order to clarify the local mechanisms of reproductive physiology and establish a non-systemic therapeutic strategy for reproductive failure as well as for endometrial cancer, we applied haemagglutinating virus of Japan envelope (HVJ-E) vector to in-vivo gene transfer into the uterine cavity of IVCS mice. Injection of HVJ-E vector into mouse uterine cavity on day 1.5 post coitum (p.c.) introduced a reporter gene approximately 120-fold more efficiently than introduction using the cationic liposome method. The expression of the introduced gene continued for at least 3 days. The plasmid vector was localized in the endometrial epithelium, whereas oligo deoxynucleotides were distributed throughout the epithelium, stromal cells and myometrium. HVJ-E vector did not affect the pregnancy rate, course of pregnancy, litter size, fetal growth in utero or parturition, and did not transfect the exogenous gene to the fetus. These results indicate that gene transfer into the uterus using HVJ-E vector is highly efficient and safe during pregnancy, and results in a well controlled distribution of the exogenous DNA. We believe that this procedure should be widely applicable for investigations of reproductive physiology as well as for methods of local gene therapy in the uterus.

  7. 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.

  8. Neuraminidase-mediated haemagglutination of recent human influenza A(H3N2) viruses is determined by arginine 150 flanking the neuraminidase catalytic site.

    PubMed

    Mögling, Ramona; Richard, Mathilde J; Vliet, Stefan van der; Beek, Ruud van; Schrauwen, Eefje J A; Spronken, Monique I; Rimmelzwaan, Guus F; Fouchier, Ron A M

    2017-06-01

    Over the last decade, an increasing proportion of circulating human influenza A(H3N2) viruses exhibited haemagglutination activity that was sensitive to neuraminidase inhibitors. This change in haemagglutination as compared to older circulating A(H3N2) viruses prompted an investigation of the underlying molecular basis. Recent human influenza A(H3N2) viruses were found to agglutinate turkey erythrocytes in a manner that could be blocked with either oseltamivir or neuraminidase-specific antisera, indicating that agglutination was driven by neuraminidase, with a low or negligible contribution of haemagglutinin. Using representative virus recombinants it was shown that the haemagglutinin of a recent A(H3N2) virus indeed had decreased activity to agglutinate turkey erythrocytes, while its neuraminidase displayed increased haemagglutinating activity. Viruses with chimeric and mutant neuraminidases were used to identify the amino acid substitution histidine to arginine at position 150 flanking the neuraminidase catalytic site as the determinant of this neuraminidase-mediated haemagglutination. An analysis of publicly available neuraminidase gene sequences showed that viruses with histidine at position 150 were rapidly replaced by viruses with arginine at this position between 2005 and 2008, in agreement with the phenotypic data. As a consequence of neuraminidase-mediated haemagglutination of recent A(H3N2) viruses and poor haemagglutination via haemagglutinin, haemagglutination inhibition assays with A(H3N2) antisera are no longer useful to characterize the antigenic properties of the haemagglutinin of these viruses for vaccine strain selection purposes. Continuous monitoring of the evolution of these viruses and potential consequences for vaccine strain selection remains important.

  9. Apoptosis of peripheral blood leukocytes from rabbits infected with non-haemagglutinating strains of rabbit haemorrhagic disease virus (RHDV).

    PubMed

    Niedźwiedzka-Rystwej, Paulina; Deptuła, Wiesław

    2012-09-15

    The report demonstrates that the induction of apoptosis in peripheral blood granulocytes and lymphocytes of rabbits infected with three non-haemagglutinating RHDV strains (English Rainham, German Frankfurt, and Spanish Asturias) is a crucial determinant of the pathogenesis of rabbit haemorrhagic disease. Apoptosis was measured by flow cytometric detection of caspase activity. These studies demonstrated that the investigated RHDV (rabbit haemorrhagic disease virus) viral strains affected leukocyte apoptosis to varying degrees. Enhanced leukocyte apoptosis was detected between 4 and 36 h after infection and was more pronounced in lymphocytes than in granulocytes. The data presented here thus provide a preliminary understanding of the kinetics of apoptosis in leukocytes of rabbits infected with RHDV.

  10. DNA testing and molecular screening for colon cancer.

    PubMed

    Carethers, John M

    2014-03-01

    Colon cancer develops and progresses as a consequence of abnormal cellular molecular changes, many of which result in mutant DNA. Modern molecular techniques allow examination of individual patient genetic data that ascribe risk, predict outcome, and/or modify an approach to therapy. DNA testing and molecular screening are in use today and are becoming a critical and necessary part of routine patient care. Assessing at-risk patients for hereditary colon cancer is predicted to move from individual gene testing that is commonly performed today to whole exome or whole genome sequencing, providing additional vast information of the patient's genome that might not be related to the colon cancer syndrome. Detecting mutant DNA from shed tumor cells in fecal material for colon cancer screening will increase in diagnostic accuracy over time, with improvements in the panel of mutant DNA being examined and through clinical testing. DNA mutations and other molecular changes detected directly from within the colon cancer help to inform and guide the physician for the best approach for optimal patient care and outcome. The use of epidermal growth factor receptor-targeted therapy in advanced colon cancer patients requires knowledge of the mutation status for KRAS and BRAF genes, and knowing the mutational status of PIK3CA may predict how patients respond to aspirin to prevent colon cancer recurrence. Biologically driven decision-making, or precision medicine, is becoming increasingly adopted for optimal care and outcome for colon cancer patients. Gastroenterologists will need to be increasingly aware.

  11. A comparison of a blocking ELISA and a haemagglutination inhibition assay for the detection of antibodies to Avibacterium (Haemophilus) paragallinarum in sera from artificially infected chickens.

    PubMed

    Sun, H; Miao, D; Zhang, P; Gong, Y; Blackall, P J

    2007-10-01

    The ability of blocking ELISAs and haemagglutination-inhibition (HI) tests to detect antibodies in sera from chickens challenged with either Avibacterium (Haemophilus) paragallinarum isolate Hp8 (serovar A) or H668 (serovar C) was compared. Serum samples were examined weekly over the 9 weeks following infection. The results showed that the positive rate of serovar A specific antibody in the B-ELISA remained at 100% from the second week to the ninth week. In chickens given the serovar C challenge, the highest positive rate of serovar C specific antibody in the B-ELISA appeared at the seventh week (60% positive) and was then followed by a rapid decrease. The B-ELISA gave significantly more positives at weeks 2, 3, 7, 8 and 9 post-infection for serovar A and at week 7 post-infection for serovar C. In qualitative terms, for both serovar A and serovar C infections, the HI tests gave a lower percentage of positive sera at all time points except at 9 weeks post-infection with serovar C. The highest positive rate for serovar A HI antibodies was 70% of sera at the fourth and fifth weeks post-infection. The highest rate of serovar C HI antibodies was 20% at the fifth and sixth weeks post-infection. The results have provided further evidence of the suitability of the serovar A and C B-ELISAs for the diagnosis of infectious coryza.

  12. The Interpretation of Lineage Markers in Forensic DNA Testing

    PubMed Central

    Buckleton, J.S.; Krawczak, M.; Weir, B.S.

    2011-01-01

    Mitochondrial DNA (mtDNA) and the non-recombining portion of the Y chromosome are inherited matrilinealy and patrilinealy, respectively, and without recombination. Collectively they are termed ‘lineage markers’. Lineage markers may be used in forensic testing of an item, such as a hair from a crime scene, against a hypothesised source, or in relationship testing. An estimate of the evidential weight of a match is usually provided by a count of the occurrence in some database of the mtDNA or Y-STR haplotype under consideration. When the factual statement of a count in the database is applied to a case, issues of relevance of the database and sampling uncertainty may arise. In this paper, we re-examine the issues of sampling uncertainty, the relevance of the database, and the combination of autosomal and lineage marker evidence. We also review the recent developments by C.H. Brenner. PMID:21397888

  13. 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...

  14. NIST physical standards for DNA-based medical testing.

    PubMed

    Barker, Peter E; Watson, Michael S; Ticehurst, John R; Colbert, Jennifer C; O'Connell, Catherine D

    2002-01-01

    As DNA and RNA become major targets for clinical laboratory analysis, benchmark reagents will play an increasingly important role in standardization. Reliable national and international nucleic acid standards promote automation and third-party reimbursement for clinical testing. Furthermore, nucleic acid standards provide materials for quality assurance and quality control (QA/QC), and proficiency testing. Standard methods and training initially evolved from consensus guidelines endorsed by professional societies and governmental agencies. The National Institute of Standards and Technology (NIST), a nonregulatory agency of the U.S. Department of Commerce, develops and certifies physical and chemical standards in support of national commerce, manufacturing, and science. In its role supporting U.S. science and industry, the NIST responds to specific standards needs, most recently for medically and biologically important analytes. Broad-based consensus developed through interdisciplinary NIST workshops initiated development of NIST-certified DNA standards. Such materials serve the diagnostic community and help manufacturers benchmark a variety of DNA diagnostic testing platforms. Here we summarize the NIST experience and programs for development of national standards for DNA-based medical diagnostic testing. Copyright 2002 Wiley-Liss, Inc.

  15. Molecular medicine: a primer for clinicians--Part VIII: Forensic DNA testing.

    PubMed

    1995-02-01

    Forensic DNA analysis or "DNA fingerprinting" represents a specific application of DNA testing methods. Previously, we have discussed DNA testing to determine carrier status or to provide confirmatory or presymptomatic diagnosis. Forensic DNA testing seeks to determine the identity of an individual to the exclusion of all others. The most common application of forensic DNA testing is in criminalistics and establishing parentage. In this paper we discuss the application of molecular biology methods to the analysis of DNA for forensic purposes. We also consider some of the controversial issues that surround such uses of DNA testing.

  16. DNA testing for malignant hyperthermia: the reality and the dream.

    PubMed

    Stowell, Kathryn M

    2014-02-01

    The advent of the polymerase chain reaction and the availability of data from various global human genome projects should make it possible, using a DNA sample isolated from white blood cells, to diagnose rapidly and accurately almost any monogenic condition resulting from single nucleotide changes. DNA-based diagnosis for malignant hyperthermia (MH) is an attractive proposition, because it could replace the invasive and morbid caffeine-halothane/in vitro contracture tests of skeletal muscle biopsy tissue. Moreover, MH is preventable if an accurate diagnosis of susceptibility can be made before general anesthesia, the most common trigger of an MH episode. Diagnosis of MH using DNA was suggested as early as 1990 when the skeletal muscle ryanodine receptor gene (RYR1), and a single point mutation therein, was linked to MH susceptibility. In 1994, a single point mutation in the α 1 subunit of the dihydropyridine receptor gene (CACNA1S) was identified and also subsequently shown to be causative of MH. In the succeeding years, the number of identified mutations in RYR1 has grown, as has the number of potential susceptibility loci, although no other gene has yet been definitively associated with MH. In addition, it has become clear that MH is associated with either of these 2 genes (RYR1 and CACNA1S) in only 50% to 70% of affected families. While DNA testing for MH susceptibility has now become widespread, it still does not replace the in vitro contracture tests. Whole exome sequence analysis makes it potentially possible to identify all variants within human coding regions, but the complexity of the genome, the heterogeneity of MH, the limitations of bioinformatic tools, and the lack of precise genotype/phenotype correlations are all confounding factors. In addition, the requirement for demonstration of causality, by in vitro functional analysis, of any familial mutation currently precludes DNA-based diagnosis as the sole test for MH susceptibility. Nevertheless

  17. Genotoxicity of refinery waste assessed by some DNA damage tests.

    PubMed

    Gupta, Amit Kumar; Ahmad, Irshad; Ahmad, Masood

    2015-04-01

    Refinery waste effluent is well known to contain polycyclic aromatic hydrocarbons, phenols and heavy metals as potentially genotoxic substances. The aim of the present study was to assess the genotoxic potential of Mathura refinery wastewater (MRWW) by various in vitro tests including the single cell gel electrophoresis, plasmid nicking assay and S1 nuclease assay. Treatment of human lymphocytes to different MRWW concentrations (0.15×, 0.3×, 0.5× and 0.78×) caused the formation of comets of which the mean tail lengths increased proportionately and differed significantly from those of unexposed controls. The toxic effect of MRWW on DNA was also studied by plasmid nicking assay and S1 nuclease assay. Strand breaks formation in the MRWW treated pBR322 plasmid confirmed its genotoxic effect. Moreover, a dose dependent increase in cleavage of calf thymus DNA in S1 nuclease assay was also suggestive of the DNA damaging potential of MRWW. A higher level of ROS generation in the test water sample was recorded which might be contributing to its genotoxicity. Interaction between the constituents of MRWW and calf thymus DNA was also ascertained by UV-visible spectroscopy. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. The Diagnostic Performance of Stool DNA Testing for Colorectal Cancer

    PubMed Central

    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

    2016-01-01

    Abstract 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. PMID:26844449

  19. DNA barcoding of the recently evolved genus Holcoglossum (Orchidaceae: Aeridinae): a test of DNA barcode candidates.

    PubMed

    Xiang, Xiao-Guo; Hu, Hao; Wang, Wei; Jin, Xiao-Hua

    2011-11-01

    Orchidaceae is one of the largest families of flowering plants. Many species of orchid are endangered, and all species are included in Conventions on International Trade of Endangered Species of Fauna and Flora (CITES) I and II, but it is very difficult to identify orchid species, even those with fertile parts. The genus Holcoglossum (Orchidaceae: Aeridinae) has long been problematic in taxonomy. It consists of both long-evolved and radiated species and is an excellent case to use for testing DNA barcodes for Orchidaceae. We investigated the power of a subset of proposed plant barcoding loci [rbcL, matK, atpF-atpH, psbK-psbI, trnH-psbA and internal transcribed spacer (ITS)] to discriminate between species in this genus. Our results showed that all these DNA regions, except psbK-psbI and atpF-atpH, can be amplified easily from Holcoglossum and sequenced with established primers. The DNA regions matK and ITS had the highest variability. Among the six loci, matK resolved eight of the 12 Holcoglossum species and had the highest discriminatory ability. However, the combination of matK and ITS showed a greater ability to identify species than matK alone. Single or combined DNA markers discriminated between Holcoglossum species distributed in tropical areas effectively, but had less ability to identify radiated species from the temperate Hengduan Mountains of China. In the study, matK proved to be a useful DNA barcode for the genus Holcoglossum; however, complementary DNA regions are still required to accelerate the investigation and preservation of radiated species of orchid. © 2011 Blackwell Publishing Ltd.

  20. The feasibility of external blind DNA proficiency testing. II. Experience with actual blind tests.

    PubMed

    Peterson, Joseph L; Lin, George; Ho, Monica; Chen, Yingyu; Gaensslen, R E

    2003-01-01

    The background and goals of a national study to determine the feasibility of blind proficiency testing in U.S. forensic DNA laboratories are discussed. Part of the project involved designing and executing a series of fifteen blind proficiency tests. Execution included biological specimen donor recruitment and case evidence manufacturing. Simulated cases were submitted to DNA laboratories by law enforcement agencies and in some cases by other forensic-science laboratories. Replicate-manufactured evidence was submitted to reference laboratories to simulate the workings of a larger-scale program. Ten tests were straightforward, and essentially tested analytical ability. Five tests involved selecting on the basis of case facts appropriate bloodstains for typing from a bloodstain pattern. We describe in detail our experience in designing and conducting these blind proficiency test trials, and relate those experiences to the overall issue of blind proficiency testing as a quality-assurance tool in forensic DNA laboratories. In this feasibility test series, one blind test was detected by a laboratory, a second one was shown to the lab by law enforcement, and a third was never completed because of lapses in communication. Turnaround times were relatively fast in the independent/commercial labs and relatively slow in the larger public laboratories. Two cross-state case-to-case CODIS "hits" were "planted" among the first series of ten blind tests. One pair was detected. One member of the second pair went to a lab that was not CODIS-ready.

  1. Testing evolutionary hypotheses for DNA barcoding failure in willows.

    PubMed

    Twyford, Alex D

    2014-10-01

    The goal of DNA barcoding is to enable the rapid identification of taxa from short diagnostic DNA sequence profiles. But how feasible is this objective when many evolutionary processes, such as hybridization and selective sweeps, cause alleles to be shared among related taxa? In this issue of Molecular Ecology, Percy et al. (2014) test the full suite of seven candidate plant barcoding loci in a broad geographic sample of willow species. They show exceptional plastid haplotype sharing between species across continents, with most taxa not possessing a unique barcode sequence. Using population genetic and molecular dating analyses, they implicate hybridization and selective sweeps, but not incomplete lineage sorting, as the historical processes causing widespread haplotype sharing among willow taxa. This study represents an exceptional case of how poorly barcoding can perform, and highlights methodological issues using universal organellar regions for species identification.

  2. Recommendations for animal DNA forensic and identity testing.

    PubMed

    Budowle, Bruce; Garofano, Paolo; Hellman, Andreas; Ketchum, Melba; Kanthaswamy, Sree; Parson, Walther; van Haeringen, Wim; Fain, Steve; Broad, Tom

    2005-09-01

    Genetic analysis in animals has been used for many applications, such as kinship analysis, for determining the sire of an offspring when a female has been exposed to multiple males, determining parentage when an animal switches offspring with another dam, extended lineage reconstruction, estimating inbreeding, identification in breed registries, and speciation. It now also is being used increasingly to characterize animal materials in forensic cases. As such, it is important to operate under a set of minimum guidelines that assures that all service providers have a template to follow for quality practices. None have been delineated for animal genetic identity testing. Based on the model for human DNA forensic analyses, a basic discussion of the issues and guidelines is provided for animal testing to include analytical practices, data evaluation, nomenclature, allele designation, statistics, validation, proficiency testing, lineage markers, casework files, and reporting. These should provide a basis for professional societies and/or working groups to establish more formalized recommendations.

  3. Copro-DNA tests for diagnosis of animal taeniid cestodes.

    PubMed

    Mathis, Alexander; Deplazes, Peter

    2006-01-01

    PCR has proven its value for the diagnosis of taeniid cestodes in animal definitive hosts, although only few specific tests are available at the moment (Echinococcus multilocularis, Echinococcus granulosus 'sheep strain'). Additional tests with specificities for further taeniids are urgently needed and new tests are currently being developed, e.g. a multiplex PCR for simultaneous detection of E. multilocularis, E. granulosus (all strains) and Taenia spp. (all species). PCR is a technically demanding and expensive technique: DNA isolation from faecal specimens remains a laborious task because of the presence of PCR-inhibitory substances, and special precautions need to be taken to avoid false-positive results due to cross-contamination of amplification reactions. PCR, therefore, is mainly used for confirmative purposes of coproantigen-positive samples or for identification of taeniid eggs recovered from faecal specimens or from environmental samples.

  4. 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.

  5. 78 FR 58514 - Availability of an Environmental Assessment for Field Testing of a DNA Immunostimulant

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-24

    ... of a DNA Immunostimulant AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION: Notice of... then to field test, an unlicensed DNA Immunostimulant recommended for reduction in morbidity and.... Product: DNA Immunostimulant. Possible Field Test Locations: Texas, Mississippi, and Georgia for poultry...

  6. Newcastle disease viruses in birds in the Atlantic flyway: isolations, haemagglutination-inhibition and elution-inhibition antibody profiles.

    PubMed

    Graves, I L

    1996-01-01

    The study involved 15 avian species with 5,012 attempts to isolate Newcastle disease viruses (NDV) from their faeces over a three-year period (1977-1979). NDV were isolated from asymptomatic adult Canada geese, nestling Royal terms, a juvenile European Mute swan, and adult Tundra swans on the Eastern flyway. Ring-billed gulls were negative for haemagglutination-inhibition (HI), elution-inhibition (EI) (anti-neuraminidase) antibodies, and NDV despite 3,403 isolation attempts. The EI antibody assay used a strain isolated from a Mute swan. The prevalence of EI antibodies in the swan and geese ranged from 3-41%, while the geometric mean titre (GMT) varied from 20-36. The prevalence of HI antibodies in the swans and geese ranged from 4-62%, while the GMT varied from 16-42. In each of three years (1977-1979), the adult Mute swans had a higher HI antibody prevalence than the juveniles (P < 0.01). Among the Mute swans the HI and EI assays detected serologic conversions and persistent antibodies over the three-year period. The HI and EI assays were effective in showing differences in antibody prevalences in populations of feral birds of different species and age. The EI assay is applicable for population studies of the anti-neuraminidase antibody.

  7. Recognition of glycoconjugates by Helicobacter pylori. Comparison of two sialic acid-dependent specificities based on haemagglutination and binding to human erythrocyte glycoconjugates.

    PubMed

    Miller-Podraza, H; Bergström, J; Milh, M A; Karlsson, K A

    1997-06-01

    Helicobacter pylori expresses separate binding characteristics depending on growth conditions, as documented by binding to human erythrocyte glycoconjugates. Cells grown in Ham's F12 liquid medium exhibited a selective sialic acid-dependent binding to polyglycosylceramides, PGCs (Miller-Podraza et al.(1996) Glycoconjugate J13:453-60). There was no binding to traditional sialylated glycoconjugates like shorter-chain gangliosides, glycophorin or fetuin. However, cells grown on Brucella agar bound both to PGCs and other sialylated glycoconjugates. Fetuin was an effective inhibitor of haemagglutination caused by agar-grown cells, but had no or a very weak inhibitory effect on haemagglutination by F12-grown bacteria. PGCs were strong inhibitors in both cases, while asialofetuin was completely ineffective. The results indicate that H. pylori is able to express two separate sialic acid-dependent specificities, one represented by binding to fetuin, as described before, and another represented by a selective binding to PGCs.

  8. White and red blood cell picture in rabbits experimentally infected with strains of the rabbit haemorrhagic disease (RHD) virus without or with variable haemagglutination capacity.

    PubMed

    Niedźwiedzka-Rystwej, P; Tokarz-Deptuła, B; Deptuła, W

    2016-12-01

    The aim of the study was to establish if haemagglutination of rabbit haemorrhagic disease virus (RHDV) affects haematological picture of peripheral blood in rabbits and the pathogenicity of the virus. The study analyzed white and red blood cell picture in rabbits experimentally infected with two non-haemagglutinating (HA-) RHDV strains (Frankfurt and Asturias) and one strain with variable haemagglutination capacity (HA+/-) (Hagenow). Studies with HA- and HA +/- are rare and relate only to 4 HA- strains (2 RHDV: BLA and Rainham; 2 RHDVa: Pv97 and 9905) and 1 HA+/- RHDV strain: ŻD, where less changes in haematological indices and less pathogenicity were observed. We found that changes caused by HA- Frankfurt strain were related to the number of neutrophils and thrombocytes, while in HA- strain Asturias, in thrombocytes and leukocytes. Changes evoked by HA+/- Hagenow strain pertained to the number of eosinophils, thrombocytes, leukocytes, monocytes, and concentration of hemoglobin. Mortality caused by the Frankfurt strain was 100% between 36 and 48 h post infection (p.i.), while that caused by Asturias strain was 100% between 24 and 36 h p.i., and that observed in case of Hagenow strain was 90% between 36 and 48 h p.i. The changes in haematological picture caused by the HA- and HA+/- RHDV strains were less intensive than those found in case of the HA+ RHDV strains, which cannot be confirmed for pathogenicity, and is not in line with the existing hypothesis suggesting higher pathogenicity in HA+ viruses.

  9. Verification of sex from harvested sea otters using DNA testing

    USGS Publications Warehouse

    Scribner, K.T.; Green, B.A.; Gorbics, C.; Bodkin, J.

    2005-01-01

    We used molecular genetic methods to determine the sex of 138 sea otters (Enhydra lutris) harvested from 3 regions of Alaska from 1994 to 1997, to assess the accuracy of post-harvest field-sexing. We also tested each of a series of factors associated with errors in field-sexing of sea otters, including male or female bias, age-class bias, regional bias, and bias associated with hunt characteristics. Blind control results indicated that sex was determined with 100% accuracy using polymerase chain reaction (PCR) amplification using primers that co-amplify the zinc finger-Y-X gene, located on both the mammalian Y- and X-chromosomes, and Testes Determining Factor (TDF), located on the mammalian Y-chromosome. DNA-based sexing revealed that 12.3% of the harvested sea otters were incorrectly sexed in the field, with most errors (13 of 17) occurring as males incorrectly reported as females. Thus, female harvest was overestimated. Using logistic regression analysis, we detected no statistical association of incorrect determination of sex in the field with age class, hunt region, or hunt type. The error in field-sexing appears to be random, at least with respect to the variables evaluated in this study.

  10. Lies, damned lies, and DNA statistics: DNA match testing Bayes' Theorem, and the criminal courts.

    PubMed

    Jowett, C

    2001-07-01

    This study explains the correct method for the interpretation of DNA matches by using Bayesian Probability, and its various traps: the DNA fallacies. The current approach of the Court of Appeal to the use and presentation of DNA evidence is outlined. A new Bayesian Fallacy is explored. Specific focus is on Norman Fenton and Martin Neil's recent research (Fenton and Neil, 2000) which suggests a new dimension in the presentation of DNA evidence in court.

  11. Gynecologists and human papillomavirus DNA testing: exploring knowledge, attitudes, and practice in Italy.

    PubMed

    Caglioti, Claudia; Pileggi, Claudia; Nobile, Carmelo G A; Pavia, Maria

    2016-11-22

    The aim of this survey was to examine the knowledge, attitudes, and behavior of gynecologists in terms of human papillomavirus (HPV) DNA testing as a primary screening tool for cervical cancer. A national cross-sectional web survey was carried out through multistage sampling using an overall sample of 1000 gynecologists. Gynecologists were asked to fill in a self-administered questionnaire exploring their knowledge, attitudes, and practice toward cervical cancer screening and HPV-DNA testing. A total of 582 gynecologists completed the web questionnaire. Of these, 24.5% were uncertain on the higher sensitivity of HPV-DNA compared with the Pap test, whereas 19% were uncertain on the role of the HPV-DNA test as a primary test in women younger than 30 years old and only 44.9% knew that a negative HPV-DNA test allows for an extension of the test interval to 5 years. Most gynecologists showed a definite positive attitude on the role of screening for cervical cancer prevention and were prepared to accept new technologies. The HPV-DNA test was considered highly effective by 86.9%, whereas 94% recommend/perform HPV-DNA tests in women older than 30 years of age; 25.5% performed HPV-DNA as a primary test, followed by a Pap test in those cases that were positive. Only 56.3% recommended/performed HPV-DNA tests 1 year after a positive HPV-DNA test, followed by a negative Pap test, whereas 42.9% recommended colposcopy. Although the use of the HPV-DNA test is very widespread among Italian gynecologists performing cervical cancer screening, there is lack of standardization of practices according to current guidelines.

  12. Genotoxicity and carcinogenicity testing of pharmaceuticals: correlations between induction of DNA lesions and carcinogenic activity.

    PubMed

    Brambilla, Giovanni; Mattioli, Francesca; Robbiano, Luigi; Martelli, Antonietta

    2010-01-01

    This survey is a compendium of the results of DNA lesions assays (DNA adducts, DNA strand breaks, DNA repair synthesis) and of the results of carcinogenicity assays of 146 pharmaceuticals. Of these drugs, 55 (37.7%) tested negative in both DNA lesions assay(s) and in carcinogenicity assay(s); 65 (44.5%) tested negative in DNA lesions assay(s), but gave a positive response in at least one carcinogenicity assay; 6 (4.1%) tested positive in at least one DNA lesions assay, but negative in carcinogenicity assay(s); 20 (13.7%) tested positive in at least one DNA lesions assay and in at least one carcinogenicity assay. Concerning the predictivity of DNA lesions assays findings for the results of long-term carcinogenesis assays performed in mice, rats or other species, concordance was found to exist for the 46.2% of pharmaceuticals in the case of DNA adducts, for 63.1% in the case of DNA strand breaks, and for 47.3% in the case of DNA repair synthesis (UDS). 2010 Elsevier B.V. All rights reserved.

  13. A Comparative Study of Tests for Homogeneity of Variances with Application to DNA Methylation Data

    PubMed Central

    Li, Xuan; Qiu, Weiliang; Morrow, Jarrett; DeMeo, Dawn L.; Weiss, Scott T.; Fu, Yuejiao; Wang, Xiaogang

    2015-01-01

    Variable DNA methylation has been associated with cancers and complex diseases. Researchers have identified many DNA methylation markers that have different mean methylation levels between diseased subjects and normal subjects. Recently, researchers found that DNA methylation markers with different variabilities between subject groups could also have biological meaning. In this article, we aimed to help researchers choose the right test of equal variance in DNA methylation data analysis. We performed systematic simulation studies and a real data analysis to compare the performances of 7 equal-variance tests, including 2 tests recently proposed in the DNA methylation analysis literature. Our results showed that the Brown-Forsythe test and trimmed-mean-based Levene's test had good performance in testing for equality of variance in our simulation studies and real data analyses. Our results also showed that outlier profiles could be biologically very important. PMID:26683022

  14. DNA methylation testing and marker validation using PCR: diagnostic applications.

    PubMed

    Egger, Gerda; Wielscher, Matthias; Pulverer, Walter; Kriegner, Albert; Weinhäusel, Andreas

    2012-01-01

    DNA methylation provides a fundamental epigenetic mechanism to establish and promote cell-specific gene-expression patterns, which are inherited by subsequent cell generations. Thus, the epigenome determines the differentiation into a cell lineage but can also program cells to become abnormal or malignant. In humans, different germline and somatic diseases have been linked to faulty DNA methylation. In this article, we will discuss the available PCR-based technologies to assess differences in DNA methylation levels mainly affecting 5-methylcytosine in the CpG dinucleotide context in hereditary syndromal and somatic pathological conditions. We will discuss some of the current diagnostic applications and provide an outlook on how DNA methylation-based biomarkers might provide novel tools for diagnosis, prognosis or patient stratification for diseases such as cancer.

  15. 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…

  16. 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…

  17. [Selection of suitable polypropylene tubes for DNA testing using real-time PCR].

    PubMed

    Shimizu, Eri; Futo, Satoshi; Masubuchi, Tomoko; Minegishi, Yasutaka; Kasahara, Masaki; Akiyama, Hiroshi; Teshima, Reiko; Hino, Akihiro; Mano, Jyunichi; Furui, Satoshi; Kitta, Kazumi

    2010-01-01

    Polypropylene microtubes (tubes) are generally used for bio-material tests in addition to PCR tests such as genetically modified organism (GMO) testings. However, the choice of suitable tubes is quite important, because it might influence the results: DNA binding and/or elution of chemical substances sometimes occurs. In this study, we established methods to select tubes with the most suitable characteristics for DNA testing.

  18. Effect of presumptive tests reagents on human blood confirmatory tests and DNA analysis using real time polymerase chain reaction.

    PubMed

    de Almeida, Juliana Piva; Glesse, Nadine; Bonorino, Cristina

    2011-03-20

    Bloodstains often constitute the major physical evidence in crime investigation. Diluted blood invisible to the naked eye can be detected through presumptive tests however such tests can damage samples and prevent further processing such as DNA analysis. In this study, we compared the effects of luminol (prepared according to Weber [15]), Luminol 16(®), Bluestar(®) and benzidine for inhibition in the human antiglobulin test and the human hemoglobin immunochromatographic test and on the total human DNA concentration up to 120 days after sample treatment. Treatment with both luminol solutions and Bluestar(®) still allowed positive results for the immunologic tests, indicating non-interference with human blood confirmatory tests. However, samples treated with benzidine could not be further analyzed by serological tests. Also, DNA quantification showed that 48h after benzidine treatment, but not luminol or Bluestar solution application, sample DNA was degraded. Luminol 16(®) caused DNA degradation already at 30 days post-application. At 120 days post treatment, all samples treated with any of the agents but not untreated samples had DNA degradation. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  19. Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing

    PubMed Central

    Medina Diaz, Inga; Nocon, Annette; Mehnert, Daniel H.; Fredebohm, Johannes; Diehl, Frank; Holtrup, Frank

    2016-01-01

    Objectives Making liquid biopsy testing widely available requires a concept to ship whole blood at ambient temperatures while retaining the integrity of the cell-free DNA (cfDNA) population and stability of blood cells to prevent dilution of circulating tumor DNA (ctDNA) with wild-type genomic DNA. The cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) were evaluated to determine if they can be utilized in combination with highly sensitive mutation detection technologies. Methods Venous blood from healthy donors or patients with advanced colorectal cancer (CRC) was collected in cfDNA BCTs and standard K2EDTA tubes. Tubes were stored at different temperatures for various times before plasma preparation and DNA extraction. The isolated cfDNA was analyzed for overall DNA yield of short and long DNA fragments using qPCR as well as for mutational changes using BEAMing and Plasma Safe-Sequencing (Safe-SeqS). Results Collection of whole blood from healthy individuals in cfDNA BCTs and storage for up to 5 days at room temperature did not affect the DNA yield and mutation background levels (n = 60). Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from CRC patients collected in cfDNA BCTs were reliably detected after 3 days of storage at room temperature. However, blood samples stored at ≤ 10°C and at 40°C for an extended period of time showed elevated normal genomic DNA levels and an abnormally large cellular plasma interface as well as lower plasma volumes. Conclusion Whole blood shipped in cfDNA BCTs over several days can be used for downstream liquid biopsy testing using BEAMing and Safe-SeqS. Since the shipping temperature is a critical factor, special care has to be taken to maintain a defined room temperature range to obtain reliable mutation testing results. PMID:27832189

  20. 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.

  1. Clinical value of DNA fragmentation evaluation tests under ART treatments.

    PubMed

    Tavukçuoğlu, Ilkay Şafak; Al-Azawi, Tahani; Khaki, Amir Afshin; Khaki, Arash; Khalil, Ahmed; Al-Hasani, Safaa

    2012-01-01

    Male reproductive health has been under scrutiny recently. Many studies in the literature have concluded that semen quality is declining and that the incidence of testicular cancers is increasing. The reason for this change has been attributed to damage in sperm chromatin. During in vivo reproduction, the natural selection process ensures that only a spermatozoon with normal genomic material can fertilize an oocyte. However, the assisted reproduction technique (ART) is our selection process, leading to the possibility that abnormal spermatozoa could be used to fertilize an oocyte. We could avoid this by quantifying the amount and type of genomic damage in sperm using well-accepted laboratory methods. The sperm deoxyribonucleic acid (DNA) integrity is important for success of natural or assisted fertilization as well as normal development of the embryo, fetus and child. Intra cytoplasmic sperm injection (ICSI) is bypassing natural sperm selection mechanisms, which increases the risk of transmitting damaged DNA. The significance of required investigations and multiple techniques is that they could evaluate DNA defects in human spermatozoa. The ability of these techniques to accurately estimate sperm DNA damage depends on many technical and biological aspects. The aim of this review is to evaluate the most commonly used methods.

  2. 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

  3. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.

    PubMed

    Fernández, Jose Luis; Muriel, Lourdes; Rivero, Maria Teresa; Goyanes, Vicente; Vazquez, Rosana; Alvarez, Juan G

    2003-01-01

    Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test

  4. 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.

  5. 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.

  6. [DNA synthesis inhibition test of INAH by cultured human fibroblasts].

    PubMed

    Nishio, K; Yanagisawa, K

    1986-03-20

    The most commonly used screening test of carcinogens is the Ames test. But this system occasionally shows false positive and false negative. Painter's method is one which has been developed to minimize false results. Now we test by Painter's method isonicotinic acid hydrazide, which shows negative in the Ames test but positive in an animal test. INAH showed positive by Painter's method. More chemicals are now under study for their carcinogenicity by Painter's method.

  7. Diffusion of DNA Testing in the Immigration Process

    DTIC Science & Technology

    2009-12-01

    identification, DNA has the potential to illuminate otherwise hidden aspects of congenital health issues, ancestry, and racial composition, which is socially...are going have a sweeping vision that is going to resolve everything all at once. He suggests that is bad government and, although he was a political...persons of all ages, regardless of physical impairment . Probably DNA’s greatest value is its utility in verifying family relationships that are the

  8. An improved test for Africanized honeybee mitochondrial DNA.

    PubMed

    Crozier, Y C; Koulianos, S; Crozier, R H

    1991-09-15

    Mitochondrial DNA derived from Apis mellifera scutellata, the ancestor of the Africanized bees of the New World, lacks a BglII restriction site found in other types of honeybee. We present primers allowing amplification of a 485-bp section of the cytochrome b gene containing this site, using the polymerase chain reaction. Digestion of the amplified product with BglII yields contrasting patterns between Africanized and other honeybees.

  9. Stool DNA Testing to Screen for Colorectal Cancer in the Medicare Population

    PubMed Central

    Lansdorp-Vogelaar, Iris; Kuntz, Karen M.; Knudsen, Amy B.; Wilschut, Janneke A.; Zauber, Ann G.; van Ballegooijen, Marjolein

    2013-01-01

    Background Centers for Medicare and Medicaid Services (CMS) considered whether to reimburse stool DNA testing for colorectal cancer screening among Medicare enrollees. Objective To evaluate the conditions under which stool DNA testing could be cost-effective compared with the colorectal cancer screening tests currently reimbursed by CMS. Design Comparative microsimulation modeling study using two independently-developed models. Data Sources Derived from literature. Target Population 65-year-old (Medicare eligible) individuals; 50-year old individuals as sensitivity analysis. Time Horizon Lifetime. Perspective Third-party payer. Interventions Stool DNA test every 3 or 5 years in comparison to currently-recommended colorectal cancer screening strategies. Outcome Measures Life expectancy, lifetime costs, incremental cost-effectiveness ratios, threshold costs. Results of Base-Case Analysis Assuming a cost of $350 per test, strategies of stool DNA testing every 3 or 5 years yielded fewer life-years and higher costs than the currently recommended colorectal cancer screening strategies. Results of Threshold Analysis Screening with the stool DNA test would be cost-effective at per-test cost of $40 to $60 for 3-yearly stool DNA testing, depending on the simulation model used. There were no levels of sensitivity and specificity for which stool DNA testing would be cost-effective at its current cost of $350 per test. Stool DNA testing at 3-yearly intervals would be cost-effective at a cost of $350 per test if the relative adherence with stool DNA testing were at least 50% better than with other screening tests. Results of Sensitivity Analysis None of the above mentioned results changed significantly when considering a 50-year old cohort. Limitations We did not model other pathways than the traditional adenoma-carcinoma sequence. Conclusions Only if a significant reduction can be made to the test cost or if its availability would entice a large fraction of otherwise unscreened

  10. A Retrospective Approach to Testing the DNA Barcoding Method

    PubMed Central

    Chapple, David G.; Ritchie, Peter A.

    2013-01-01

    A decade ago, DNA barcoding was proposed as a standardised method for identifying existing species and speeding the discovery of new species. Yet, despite its numerous successes across a range of taxa, its frequent failures have brought into question its accuracy as a short-cut taxonomic method. We use a retrospective approach, applying the method to the classification of New Zealand skinks as it stood in 1977 (primarily based upon morphological characters), and compare it to the current taxonomy reached using both morphological and molecular approaches. For the 1977 dataset, DNA barcoding had moderate-high success in identifying specimens (78-98%), and correctly flagging specimens that have since been confirmed as distinct taxa (77-100%). But most matching methods failed to detect the species complexes that were present in 1977. For the current dataset, there was moderate-high success in identifying specimens (53-99%). For both datasets, the capacity to discover new species was dependent on the methodological approach used. Species delimitation in New Zealand skinks was hindered by the absence of either a local or global barcoding gap, a result of recent speciation events and hybridisation. Whilst DNA barcoding is potentially useful for specimen identification and species discovery in New Zealand skinks, its error rate could hinder the progress of documenting biodiversity in this group. We suggest that integrated taxonomic approaches are more effective at discovering and describing biodiversity. PMID:24244283

  11. 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.

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

    PubMed Central

    2011-01-01

    Background 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. Methods 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. Results 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. Conclusion 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Belgian recommendations on ANA, anti-dsDNA and anti-ENA antibody testing.

    PubMed

    Van Blerk, M; Bossuyt, X; Humbel, R; Mewis, A; Servais, G; Tomasi, J P; Van Campenhout, C; Van Hoovels, L; Vercammen, M; Damoiseaux, J; Coucke, W; Van de Walle, P

    2014-04-01

    Autoantibodies to nuclear antigens, i.e. antinuclear antibodies (ANA), antibodies to double-stranded DNA (dsDNA) and extractable nuclear antigens (ENA), are useful as diagnostic markers for a variety of autoimmune diseases. In March 2010, the Belgian national External Quality Assessment Scheme sent a questionnaire on ANA, anti-dsDNA and anti-ENA antibody testing designed by the Dutch EASI (European Autoimmunity Standardization Initiative) team, to all clinical laboratories performing ANA testing. Virtually all laboratories completed the questionnaire (97·7%, 127/130). This paper discusses the results of this questionnaire and provides valuable information on the state-of-the-art of ANA, anti-dsDNA and anti-ENA antibody testing as practiced in the Belgian laboratories. In addition, this work presents practical recommendations developed by the members of the advisory board of the scheme as a result of the outcome of this study.

  18. 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.

  19. Application of Optical Fibers to DNA’s Testing Program.

    DTIC Science & Technology

    1980-10-15

    underground testing environment. The success of these early measurements has developed confidence in fibers to the point of fielding non-redundant fiber optic...large quantities of data required, many highly developed technologies often unique to this program, are needed to ensure a successful test. Further...cable. For example, the radiation dose rates vary from approximately 1013 rad/ sec (prompt gamma) at 3 meters from the source to background at the

  20. Methods of DNA adduct determination and their application to testing compounds for genotoxicity.

    PubMed

    Phillips, D H; Farmer, P B; Beland, F A; Nath, R G; Poirier, M C; Reddy, M V; Turteltaub, K W

    2000-01-01

    At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25-26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by (32)P-postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case-by-case basis, depending on the compound's profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance. Copyright 2000 Wiley-Liss, Inc.

  1. 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.

  2. An improved method for the diatom test utilizing DNA binding ability of silica.

    PubMed

    Seo, Yasuhisa; Ichida, Daisuke; Sato, Shingo; Kuroki, Kohji; Kishida, Tetsuko

    2014-05-01

    In order to devise a better forensic test for diatoms, the DNA binding ability of the diatom frustule constructing by silica, in the presence of chaotropic ions were utilized. It was proved that the diatoms were able to be captured via λDNA using silica-coated magnetic beads (Mag beads), followed by isolation and purification from the Mag beads as a solid phase by substituting the chaotropic agent with ultrapure water. Five cases of drowning, three in freshwater and two in seawater, were applied to the present method and similar results as the usual diatom test were obtained. Specimens of lung and other organs were rendered clearly visible, with elimination of foreign impurities. The present method appears applicable for detection of diatoms indirectly using PCR amplification of bound DNA or directly staining of the DNA. © 2014 American Academy of Forensic Sciences.

  3. Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay

    SciTech Connect

    Delahunty, C.; Ankener, W.; Deng, Qiang

    1996-06-01

    The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a calorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed. 62 refs., 2 figs., 4 tabs.

  4. Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay.

    PubMed Central

    Delahunty, C.; Ankener, W.; Deng, Q.; Eng, J.; Nickerson, D. A.

    1996-01-01

    The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a colorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled; and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed. Images Figure 2 PMID:8651301

  5. 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

  6. 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.

  7. Comparative testing of DNA segmentation algorithms using benchmark simulations.

    PubMed

    Elhaik, Eran; Graur, Dan; Josic, Kresimir

    2010-05-01

    Numerous segmentation methods for the detection of compositionally homogeneous domains within genomic sequences have been proposed. Unfortunately, these methods yield inconsistent results. Here, we present a benchmark consisting of two sets of simulated genomic sequences for testing the performances of segmentation algorithms. Sequences in the first set are composed of fixed-sized homogeneous domains, distinct in their between-domain guanine and cytosine (GC) content variability. The sequences in the second set are composed of a mosaic of many short domains and a few long ones, distinguished by sharp GC content boundaries between neighboring domains. We use these sets to test the performance of seven segmentation algorithms in the literature. Our results show that recursive segmentation algorithms based on the Jensen-Shannon divergence outperform all other algorithms. However, even these algorithms perform poorly in certain instances because of the arbitrary choice of a segmentation-stopping criterion.

  8. Hypo-osmotic swelling test identifies individual spermatozoa with minimal DNA fragmentation.

    PubMed

    Stanger, James D; Vo, Long; Yovich, John L; Almahbobi, Ghanim

    2010-10-01

    One concern during intracytoplasmic sperm injection (ICSI) is that selected spermatozoa may have increased levels of DNA damage; however, the available testing for this is largely destructive in nature and therefore unsuitable as a tool for sperm selection. One alternative selection process that has previously achieved pregnancies is the hypo-osmotic swelling test (HOST). This study reports that low HOST values of neat semen samples were significantly (P<0.001) associated with increased DNA damage identified by the DNA fragmentation index (DFI) from the sperm chromatin structure assay as well as the TdT-mediated dUTP nick-end labelling (TUNEL) assay. The HOST value was highly predictive of an abnormal DFI value by receiver operating characteristic curve analysis (P<0.001). Furthermore, when individual spermatozoa were assessed for both HOST status and DNA fragmentation by TUNEL, the key HOST-induced tail-swelling grades D, E and F were most commonly associated with high HOST values and were significantly (P<0.001) associated with minimal DNA damage regardless of the DNA status of the ejaculate. The application of HOST may be a valuable tool in the routine identification and selection of viable, DNA-intact individual spermatozoa for ICSI after further research to demonstrate its efficacy and safety. Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  9. Development of a diagnostic test for Johne's disease using a DNA hybridization probe.

    PubMed Central

    Hurley, S S; Splitter, G A; Welch, R A

    1989-01-01

    A DNA probe, M13 mpHAW71, that detects Mycobacterium paratuberculosis in the fecal material of infected animals was developed for use in the diagnosis of Johne's disease. The probe detected as few as 10(5) M. paratuberculosis when hybridized under stringent conditions to total genomic DNA purified from bovine fecal material. When the probe was used diagnostically, it did not differentiate members of the Mycobacterium avium-M. intracellulare-M. paratuberculosis complex. Compared with culturing, the DNA probe identified 34.4% more mycobacterium-containing fecal samples, and testing took only 72 h to complete. Images PMID:2768445

  10. 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...

  11. Sperm chromatin dispersion test in the assessment of DNA fragmentation and aneuploidy in human spermatozoa.

    PubMed

    Balasuriya, A; Speyer, B; Serhal, P; Doshi, A; Harper, J C

    2011-05-01

    Sperm DNA damage is thought to be increased in men with male factor infertility. Previous studies suggest a correlation between sperm DNA fragmentation and aneuploidy. The sperm chromatin dispersion (SCD) test was modified to produce the Halosperm Kit. The SCD-fluorescent in-situ hybridization (FISH) test allows the simultaneous detection of DNA fragmentation and aneuploidy on the same sperm cell. The objectives of this study were to validate the SCD, SCD-FISH and Halosperm tests for the analysis of sperm DNA fragmentation and compare them to the sperm chromatin structure assay (SCSA). Semen samples from 20 males undergoing IVF/intracytoplasmic sperm injection were processed using FISH, SCD-FISH, SCD and Halosperm, and compared with SCSA results. There was a significant difference between FISH and SCD-FISH results in the detection of aneuploidy (P=0.000) and the level of sperm DNA fragmentation in the samples subjected to SCSA and SCD (P=0.001) or SCSA and SCD-FISH (P=0.001). There was no significant correlation between DNA fragmentation and aneuploidy. If sperm aneuploidy is to be determined, more reliable results will be obtained if FISH is performed rather than SCD-FISH. A lack of validation and unknown clinical significance question the value of DNA fragmentation assays. DNA damage in the male germ line may result in adverse clinical outcomes and the pathophysiology and clinical consequences of sperm DNA damage are being actively researched. Many DNA fragmentation assays such as the Halosperm Kit have been developed recently and are now available at a commercial level. Unfortunately, aimed at vulnerable couples with difficulty conceiving, many of these tests have not been clinically validated. Despite its plausible appeal and fervour of its supporters, the benefits of widespread DNA testing that only achieves the distressing of couples with the knowledge that effectual therapeutic strategies are absent are questionable. Commercially, however, it is no doubt

  12. Comparative evaluation of Amplicor HIV-1 DNA test, version 1.5, by manual and automated DNA extraction methods using venous blood and dried blood spots for HIV-1 DNA PCR testing.

    PubMed

    Nsojo, Anthony; Aboud, Said; Lyamuya, Eligius

    2010-10-01

    Human immunodeficiency virus (HIV) DNA polymerase chain reaction (PCR) test using venous blood sample has been used for many years in low resource settings for early infant diagnosis of HIV infection in children less than 18 months. The aim of this study was to evaluate and compare the performance characteristics of Amplicor HIV-1 DNA assay version 1.5 following processing of venous blood and dried blood spot (DBS) samples by Roche manual DNA extraction and automated Roche MagNA Pure LC instrument (MP) for HIV-1 DNA PCR testing in Dar es Salaam, Tanzania, in order to scale up early infant diagnosis of HIV infection in routine practice. Venous blood samples from children under 18 months born to HIV-infected mothers between January and April 2008 were collected. Venous blood was used to prepare cell pellet and DBS samples. DNA extractions by manual procedure and MP were performed each on cell pellet, venous blood and DBS samples and tested by Amplicor HIV-1 DNA assay. Of 325 samples included, 60 (18.5%) were confirmed HIV-infected by manual extraction performed on cell pellets. Sensitivity of the assay following MP processing of venous blood was 95% (95% CI; 86.1-99.0%) and 98.3% (95% CI; 91.1 to 99.9%) for the manual extraction and processing by MP performed on DBS samples. Specificity of the assay with all DNA extraction methods was 99.6% (95% CI; 97.9 to 100%). Performance of the assay with Roche manual extraction and processing by MP on DBS samples compared well with Roche manual extraction performed on cell pellet samples. The choice of DNA extraction method needs to be individualized based on the level of laboratory facility, volume of testing and cost benefit analysis before it is adopted for use.

  13. HPV DNA testing of the residual sample of liquid-based Pap test: utility as a quality assurance monitor.

    PubMed

    Zuna, R E; Moore, W; Dunn, S T

    2001-03-01

    HPV DNA testing of the residual sample volume of liquid-based Pap tests has been recommended as a way to determine the appropriate follow-up for women who have equivocal results in routine clinical screening. A major aspect of quality assurance in the cytopathology laboratory consists of correlation of smear interpretation with biopsy or conization results as mandated by CLIA '88. However, the use of histology as the gold standard suffers from similar problems of subjectivity and sampling as the Pap smear. In this study we explore the potential use of HPV DNA testing of the residual volume from the ThinPrep Pap Test (Cytyc Corporation, Boxborough, Massachusetts) as a substitute gold standard in quality assurance monitoring of a cervical cytology screening program. The residual samples from 397 ThinPrep Pap cases were retrospectively analyzed for high-risk HPV DNA using the Hybrid Capture II technique. Sensitivity (71.8%), specificity (86.5%), predictive value of positive (77.1%) and negative (82.9%) ThinPrep Pap interpretations were calculated on the basis of HPV DNA results for 266 cases classed as either squamous intraepithelial lesion (SIL) or negative. Overall, there was agreement between the two tests in 80.8% of cases (Cohen's kappa =.59). The percentage of HPV DNA-positive cases interpreted as atypical squamous cells of uncertain significance (ASCUS) was 43.7%, and the percentage of negative cases was 17.1%. We believe that this approach is an objective adjunct to the traditional quality assurance protocol, with the added benefit that it includes cases interpreted as negative, as well as abnormal cases that do not come to biopsy.

  14. The effect of luminol on presumptive tests and DNA analysis using the polymerase chain reaction.

    PubMed

    Gross, A M; Harris, K A; Kaldun, G L

    1999-07-01

    This study was designed to test the following factors involved with processing luminol treated bloodstained evidence: 1) The reactivity of other presumptive chemical color tests, phenolphthalin (PT) and tetramethylbenzidine (TMB), following the application of the light emitting luminol presumptive test. 2) The effect of different cleanings of various bloody substrates on the luminol test. 3) The effect of different cleanings of various bloody substrates on the ability to obtain DNA suitable for PCR testing. 4) The ability to extract DNA from luminol treated bloodstained substrates using three extraction techniques. 5) The effect of spraying washed and unwashed bloodstains on various substrates with luminol on the ability to correctly type the DNA using PCR. Our findings indicated that luminol did not adversely effect the PCR testing and did not interfere with the PT and TMB presumptive tests for blood. It was determined that the substrate and the method of cleaning were the major factors affecting DNA yield and the ability to type the bloodstains using PCR based technologies.

  15. [Parvovirus B19 DNA testing in Polish blood donors, 2004-2010].

    PubMed

    Grabarczyk, Piotr; Korzeniowska, Jolanta; Liszewski, Grzegorz; Kalińska, Aleksandra; Sulkowska, Ewa; Krug-Janiak, Maria; Kopacz, Aneta; Łetowska, Magdalena; Brojer, Ewa

    2012-01-01

    Since 2004 Polish blood donors have been tested for parvovirus B19 (B19V) DNA. The screening testing has been performed in donors of plasma for fractionation and anti-D and anti-HBs production and donors of erythrocytes used for immunization. AIM is to present methods of the testing, quality control and results in period 2004-2010. Testing was performed in individual donation testing (IDT) in Regional Blood Transfusion Center (RBTC) in Lublin or in pools of 24 in Institute of Haematology and Transfusion Medicine in Warsaw (IHTM). Quantitative testing with real-time PCR was preceded with nucleic acid isolation on silica based methods (Prepito Viral DNA/RNA, Chemagen and QIAamp DNA Mini Kit, QIAGEN). Amplification was performed initially with home made method and later with commercial assay (Artus Parvo B19 RG PCR Kit on Rotor Gene 6 000). In total 17 625 donations were tested: 8 539 in pools and 9 090 individually. Beside routine external quality control programmes in which both laboratories participated (Proficiency Study VQC,Amsterdam, Holand; EQA Programe, Glasgow, Scotland), panel containing negative samples, positive with very high DNA B 19V level and plasma infected with genotype 2 was prepared for RBTC in Lublin. B19V infection frequency was 1:980 donations, low viraemic donations were detected most frequently (1:1 037). It was identified only one donation with DNA load that could cause potential health risk for plasma product recipients (1:17 625). In one of the donors B 19V DNA was observed for 3 years and 3 months. In acute or persistent phase of infection no clinical or laboratory symptoms (morphology of peripheral blood, ALT) were observed. Due to risk of underestimation of viral load connected with viral genome polymorphism all donations with B19V positive result were not allowed to be clinically used.

  16. Genetic citizenship: DNA testing and the Israeli Law of Return.

    PubMed

    McGonigle, Ian V; Herman, Lauren W

    2015-07-01

    The Israeli State recently announced that it may begin to use genetic tests to determine whether potential immigrants are Jewish or not. This development would demand a rethinking of Israeli law on the issue of the definition of Jewishness. In this article, we discuss the historical and legal context of secular and religious definitions of Jewishness and rights to immigration in the State of Israel. We give a brief overview of different ways in which genes have been regarded as Jewish, and we discuss the relationship between this new use of genetics and the society with which it is co-produced. In conclusion, we raise several questions about future potential impacts of Jewish genetics on Israeli law and society.

  17. Genetic citizenship: DNA testing and the Israeli Law of Return

    PubMed Central

    McGonigle, Ian V.; Herman, Lauren W.

    2015-01-01

    The Israeli State recently announced that it may begin to use genetic tests to determine whether potential immigrants are Jewish or not. This development would demand a rethinking of Israeli law on the issue of the definition of Jewishness. In this article, we discuss the historical and legal context of secular and religious definitions of Jewishness and rights to immigration in the State of Israel. We give a brief overview of different ways in which genes have been regarded as Jewish, and we discuss the relationship between this new use of genetics and the society with which it is co-produced. In conclusion, we raise several questions about future potential impacts of Jewish genetics on Israeli law and society. PMID:27774208

  18. The feasibility of external blind DNA proficiency testing. I. Background and findings.

    PubMed

    Peterson, Joseph L; Lin, George; Ho, Monica; Chen, Yingyu; Gaensslen, R E

    2003-01-01

    We describe the origins, purposes, and findings of a national study to determine whether a large-scale program of blind proficiency testing in U.S. DNA laboratories is feasible and/or practical. Proficiency testing in clinical laboratories is reviewed, particularly as mandated by the Clinical Laboratory Improvement Acts and its role in the regulation of those laboratories. Proficiency testing in forensic urine drug testing labs is also briefly reviewed. Studies involving comparisons between open and blind proficiency testing are discussed. The clinical laboratory proficiency testing and regulation landscape provides the background for the DNA Act of 1994, and the congressional mandate to investigate blind proficiency testing in forensic DNA laboratories. Four models of blind proficiency testing are defined and discussed, along with the advantages and disadvantages of each and estimates of the costs of a large-scale program. The purposes of proficiency testing in a quality-assurance context are likewise discussed and related to the models and the arguments generally proffered for and against blind vs. open proficiency testing.

  19. Value and feasibility of HPV DNA test in cervical scraping smears.

    PubMed

    Wu, Sufang; Chen, Gang; Wang, Wei; Xu, Qian; Gu, Hainian; Lu, Yunping; Zhou, Liping; Du, Juan; Li, Fujun; Liao, Guoning; Ma, Ding

    2005-01-01

    To investigate the reliability and feasibility of human papillomavirus (HPV) DNA test in cervical scraping smears with polymerase chain reaction (PCR), 131 cases of cervical scraping specimens were collected, and the positive rates and accuracy of HPV infection were determined in normal subjects and cervical cancer patients. GP5+/GP6+ and E7 primer pairs designed for detecting HPV L1 and HPV type 16 E7 were tested in this study. Our results showed that positive rates of HPV DNA in normal population and cervical cancer patients were 32.99% and 73.53% respectively and there was significant difference between them (P < 0.001). In normal subjects, detection rates of HPV DNA with GP5+/GP6+ and E7 primer pairs were 27.84% and 16.49% respectively, with statistically significant difference between them (P > 0.05). However the detection rates in cervical cancer patients were 38.24% and 67.65% for the two markers, with a significant difference found between them (P < 0.05). It is concluded that HPV DNA test with PCR for cervical scraping smears was feasible. GP5+/GP6+ primer pairs may be a useful probe to screen HPV infection in normal population, but they are not sensitive enough in cervical cancer patients. It is suggested that high risk type HPV DNA test was very useful in population with high risk of cervical cancer.

  20. 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.

  1. An evaluation of the ToxHA test for the detection of antibodies to Toxoplasma gondii in human serum.

    PubMed Central

    Balfour, A H; Bridges, J B; Harford, J P

    1980-01-01

    The titre of antibodies to Toxoplasma gondii was compared in 1985 sera by use of an indirect haemagglutination test (ToxHA test--Wellcome Reagents Ltd) and the dye test. Sera from 42 patients with clinical or serological indications of recent toxoplasmosis were also examined to determine if the ToxHA test would be of value in detecting early stages of the disease. Nine recent cases of lymphadenopathy and four cases of eye infection were not detected by the test. In addition, a large number of sera found negative in the dye test gave a positive result in the haemagglutination test. PMID:7430370

  2. DNA vaccine encoding avian influenza virus H5 and Esat-6 of Mycobacterium tuberculosis improved antibody responses against AIV in chickens.

    PubMed

    Oveissi, Sara; Omar, Abdul Rahman; Yusoff, Khatijah; Jahanshiri, Fatemeh; Hassan, Sharifah Syed

    2010-12-01

    The H5 gene of avian influenza virus (AIV) strain A/chicken/Malaysia/5744/2004(H5N1) was cloned into pcDNA3.1 vector, and Esat-6 gene of Mycobacterium tuberculosis was fused into downstream of the H5 gene as a genetic adjuvant for DNA vaccine candidates. The antibody level against AIV was measured using enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI) test. Sera obtained from specific-pathogen-free chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5/Esat-6 demonstrated antibody responses as early as 2 weeks after the first immunization. Furthermore, the overall HI antibody titer in chickens immunized with pcDNA3.1/H5/Esat-6 was higher compared to the chickens immunized with pcDNA3.1/H5 (p<0.05). The results suggested that Esat-6 gene of M. tuberculosis is a potential genetic adjuvant for the development of effective H5 DNA vaccine in chickens. Crown Copyright © 2009. Published by Elsevier Ltd. All rights reserved.

  3. Detection of Colorectal Serrated Polyps by Stool DNA Testing: Comparison with Fecal Immunochemical Testing for Occult Blood (FIT)

    PubMed Central

    Heigh, Russell I.; Yab, Tracy C.; Taylor, William R.; Hussain, Fareeda T. N.; Smyrk, Thomas C.; Mahoney, Douglas W.; Domanico, Michael J.; Berger, Barry M.; Lidgard, Graham P.; Ahlquist, David A.

    2014-01-01

    Objectives Precursors to 1/3 of colorectal cancer (CRC), serrated polyps have been under-detected by screening due to their inconspicuous, non-hemorrhagic, and proximal nature. A new multi-target stool DNA test (multi-target sDNA) shows high sensitivity for both CRC and advanced adenomas. Screen detection of serrated polyps by this approach requires further validation. We sought to assess and compare noninvasive detection of sessile serrated polyps (SSP) ≥1 cm by sDNA and an occult blood fecal immunochemical test (FIT). Methods In a blinded prospective study, a single stool sample used for both tests was collected from 456 asymptomatic adults prior to screening or surveillance colonoscopy (criterion standard). All 29 patients with SSP≥1 cm were included as cases and all 232 with no neoplastic findings as controls. Buffered stool samples were processed and frozen on receipt; Exact Sciences performed sDNA in batches using optimized analytical methods. The sDNA multi-marker panel targets methylated BMP3 (mBMP3) and NDRG4, mutant KRAS, β-actin, and hemoglobin. FIT (Polymedco OC-FIT Check) was performed in separate lab ≤2 days post defecation and evaluated at cutoffs of 50 (FIT-50) and 100 ng/ml (FIT-100). Results Median ages: cases 61 (range 57–77), controls 62 (52–70), p = NS. Women comprised 59% and 51%, p = NS, respectively. SSP median size was 1.2 cm (1–3 cm), 93% were proximal, and 64% had synchronous diminutive polyps. Among multi-target sDNA markers, mBMP3 proved highly discriminant for detection of SSP≥1 cm (AUC = 0.87, p<0.00001); other DNA markers provided no incremental sensitivity. Hemoglobin alone showed no discrimination (AUC = 0.50, p = NS). At matched specificities, detection of SSP≥1 cm by stool mBMP3 was significantly greater than by FIT-50 (66% vs 10%, p = 0.0003) or FIT-100 (63% vs 0%, p<0.0001). Conclusions In a screening and surveillance setting, SSP≥1 cm can be detected noninvasively by stool assay of

  4. Molecular organisation of the genes involved in the production of F7(2) fimbriae, causing mannose-resistant haemagglutination, of a uropathogenic Escherichia coli 06:K2:H1:F7 strain.

    PubMed

    van Die, I; van Megen, I; Hoekstra, W; Bergmans, H

    1984-01-01

    The genes responsible for the formation of the F7 (2) fimbriae of the uropathogenic E. coli strain AD110 (O6:K2:H1: F7 ) have been cloned on the recombinant plasmid pPIL110 -35 (Van Die et al. 1983). The F7 (2) fimbriae, like the F7 (1) fimbriae of AD110 , are responsible for mannose resistant haemagglutination ( MRHA ). The molecular organisation of the genes of pPIL110 -35 involved in the expression of MRHA was studied by: (a) analysis of transposon gamma delta and Tn5 insertion mutants. Mutations that cause an MRHA -deficient phenotype were located in discrete groups within an 11.5 kb restriction fragment of pPIL110 -35, separated by insertion mutations that do not inactivate MRHA . (b) complementation experiments. Restriction fragments of pPIL110 -35 subcloned in the vector pBR322 were tested for their ability to complement transposon insertion mutations in the corresponding regions of pPIL110 -35. Five complementation groups were distinguished. Five genes (designated A-E) involved in the expression of MRHA can be distinguished by these results. The products of these genes were analysed in minicells. The results indicate that gene B codes for a 75 K dalton protein, gene C for a 23 K dalton protein and gene E for a 36 K dalton protein. No product of gene D was observed. Gene A probably codes for the 17 K dalton subunit polypeptide of the F7 (2) fimbriae, as will be discussed.

  5. Implications of fetoplacental mosaicism on cell-free DNA testing for sex chromosome aneuploidies.

    PubMed

    Grati, Francesca Romana; Bajaj, Komal; Zanatta, Valentina; Malvestiti, Francesca; Malvestiti, Barbara; Marcato, Livia; Grimi, Beatrice; Maggi, Federico; Simoni, Giuseppe; Gross, Susan J; Ferreira, Jose

    2017-08-11

    The unique biological behavior of sex chromosomes has implications for cell-free DNA (cfDNA) testing. Our purpose is to predict the (1) false positive/negative rates of cfDNA testing consequent to fetoplacental mosaicism for any sex chromosome aneuploidies (SCA) and (2) positive predictive value (PPV) and negative predictive values of a high-risk and low-risk cfDNA result for any SCA. This is a retrospective analysis of 67 030 chorionic villus sampling karyotypes, including fetoplacental mosaicism cases. Non-mosaic 45, X is associated with cystic hygroma/increased nuchal translucency and fetal anomalies. The false positive rate consequent to confined placental mosaicism is predicted to be 0.05%. The estimated false negative rate is in the range of 0% to 5.7% for all non-mosaic SCAs; it is 70% for mosaic 45, X with normal ultrasound. The predicted PPV on amniocytes is very high for most SCAs (94.4-99.4%). However, the stratified analysis shows that the PPV is much lower for 45, X without ultrasound anomalies compared with 45, X with abnormal scan (51% or 71%, vs 99%, respectively). Mosaicism is a major issue for SCA cfDNA testing, and prenatal confirmation, preferentially with amniocentesis if there are no ultrasound anomalies, remains important in counseling. As PPV varies on the basis of the presence of an ultrasound anomaly, skilled evaluation is critical. © 2017 John Wiley & Sons, Ltd. © 2017 John Wiley & Sons, Ltd.

  6. 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

  7. Unique DNA-barcoded aerosol test particles for studying aerosol transport

    SciTech Connect

    Harding, Ruth N.; Hara, Christine A.; Hall, Sara B.; Vitalis, Elizabeth A.; Thomas, Cynthia B.; Jones, A. Daniel; Day, James A.; Tur-Rojas, Vincent R.; Jorgensen, Trond; Herchert, Edwin; Yoder, Richard; Wheeler, Elizabeth K.; Farquar, George R.

    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 use 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.

  8. 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

  9. Testing a proposed paradigm shift in analysis of phage DNA packaging

    PubMed Central

    Serwer, Philip; Wright, Elena T.

    2016-01-01

    ABSTRACT We argue that a paradigm shift is needed in the analysis of phage DNA packaging. We then test a prediction of the following paradigm shift-engendering hypothesis. The motor of phage DNA packaging has two cycles: (1) the well-known packaging ATPase-driven (type 1) cycle and (2) a proposed back-up, shell expansion/contraction-driven (type 2) cycle that reverses type 1 cycle stalls by expelling accidentally packaged non-DNA molecules. We test the prediction that increasing the cellular concentration of all macromolecules will cause packaging-active capsids to divert to states of hyper-expansion and contraction. We use a directed evolution-derived, 3-site phage T3 mutant, adapted to propagation in concentrated bacterial cytoplasm. We find this prediction correct while discovering novel T3 capsids previously obscure. PMID:28090387

  10. Testing a proposed paradigm shift in analysis of phage DNA packaging.

    PubMed

    Serwer, Philip; Wright, Elena T

    2016-01-01

    We argue that a paradigm shift is needed in the analysis of phage DNA packaging. We then test a prediction of the following paradigm shift-engendering hypothesis. The motor of phage DNA packaging has two cycles: (1) the well-known packaging ATPase-driven (type 1) cycle and (2) a proposed back-up, shell expansion/contraction-driven (type 2) cycle that reverses type 1 cycle stalls by expelling accidentally packaged non-DNA molecules. We test the prediction that increasing the cellular concentration of all macromolecules will cause packaging-active capsids to divert to states of hyper-expansion and contraction. We use a directed evolution-derived, 3-site phage T3 mutant, adapted to propagation in concentrated bacterial cytoplasm. We find this prediction correct while discovering novel T3 capsids previously obscure.

  11. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  12. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  13. 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.

  14. Role of intercalation and redox potential in DNA photosensitization by ruthenium(II) polypyridyl complexes: assessment using DNA repair protein tests.

    PubMed

    Gicquel, Etienne; Souchard, Jean-Pierre; Magnusson, Fay; Chemaly, Jad; Calsou, Patrick; Vicendo, Patricia

    2013-08-01

    Here we report that the photoreactivity of ruthenium(II) complexes with nucleobases may not only be modulated by their photoredox properties but also by their DNA binding mode. The damage resulting from photolysis of synthetic oligonucleotides and plasmid DNA by [Ru(bpz)3](2+), [Ru(bipy)3](2+) and the two DNA intercalating agents [Ru(bpz)2dppz](2+) and [Ru(bipy)2dppz](2+) has been monitored by polyacrylamide gel electrophoresis and by tests using proteins involved in DNA repair processes (DNA-PKCs, Ku80, Ku70, and PARP-1). The data show that intercalation controls the nature of the DNA damage photo-induced by ruthenium(II) complexes reacting with DNA via an electron transfer process. The intercalating agent [Ru(bpz)2dppz](2+) is a powerful DNA breaker inducing the formation of both single and double (DSBs) strand breaks which are recognized by the PARP-1 and DNA-PKCs proteins respectively. [Ru(bpz)2dppz](2+) is the first ruthenium(II) complex described in the literature that is able to induce DSBs by an electron transfer process. In contrast, its non-intercalating parent compound, [Ru(bpz)3](2+), is mostly an efficient DNA alkylating agent. Photoadducts are recognized by the proteins Ku70 and Ku80 as with cisplatin adducts. This result suggests that photoaddition of [Ru(bpz)2dppz](2+) is strongly affected by its DNA intercalation whereas its photonuclease activity is exalted. The data clearly show that DNA intercalation decreases drastically the photonuclease activity of ruthenium(II) complexes oxidizing guanine via the production of singlet oxygen. Interestingly, the DNA sequencing data revealed that the ligand dipyridophenazine exhibits on single-stranded oligonucleotides a preference for the 5'-TGCGT-3' sequence. Moreover the use of proteins involved in DNA repair processes to detect DNA damage was a powerful tool to examine the photoreactivity of ruthenium(II) complexes with nucleic acids.

  15. Demand for DNA probe testing in three genetic centres in Britain (August 1986 to July 1987).

    PubMed Central

    Rona, R J; Swan, A V; Beech, R; Prentice, L; Reynolds, A; Wilson, O; Mole, G; Vadera, P

    1989-01-01

    We report a preliminary analysis of the data collected during the first year of the evaluation of clinical genetics in the context of DNA probes in three genetic centres, to show the pattern of the demand for genetic services in the three centres and the services used in meeting that demand. The analysis includes information on 10,185 persons from 2852 families. The results are presented according to mode of inheritance and according to the most common disorders for which DNA probes have been used in the three centres. The results indicate that the use of DNA probes is now a major element of activity in genetic departments, and that as long as indirect DNA probe testing is the predominant manner of using recombinant technology, the clinical input will be an important element of the costs, probably more so than that of the DNA laboratories, as a large number of family members needs to be tested. In most cases centres have concentrated activity on DNA testing for common and severe genetic disorders. However, there are disorders, such as familial hypercholesterolaemia, which have not been part of the established pattern of services. Conversely, a relatively high number of families have been studied for some disorders of very low incidence. This suggests that the number of DNA laboratories should be limited. The precise arrangements will need to be established. With such services the distribution of DNA testing facilities for different disorders can be controlled to limit duplication. The model followed in Scotland based on collaboration between centres is worth considering. We have detected very large differences in take up rate of services within and between regions. Although many factors may contribute to these differences, ease of access and lay and professional awareness are probably the most important. This is supported by the fact that more patients from the same or neighbouring DHAs attend the genetic centre than from those further away. We also concluded that

  16. 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...

  17. 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...

  18. 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...

  19. Should sperm DNA fragmentation testing be included in the male infertility work-up?

    PubMed

    Lewis, Sheena E M

    2015-08-01

    A response to the editorial 'Are we ready to incorporate sperm DNA fragmentation testing into our male infertility work-up? A plea for more robust studies' by Erma Drobnis and Martin Johnson. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  20. 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 ...

  1. 78 FR 29698 - Availability of an Environmental Assessment for Field Testing a Canine Lymphoma Vaccine, DNA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-21

    ... Animal and Plant Health Inspection Service Availability of an Environmental Assessment for Field Testing a Canine Lymphoma Vaccine, DNA AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION: Notice of availability. SUMMARY: We are advising the public that the Animal and Plant Health Inspection...

  2. 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.

  3. 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...

  4. Application of the DNA adductome approach to assess the DNA-damaging capability of in vitro micronucleus test-positive compounds.

    PubMed

    Kato, Kyoko; Yamamura, Eiji; Kawanishi, Masanobu; Yagi, Takashi; Matsuda, Tomonari; Sugiyama, Akio; Uno, Yoshifumi

    2011-03-18

    The in vitro micronucleus (MN) test is widely used for screening genotoxic compounds, but it often produces false-positive results. To consider the significance of positive results, it is important to know whether DNA adducts are formed in the cells treated with the test compound. Recently, Matsuda et al. developed the DNA adductome approach to detect DNA adducts comprehensively ([4] Kanaly, et al., Antioxid. Redox Signal., 2006, 8, 993-1001). We applied this method to assess the DNA-damaging capability of in vitro MN test-positive compounds. CHL/IU cells were treated with compounds from three categories: (1) carcinogens causing DNA alkylation, ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine; (2) carcinogens producing DNA bulky adducts, 2-amino-6-phenyl-1-methylimidazo[4,5-b]pyrene, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and 4-nitroquinoline-1-oxide, and (3) non-carcinogens, caffeine, maltol, and sodium chloride, with or without metabolic activation. With the conditions in which all test compounds gave positive results in the MN tests, DNA was extracted from the cells and hydrolyzed to deoxyribonucleosides, which were subsequently subjected to LC/ESI-MS/MS analysis. All carcinogens (categories 1 and 2) produced various DNA adduct peaks, and some of the m/z peak values corresponded to known adducts. No non-carcinogens produced DNA adducts, indicating that these compounds produced MN through different mechanisms from the adduct formation. These results indicate that the adductome approach is useful to demonstrate DNA damage formation of MN test-positive compounds and to understand their mechanisms of action.

  5. Innovative methods for soil DNA purification tested in soils with widely differing characteristics.

    PubMed

    Sagova-Mareckova, Marketa; Cermak, Ladislav; Novotna, Jitka; Plhackova, Kamila; Forstova, Jana; Kopecky, Jan

    2008-05-01

    Seven methods of soil DNA extraction and purification were tested in a set of 14 soils differing in bedrock, texture, pH, salinity, moisture, organic matter content, and vegetation cover. The methods introduced in this study included pretreatment of soil with CaCO(3) or purification of extracted DNA by CaCl(2). The performance of innovated methods was compared to that of the commercial kit Mo Bio PowerSoil and the phenol-chloroform-based method of D. N. Miller, J. E. Bryant, E. L. Madsen, and W. C. Ghiorse (Appl. Environ. Microbiol. 65:4715-4724, 1999). This study demonstrated significant differences between the tested methods in terms of DNA yield, PCR performance, and recovered bacterial diversity. The differences in DNA yields were correlated to vegetation cover, soil pH, and clay content. The differences in PCR performances were correlated to vegetation cover and soil pH. The innovative methods improved PCR performance in our set of soils, in particular for forest acidic soils. PCR was successful in 95% of cases by the method using CaCl(2) purification and in 93% of cases by the method based on CaCO(3) pretreatment, but only in 79% by Mo Bio PowerSoil, for our range of soils. Also, the innovative methods recovered a higher percentage of actinomycete diversity from a subset of three soils. Recommendations include the assessment of soil characteristics prior to selecting the optimal protocol for soil DNA extraction and purification.

  6. 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.

  7. Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test.

    PubMed

    Carretero, M I; Lombardo, D; Arraztoa, C C; Giuliano, S M; Gambarotta, M C; Neild, D M

    2012-03-01

    The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm.

  8. 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.

  9. 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

  10. 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

  11. Comparison of molecular tests for detection and quantification of cell-associated cytomegalovirus DNA.

    PubMed

    Caliendo, Angela M; Yen-Lieberman, Belinda; Baptista, Jovana; Andersen, Janet; Crumpacker, Clyde; Schuurman, Rob; Spector, Stephen A; Bremer, James; Lurain, Nell S

    2003-08-01

    A cell-based standard was developed to compare the COBAS Amplicor CMV Monitor test, the Hybrid Capture System CMV DNA test, and the NucliSens CMV test. The standard was prepared by infecting human foreskin fibroblasts (HFFs) with cytomegalovirus (CMV) strain AD169 at low multiplicity of infection (0.03) and harvesting the cells at 6 h postinfection. Buffy coat cells were added to produce concentrations of from 0 to 10(5) HFFs per 10(6) total cells. Five laboratories performed the Amplicor PCR test and two laboratories performed the NucliSens and Hybrid Capture tests. The Amplicor PCR test was 1.5 to 2.0 log(10) more sensitive than the Hybrid Capture test. The specificities of the Amplicor PCR and Hybrid Capture tests were 100 and 93.8%, respectively. The linear range of the Amplicor PCR and Hybrid Capture tests were 2 to 4.48 log(10) and 3.48 to at least 5.0 log(10) CMV target cells, respectively. The standard deviations of the Amplicor PCR and Hybrid Capture tests varied throughout their linear range, and for both tests the variability was greater for lower concentrations of input CMV DNA. These data allow the direct comparison of viral load values between the Amplicor and Hybrid Capture tests. The analytical sensitivity of the NucliSens test could not be determined by using the 6-h standard, because the low multiplicity of infection and short culture time did not allow for adequate transcription of pp67 late mRNA measured in the test. Extending the incubation time of the standard to 24 h increased the analytical sensitivity of the NucliSens test to 3.0 log(10) target cells.

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

    PubMed

    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

    2017-06-07

    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.

  13. 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

  14. 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

  15. Swabbing students: should universities be allowed to facilitate educational DNA testing?

    PubMed

    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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. Importance sampling allows Hd true tests of highly discriminating DNA profiles.

    PubMed

    Taylor, Duncan; Curran, James M; Buckleton, John

    2017-03-01

    Hd true testing is a way of assessing the performance of a model, or DNA profile interpretation system. These tests involve simulating DNA profiles of non-donors to a DNA mixture and calculating a likelihood ratio (LR) with one proposition postulating their contribution and the alternative postulating their non-contribution. Following Turing it is possible to predict that "The average LR for the Hd true tests should be one"[1]. This suggests a way of validating softwares. During discussions on the ISFG software validation guidelines [2] it was argued by some that this prediction had not been sufficiently examined experimentally to serve as a criterion for validation. More recently a high profile report [3] has emphasised large scale empirical examination. A limitation with Hd true tests, when non-donor profiles are generated at random (or in accordance with expectation from allele frequencies), is that the number of tests required depends on the discrimination power of the evidence profile. If the Hd true tests are to fully explore the genotype space that yields non-zero LRs then the number of simulations required could be in the 10s of orders of magnitude (well outside practical computing limits). We describe here the use of importance sampling, which allows the simulation of rare events to occur more commonly than they would at random, and then adjusting for this bias at the end of the simulation in order to recover all diagnostic values of interest. Importance sampling, whilst having been employed by others for Hd true tests, is largely unknown in forensic genetics. We take time in this paper to explain how importance sampling works, the advantages of using it and its application to Hd true tests. We conclude by showing that employing an importance sampling scheme brings Hd true testing ability to all profiles, regardless of discrimination power. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Tested at Last: How DNA Evidence in Untested Rape Kits Can Identify Offenders and Serial Sexual Assaults.

    PubMed

    Campbell, Rebecca; Feeney, Hannah; Pierce, Steven J; Sharma, Dhruv B; Fehler-Cabral, Giannina

    2016-03-27

    An increasing number of U.S. law enforcement agencies have disclosed that they have large numbers of untested sexual assault kits (SAKs; also called "rape kits") in police property storage. Whether previously untested SAKs should be tested for DNA evidence has been the subject of considerable public debate. To inform policy and practice regarding rape kit testing, the current study tested a sample of 900 previously unsubmitted SAKs from Detroit, Michigan, and documented the DNA forensic testing outcomes associated with those kits. We assessed how many SAKs yielded DNA profiles eligible for upload into CODIS (Combined DNA Index System), the federal DNA criminal database; how many resulted in a DNA match (termed a "CODIS hit"); and how many of those hits were associated to other sexual assault crimes (i.e., serial sexual assault hits). Overall, there were 259 CODIS hits, 69 of which had DNA matches to another sexual assault case. The potential utility of a DNA profile and CODIS hit may vary depending on whether offender was known or unknown to the victim, so we examined these outcomes separately for SAKs associated with stranger- and non-stranger-perpetrated sexual assaults. We also present six case study examples of how DNA testing and CODIS hits helped identify serial sexual assaults in both stranger and non-stranger sexual assault cases. Implications for rape kit testing policies are discussed.

  3. 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.

  4. 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.

  5. Current State of PCR-Based Epstein-Barr Virus DNA Testing for Nasopharyngeal Cancer.

    PubMed

    Kim, Kelly Y; Le, Quynh-Thu; Yom, Sue S; Pinsky, Benjamin A; Bratman, Scott V; Ng, Raymond H W; El Mubarak, Haja S; Chan, K C Allen; Sander, Miriam; Conley, Barbara A

    2017-04-01

    Clinical studies have shown plasma Epstein-Barr virus (EBV) DNA level to be an independent prognostic biomarker for nasopharyngeal carcinoma (NPC). However, the proportion of NPC patients whose tumors are associated with EBV vary with geographic location, and there are a variety of assays for plasma EBV. To develop the level of evidence needed to demonstrate the clinical utility of plasma EBV DNA detection for NPC patients and encourage widespread adoption of this biomarker test in clinical laboratories, validated harmonized assays are needed. In 2015, the National Cancer Institute (NCI) convened a Workshop on Harmonization of EBV Testing for Nasopharyngeal Cancer, where experts in head and neck oncology and laboratory medicine addressed the limitations of currently available polymerase chain reaction-based EBV DNA quantitation assays and discussed strategies for advancing the development of harmonized EBV DNA assays and their appropriate clinical use. This article presents the key recommendations to direct future efforts in assay harmonization and validation. Published by Oxford University Press 2017. This work is written by US Government employees and is in the public domain in the US.

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

    PubMed Central

    Zeng, Zhao-cheng; Chang, Qing; Sun, Zhi-wei; Song, Ming-mei; Jin, Xin-ling; Jiang, Shu-ya; Yang, Xia

    2017-01-01

    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/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. PMID:28283676

  7. An observational study of women with positive HPV-DNA tests and normal cytology and colposcopy.

    PubMed

    Paraskevaidis, E; Davidson, E J; Malamou-Mitsi, V; Hirsch, P M; Pappa, L; Koliopoulos, G; Lolis, E; Zikopoulos, K; Paschopoulos, M; Doussias, V; Agnantis, N

    2002-01-01

    High risk human papillomaviruses (HPV) are implicated in the aetiology of malignant cervical disease. The usefulness of HPV DNA tests in identifying women at risk of cervical cancer as an adjunct to cervical cytology is under evaluation. This is a retrospective analysis of 47 women positive for high risk HPV but with negative cytology and negative colposcopy at the start of the study. Women were observed for three years or more (in 96% cases) using six-monthly combined HPV DNA tests, cytological and colposcopic evaluation. At the end of follow-up, 29/47 (62%) women were still positive for high risk HPV, 45/47 (96%) women had normal cytology and 47/47 (100%) women continued to have normal colposcopy. Normal colposcopy has an excellent negative predictive value for HPV positive women with normal cytology. These women can be safely screened cytologically on a three-yearly basis.

  8. Sex and strain differences in the hepatocyte primary culture/DNA repair test

    SciTech Connect

    McQueen, C.A.; Way, B.M. )

    1991-01-01

    The hepatocyte primary culture (HPC)/DNA repair test was developed using hepatocytes isolated from male F-344 rats. A number of genetic polymorphisms have been shown to occur in inbred strains of rats, which may lead to variation in biotransformation of xenobiotics resulting in differences in susceptibility to genotoxins. The effect of the strain utilized as a source of hepatocytes was investigated with female Lewis, F-344, and DA rats. Variation was observed when hepatocytes from the three strains were exposed to aflatoxin B{sub 1} (AFB{sub 1}). No clearcut strain differences were seen when cells were exposed to diethylnitrosamine (DEN) or 2-acetylaminofluorene. These results demonstrate that both the strain and the sex of the animal used as a source of hepatocytes can affect the HPC/DNA repair test.

  9. Small-molecule diagnostics based on functional DNA nanotechnology: a dipstick test for mercury

    PubMed Central

    Torabi, Seyed-Fakhreddin

    2011-01-01

    Detecting small molecular targets such as metal ions is just as important as detecting large molecules such as DNA, RNA and proteins, but the field of metal ion sensors has not yet been well developed. A good example of a metal ion target is mercury, which is highly toxic, widely distributed in the environment and affects human health. To develop a diagnostic platform for metal ions, we demonstrate that functional DNA-linked gold nanoparticles (AuNPs) can quickly and simply detect and quantify Hg2+ ions in aqueous solution, with high sensitivity and selectivity over competing metal ions. A linker DNA molecule containing thymine residues and sequences complementary to the DNA on the AuNPs was designed to aggregate DNA-functionalized AuNPs. When Hg2+ ions were introduced into this system, they induced the linker DNA to fold by forming thymine–Hg2+–thymine bonds. The linker DNA’s folding caused the AuNPs to rapidly disassemble, which caused a discernable color change in the solution from purple to red. The limit of detection for Hg2+ in the present method is 5.4 nM, which is below the 10 nM maximum contaminant level defined by the US Environmental Protection Agency (EPA) for drinking water. Our results show that this Hg2+ detection method has excellent selectivity over other divalent metal ions (e.g. Pb2+, Cu2+, Mn2+, Co2+, Zn2+, Cd2+, Mg2+, Ca2+, and Ba2+). This system has been converted into a dipstick test using lateral-flow devices, making it even more practical for point-of-care diagnostics. PMID:21413179

  10. Evaluation of a prototype DNA probe test for the noncultural diagnosis of gonorrhea.

    PubMed Central

    Granato, P A; Franz, M R

    1989-01-01

    A prototype, nonisotopic, chemiluminescent DNA probe test called the Gen-Probe PACE (Probe Assay-Chemiluminescence Enhanced) system for Neisseria gonorrhoeae (Gen-Probe, San Diego, Calif.) was compared with conventional Martin-Lewis culture medium in JEMBEC plates for the laboratory diagnosis of gonorrhea. This 2-h noncultural assay is based upon the use of an acridinium ester-labeled DNA probe. The rRNA-directed DNA probe hybridizes with the target rRNA, and the hybridized probe is separated from the unhybridized probe through the use of magnetic microparticles. The esterified acridinium is hydrolyzed from the hybridized probe by the addition of an alkaline hydrogen peroxide solution, resulting in the production of visible light which is measured in a luminometer. The amount of light generated is directly proportional to the amount of gonococcal target rRNA present in the sample. A total of 407 clinical specimens (203 urethral and 204 endocervical) were collected from high-risk walk-in patients attending a sexually transmitted disease clinic. Separate patient specimens were collected for culture on Martin-Lewis medium in JEMBEC plates and for DNA probe assay. Statistical analysis of the overall comparative results showed that the DNA probe assay had a sensitivity, specificity, and positive and negative predictive values of 93, 99, 97, and 99%, respectively, in a patient population with a gonococcal disease prevalence of 21%. The results of this comparative study showed that the prototype chemiluminescent DNA probe assay is a rapid and reliable noncultural alternative for the laboratory diagnosis of gonorrhea. PMID:2498388

  11. Model selection and efficiency testing for normalization of cDNA microarray data

    PubMed Central

    Futschik, Matthias; Crompton, Toni

    2004-01-01

    In this study we present two novel normalization schemes for cDNA microarrays. They are based on iterative local regression and optimization of model parameters by generalized cross-validation. Permutation tests assessing the efficiency of normalization demonstrated that the proposed schemes have an improved ability to remove systematic errors and to reduce variability in microarray data. The analysis also reveals that without parameter optimization local regression is frequently insufficient to remove systematic errors in microarray data. PMID:15287982

  12. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  13. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  14. Attitudes of obstetrician-gynecologists toward DNA testing for a genetic susceptibility to breast cancer.

    PubMed

    Rowley, P T; Loader, S

    1996-10-01

    To assess knowledge and attitudes of area obstetrician-gynecologists toward DNA testing for genetic susceptibility to breast cancer. At a staff meeting of each of the Rochester area's hospitals that had an obstetric service, we assessed knowledge of inherited predisposition to breast cancer, presented the current status of testing for genetic susceptibility, and assessed attitudes toward such testing. The majority of the physicians surveyed believed that current BRCA1 testing can detect a genetic predisposition to breast cancer accurately enough to be clinically useful (81%) and that a young woman with a family history of breast cancer not currently having regular mammography is likely to benefit from DNA testing because a positive result may motivate her to start mammography earlier (88%). However, most respondents thought that women who test positive are likely to be excessively anxious (87%) and may be discriminated against for insurance purposes (75%). In response to an invitation to participate in a clinical trial of free BRCA1 screening, 74 (70%) desired to participate. Obstetrician-gynecologists expect women detected to have a BRCA1 mutation to be motivated to conduct surveillance, but also to experience anxiety and possible discrimination.

  15. Joseon funerary texts tested using ancient DNA analysis of a Korean mummy.

    PubMed

    Oh, Chang Seok; Koh, Bou-Ja; Yoo, Dong Soo; Park, Jun Bum; Min, So Ri; Kim, Yi-Suk; Lee, Sang Sup; Ge, Jianye; Seo, Seung Bum; Shin, Dong Hoon

    2015-06-01

    In Korea, ancient DNA (aDNA) analysis has been applied to investigations into the genetic affiliations of mummies found in Joseon Dynasty tombs (1392-1910 CE), becoming now indispensable tool for researches studying human remains from archaeological sites. In the course of our recent examinations on a Korean mummy of Joseon Dynasty, we discovered many teeth contained in a pouch. And in fact, the historical literature on the topic of Joseon funerals contain general accounts of pouches in which an individual's lost teeth were collected over the course of a lifetime and, after death, placed in the coffin with the body. To test the veracity of the historical texts, the present study undertook aDNA analyses and compared the results between specifically questioned (Q) samples (teeth) and known (K) samples (brain and bone) from the mummy to ensure that they came from the same individual. Although the Q-K comparison of autosomal short tandem repeat results did not show full concordance due to allelic drop-outs in some loci, our statistical calculation indicated that the teeth in the pouch are highly likely those of the mummy. Additionally, Q-K comparison of mitochondrial DNA sequence results showed 100% matches between samples. There results, in short, could not gainsay the conjecture that the teeth samples originated from the person buried in the tomb; and if so, he must have kept his teeth for a long time after their loss. As the application of aDNA analysis to Korean mummy studies develops, there will be other opportunities to test historical documents, particularly those referring to funerary rites.

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

    PubMed Central

    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. PMID:27631491

  17. Screening for Colorectal Cancer Using a Multitarget Stool DNA Test: Modeling the Effect of the Intertest Interval on Clinical Effectiveness.

    PubMed

    Berger, Barry M; Schroy, Paul C; Dinh, Tuan A

    2016-09-01

    A multitarget stool DNA (mt-sDNA) test was recently approved for colorectal cancer (CRC) screening for men and women, aged ≥ 50 years, at average risk of CRC. The guidelines currently recommend a 3-year interval for mt-sDNA testing in the absence of empirical data. We used clinical effectiveness modeling to project decreases in CRC incidence and related mortality associated with mt-sDNA screening to help inform interval setting. The Archimedes model (Archimedes Inc., San Francisco, CA) was used to conduct a 5-arm, virtual, clinical screening study of a population of 200,000 virtual individuals to compare the clinical effectiveness of mt-sDNA screening at 1-, 3-, and 5-year intervals compared with colonoscopy at 10-year intervals and no screening for a 30-year period. The study endpoints were the decrease in CRC incidence and related mortality of each strategy versus no screening. Cost-effectiveness ratios (US dollars per quality-adjusted life year [QALY]) of mt-sDNA intervals were calculated versus no screening. Compared with 10-year colonoscopy, annual mt-sDNA testing produced similar reductions in CRC incidence (65% vs. 63%) and related mortality (73% vs. 72%). mt-sDNA testing at 3-year intervals reduced the CRC incidence by 57% and CRC mortality by 67%, and mt-sDNA testing at 5-year intervals reduced the CRC incidence by 52% and CRC mortality by 62%. At an average price of $600 per test, the annual, 3-year, and 5-year mt-sDNA screening costs would be $20,178, $11,313, and $7388 per QALY, respectively, compared with no screening. These data suggest that screening every 3 years using a multitarget mt-sDNA test provides reasonable performance at acceptable cost. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Immunomodulation of bivalent Newcastle disease DNA vaccine induced immune response by co-delivery of chicken IFN-γ and IL-4 genes.

    PubMed

    Sawant, P M; Verma, P C; Subudhi, P K; Chaturvedi, U; Singh, M; Kumar, Rajeev; Tiwari, A K

    2011-11-15

    The basic objective of this study was to enumerate whether co-administration of interferon-γ (IFN-γ) and/or interleukin-4 (IL-4) gene along with a bivalent Newcastle disease (ND) DNA vaccine construct could modulate the immune response to the DNA vaccine in chickens. pVIVO2 vector carrying Haemaglutinin-Neuraminidase (HN) and Fusion (F) genes of Newcastle disease virus (NDV) at its two cloning sites was used as a DNA vaccine. The same vector was used to clone the chicken IFN-γ and IL-4 genes at the multiple cloning site-1 separately. In vitro expression of IFN-γ and IL-4 gene constructs was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and that of HN and F genes by indirect fluorescent antibody technique (IFAT) in addition to RT-PCR. The chickens were immunized thrice intramuscularly at 21, 36 and 46 days of age with the bivalent DNA vaccine alone, or in combination with IFN-γ/IL-4 or both cytokine gene constructs. The bivalent DNA vaccine led to increase in both NDV specific antibodies as assessed by enzyme linked immunosorbent assay (ELISA) and haemagglutination inhibition test (HI) and cell mediated immune (CMI) response as assessed by lymphocyte transformation test (LTT) employing MTT assay. Co-administration of the DNA vaccine with IL-4 gene resulted in highest IgY levels while IFN-γ produced highest CMI response. The DNA vaccine alone could afford only 10% protection against challenge infection by velogenic NDV. This protection was increased to 40% when IL-4 gene construct was co-administered with the DNA vaccine. Co-injection of IFN-γ as well as the combination of IFN-γ and IL-4 gene constructs with the DNA vaccine yielded 20% protection. Our study suggests that IL-4 may prove to be more appropriate as a genetic adjuvant than IFN-γ for ND DNA vaccine.

  19. Testing the Effect of Metabolic Rate on DNA Variability at the Intra-Specific Level

    PubMed Central

    McGaughran, Angela; Holland, Barbara R.

    2010-01-01

    We tested the metabolic rate hypothesis (whereby rates of mtDNA evolution are postulated to be mediated primarily by mutagenic by-products of respiration) by examining whether mass-specific metabolic rate was correlated with root-to-tip distance on a set of mtDNA trees for the springtail Cryptopygus antarcticus travei from sub-Antarctic Marion Island. Using Bayesian analyses and a novel application of the comparative phylogenetic method, we did not find significant evidence that contemporary metabolic rates directly correlate with mutation rate (i.e., root-to-tip distance) once the underlying phylogeny is taken into account. However, we did find significant evidence that metabolic rate is dependent on the underlying mtDNA tree, or in other words, lineages with related mtDNA also have similar metabolic rates. We anticipate that future analyses which apply this methodology to datasets with longer sequences, more taxa, or greater variability will have more power to detect a significant direct correlation between metabolic rate and mutation rate. We conclude with suggestions for future analyses that would extend the preliminary approach applied here, in particular highlighting ways to tease apart oxidative stress effects from the effects of population size and/or selection coefficients operating on the molecular evolutionary rate. PMID:20300626

  20. Utilization of TIFF files in the analysis and interpretation of DNA tests

    SciTech Connect

    Yanamandra, K.; Krueger, S.; Thurmon, T.F.

    1994-09-01

    With the advent of DNA diagnostic testing in clinical practice, molecular geneticists in the genetic testing laboratories are routinely facing the task of interpreting electrophoretic banding patterns of DNA or RNA to diagnose genetic conditions. However, the DNA diagnosticians quite often experience difficulties in diagnoses while interpreting faint bands and smears from the blots, especially when smears are indicative of diagnosis in some conditions such as in fragile X syndrome. With the recent advances in computer imaging, we have been able to simplify this task of interpretation by converting these autoradiograms and Polaroid photographs from PCR gels into TIFF (Tagged Image Formal Files) files using HP ScanJet IIC for autoradiograms and a KODAK DCS 200 camera for PCR gels and using SigmaPlot software to analyze these TIFF files. Analysis of DNA from fragile X patients revealed poorly visible bands and smears from autoradiograms very well on TIFF files. Utilizing these files, nondiscrete bands and smears have been characterized and converted into meaningful peaks. Comparisons have been made to the respective chromosomal bands from patients and from controls and facilitated easy calculation of gene dosages. A major advantage of TIFF files is the archiving of innumerable nucleic acid blots on convenient floppy disks. These stored files could serve as a ready source of review and recall and aid in the proficiency testing and in laboratory inspections such as CAP or state inspections. They are also a ready source for publication and interlaboratory comparisons using a modem or transport by floppy without jeopardizing the quality. The TIFF patient data from our laboratory of different genetic conditions and their supplemental diagnostic value will be presented.

  1. 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

  2. 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.

  3. Integration of clinical point-of-care requirements in a DNA microarray genotyping test.

    PubMed

    Van Dorst, Bieke; Cremers, Amelieke; Jans, Karolien; Van Domburg, Trees; Steegen, Kim; Huang, Chengjun; Dorrer, Christian; Lagae, Liesbet; Ferwerda, Gerben; Stuyver, Lieven J

    2014-11-15

    Various proof-of-concept studies have shown the potential of biosensors with a high multiplex detection capability for the readout of DNA microarrays in a lab-on-a-chip. This is particularly interesting for the development of point-of-care genotyping tests, to screen for multiple pathogens and/or antibiotic resistance patterns. In this paper, an assay workflow is presented, suited for the development of novel lab-on-a-chips with an integrated DNA microarray. Besides the description of the different assay steps (DNA purification, amplification and detection), a control strategy is presented according to recommendations of the US Food and Drug Administration (FDA). To use a lab-on-a-chip for diagnostic applications, the optimization and evaluation of the assay performance with clinical samples is very important. Therefore, appropriate quantification methods are described, which allow optimization and evaluation of the separate assay steps, as well as total assay performance. In order to demonstrate and evaluate the total workflow, blood samples spiked with Streptococcus pneumoniae were tested. All blood samples with ≥ 10(3)CFU S. pneumoniae per ml of human blood were successfully detected by this genotyping assay.

  4. Emotional and functional impact of DNA testing on patients with symptoms of Huntington's disease.

    PubMed

    Jankovic, J; Beach, J; Ashizawa, T

    1995-07-01

    The potential impact of DNA testing on asymptomatic subjects at risk for Huntington's disease (HD) has been addressed by numerous studies, but the effect of revealing the genetic results to patients with a clinically established diagnosis of HD has not been previously evaluated. We studied 36 patients, with equal distribution of men and women, mean age 53.9 (SD 12.3) years (range 25-76) and mean duration of symptoms of 11.2 (SD 7.7) years (range 2-33), whose clinical diagnosis of HD was confirmed by expanded CAG repeats (> 40). Coping strategies and depression levels were assessed before the results of DNA testing were imparted. The assessments were repeated two weeks and three months after the results were explained to the patients and their relatives and were compared to the baseline assessments. This group of HD patients was compared with 10 patients who had similar symptoms but the diagnosis of HD was excluded by normal CAG repeats (< 30). Although some patients with HD expressed a subjective reaction to the positive result (four were "surprised", one was "frustrated", and one "devastated"), there were no differences in any psychological scores including Beck Depression Inventory, functional capacity, symptom interference, independence scale, and other measures of mood and behaviour two weeks and three months later. Similarly, no change was noted in any of these measures in the non-HD group. These results suggest that mood and coping strategies are unaffected by DNA confirmation of diagnosis in symptomatic patients with HD.

  5. The association between anticoagulation therapy, maternal characteristics, and a failed cfDNA test due to a low fetal fraction.

    PubMed

    Burns, Whitney; Koelper, Nathanael; Barberio, Andrea; DeAgostino-Kelly, Mary; Mennuti, Michael; Sammel, Mary D; Dugoff, Lorraine

    2017-09-07

    The objective of this study was to identify maternal characteristics associated with a failed cell-free DNA (cfDNA) test due to a low fetal fraction (FF). Retrospective cohort study of women with singleton pregnancies who had cfDNA screening at 10-25 weeks gestation between October 2011 and January 2016. cfDNA screening was performed using methylation techniques until October 2013; thereafter, samples were run with massively parallel sequencing. Multivariable logistic regression was performed to identify maternal characteristics associated with no cell free DNA result secondary to low FF. Thirty-three (1.2%) of 2890 eligible women had a failed cfDNA test, including 18 (0.6%) cases with a low FF. A failed cfDNA test due to a low FF was associated with obesity (aOR 1.11, CI 1.05-1.18, p = 0.0003) and treatment with enoxaparin (aOR 37.5, 11.19 - 125.87, p <0.0001). 5 of 28 (18%, 95% CI: 6.1%-36.9%) women on enoxaparin had a failed cfDNA test secondary to a low FFx. Enoxaparin therapy and obesity were associated with an increased incidence of a failed cfDNA test due to low FF. Further research is needed to determine the mechanism by which anticoagulation therapy alters cfDNA test functionality and identify approaches to improve test performance in these women. This article is protected by copyright. All rights reserved.

  6. Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing

    PubMed Central

    Pérez-Barrios, Clara; Nieto-Alcolado, Irene; Torrente, María; Jiménez-Sánchez, Carolina; Calvo, Virginia; Gutierrez-Sanz, Lourdes; Palka, Magda; Donoso-Navarro, Encarnación; Provencio, Mariano

    2016-01-01

    Background The implementation of liquid biopsy for biomarker testing and response to treatment monitoring in cancer patients would presumable increase laboratory throughput, requiring the development of automated methods for circulating free DNA (cfDNA) isolation. Methods The present study compares the MagNA Pure Compact (MPC) Nucleic Acid Isolation Kit I and Maxwell® RSC (MR) ccfDNA Plasma Kit and the later with QIAamp Circulating Nucleid Acid (QCNA) Kit using 57 plasma samples from cancer patients. cfDNA concentration was measured using the Qubit fluorometer. DNA fragments lengt were assessed using the Agilent 2100 Bioanalyzer. Circulating tumor DNA (ctDNA) was quantified by digital PCR (dPCR). Results Firstly, we observed that MPC method significantly extracted less cfDNA than MR (P<0.0001). However, there were no significant differences in extraction yields of QCNA and MR kits. cfDNA isolation yield was also associated with tumor stage but not with tumor location. Secondly, an oligonucleosomal DNA ladder pattern was observed in 88% of the samples and significant differences in the recovery of mono-, di- and tri-nucleosomes DNA fragments were observed between MPC and MR methodologies. Finally, tumor mutation quantification on cfDNA was performed on 38 paired samples using digital PCR. Mutant allele fractions (MAFs) between paired samples were not significantly different. Conclusions Methods for isolation of cfDNA can affect DNA yield and molecular weight fractions recovery. These observations should be taken into account for cfDNA analysis in routine clinical practice. PMID:28149760

  7. Molecular hybridization with DNA-probes as a laboratory diagnostic test for influenza viruses.

    PubMed

    Pljusnin, A Z; Rozhkova, S A; Nolandt, O V; Bryantseva, E A; Kuznetsov, O K; Noskov, F S

    1987-01-01

    The possibilities of using DNA-copies of different influenza A virus genes cloned with recombinant bacterial plasmids for the detection of virus-specific RNA by molecular dot-hybridization were analyzed. High specificity of RNA identification has been demonstrated and it has been shown expedient to use DNA-probes with high-conservative virus genes (polymerase, nucleoprotein, or matrix) for the detection of influenza A virus subtypes (H1N1, H2N2, H3N2) and probes with corresponding hemagglutinin genes for the differentiation of the subtypes H3N2 and H1N1. The results of nasopharyngeal specimens testing proved the effectiveness of molecular dot-hybridization in epidemiological studies of influenza outbreaks, especially of mixed etiology.

  8. 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.

  9. New DNA Methylation Markers for Pancreatic Cancer: Discovery, Tissue Validation, and Pilot Testing in Pancreatic Juice.

    PubMed

    Kisiel, John B; Raimondo, Massimo; Taylor, William R; Yab, Tracy C; Mahoney, Douglas W; Sun, Zhifu; Middha, Sumit; Baheti, Saurabh; Zou, Hongzhi; Smyrk, Thomas C; Boardman, Lisa A; Petersen, Gloria M; Ahlquist, David A

    2015-10-01

    Discriminant markers for pancreatic cancer detection are needed. We sought to identify and validate methylated DNA markers for pancreatic cancer using next-generation sequencing unbiased by known targets. At a referral center, we conducted four sequential case-control studies: discovery, technical validation, biologic validation, and clinical piloting. Candidate markers were identified using variance-inflated logistic regression on reduced-representation bisulfite DNA sequencing results from matched pancreatic cancers, benign pancreas, and normal colon tissues. Markers were validated technically on replicate discovery study DNA and biologically on independent, matched, blinded tissues by methylation-specific PCR. Clinical testing of six methylation candidates and mutant KRAS was performed on secretin-stimulated pancreatic juice samples from 61 patients with pancreatic cancer, 22 with chronic pancreatitis, and 19 with normal pancreas on endoscopic ultrasound. Areas under receiver-operating characteristics curves (AUC) for markers were calculated. Sequencing identified >500 differentially hyper-methylated regions. On independent tissues, AUC on 19 selected markers ranged between 0.73 and 0.97. Pancreatic juice AUC values for CD1D, KCNK12, CLEC11A, NDRG4, IKZF1, PKRCB, and KRAS were 0.92*, 0.88, 0.85, 0.85, 0.84, 0.83, and 0.75, respectively, for pancreatic cancer compared with normal pancreas and 0.92*, 0.73, 0.76, 0.85*, 0.73, 0.77, and 0.62 for pancreatic cancer compared with chronic pancreatitis (*, P = 0.001 vs. KRAS). We identified and validated novel DNA methylation markers strongly associated with pancreatic cancer. On pilot testing in pancreatic juice, best markers (especially CD1D) highly discriminated pancreatic cases from controls. ©2015 American Association for Cancer Research.

  10. Cell-free fetal DNA testing for fetal aneuploidy and beyond: clinical integration challenges in the US context

    PubMed Central

    Allyse, Megan; Sayres, Lauren C.; King, Jaime S.; Norton, Mary E.; Cho, Mildred K.

    2012-01-01

    The recent release of new, non-invasive prenatal tests for fetal aneuploidy using cell-free fetal DNA (cffDNA) has been hailed as a revolution in prenatal testing and has triggered significant commercial interest in the field. Ongoing research portends the arrival of a wide range of cffDNA tests. However, it is not yet clear how these tests will be integrated into well-established prenatal testing strategies in the USA, as the timing of such testing and the degree to which new non-invasive tests will supplement or replace existing screening and diagnostic tools remain uncertain. We argue that there is an urgent need for policy-makers, regulators and professional societies to provide guidance on the most efficient and ethical manner for such tests to be introduced into clinical practice in the USA. PMID:22863603

  11. HPV status of oropharyngeal cancer by combination HPV DNA/p16 testing: biological relevance of discordant results.

    PubMed

    Hong, Angela; Jones, Deanna; Chatfield, Mark; Lee, C Soon; Zhang, Mei; Clark, Jonathan; Elliott, Michael; Harnett, Gerald; Milross, Christopher; Rose, Barbara

    2013-12-01

    Human papillomavirus (HPV) causes up to 70 % of oropharyngeal cancers (OSCC). HPV positive OSCC has a more favorable outcome, thus HPV status is being used to guide treatment and predict outcome. Combination HPV DNA/p16(ink4) (p16) testing is commonly used for HPV status, but there are no standardized methods, scoring or interpretative criteria. The significance of discordant (HPV DNA positive/p16 negative and HPV DNA negative/p16 positive) cancers is controversial. In this study, 647 OSCCs from 10 Australian centers were tested for HPV DNA/p16 expression. Our aims are to determine p16 distribution by HPV DNA status to inform decisions on p16 scoring and to assess clinical significance of discordant cancers. HPV DNA was identified using a multiplex tandem HPV E6 polymerase chain reaction (PCR) assay and p16 expression by semiquantitative immunohistochemistry. p16 distribution was essentially bimodal (42 % of cancers had ≥ 70 % positive staining, 52 % <5 % positive, 6 % between 5 and 70 %). Cancers with 5 to <50 % staining had similar characteristics to the p16 negative group, and cancers with 50 to <70 % staining were consistent with the ≥ 70 % group. Using a p16 cut-point of 50 %, there were 25 % HPV DNA positive/p16 negative cancers and 1 % HPV DNA negative/p16 positive cancers. HPV DNA positive/p16 negative cancers had outcomes similar to HPV DNA negative/p16 negative cancers. 50 % is a reasonable cut-point for p16; HPV DNA positive/p16 negative OSCCs may be treated as HPV negative for clinical purposes; HPV DNA/p16 testing may add no prognostic information over p16 alone.

  12. Loop-mediated isothermal amplification test for Trypanosoma vivax based on satellite repeat DNA.

    PubMed

    Njiru, Z K; Ouma, J O; Bateta, R; Njeru, S E; Ndungu, K; Gitonga, P K; Guya, S; Traub, R

    2011-08-25

    Trypanosoma vivax is major cause of animal trypanosomiasis and responsible for enormous economic burden in Africa and South America animal industry. T. vivax infections mostly run low parasitaemia with no apparent clinical symptoms, making diagnosis a challenge. This work reports the design and evaluation of a loop-mediated isothermal amplification (LAMP) test for detecting T. vivax DNA based on the nuclear satellite repeat sequence. The assay is rapid with results obtained within 35 min. The analytical sensitivity is ∼ 1 trypanosome/ml while that of the classical PCR tests ranged from 10 to 10(3)trypanosomes/ml. The T. vivax LAMP test reported here is simple, robust and has future potential in diagnosis of animal trypanosomiasis in the field.

  13. Stool Investigations for Colorectal Cancer Screening: From Occult Blood Test to DNA Analysis.

    PubMed

    Iannone, Andrea; Losurdo, Giuseppe; Pricci, Maria; Girardi, Bruna; Massaro, Antonio; Principi, Mariabeatrice; Barone, Michele; Ierardi, Enzo; Di Leo, Alfredo

    2016-06-01

    We report an update of current methods for colorectal cancer (CRC) screening based on fecal sample analysis. A systematic review of the literature was performed in MEDLINE, EMBASE, and Science Direct electronic databases. Blood in the stools is the first and most used strategy. Fecal occult blood test (FOBT) and fecal immunochemical test (FIT) are the main methods. Both are economic, easy to perform with high specificity, and low sensitivity. Based on CRC multi-step process with genetic and epigenetic alterations in large bowel cell DNA, single mutations or panels of alterations have been detected. These tests have the advantage of a marked improvement of the sensitivity when compared to fecal blood. However, high costs, poor availability, and correct choice of marker panel represent the major limits. A specific sDNA panel including aberrantly methylated BMP3 and NDRG4 promoter regions, mutant k-ras and β-actin (a reference gene for human DNA quantity), and an immunochemical assay for human hemoglobin has been recently approved by Food and Drug Administration. Novel promising biomarkers for CRC screening are represented by microRNAs (miRNAs), a group of 18-25 nucleotide non-coding RNA molecules that regulate gene expression. Reports on these fecal biomarkers are case-control studies, and each of them evaluates single miRNAs or multi-target panels. On the other hand, some fecal proteins have been studied as possible CRC screening markers, even though they demonstrated poor results. Finally, alterations of estrogen receptor-beta (i.e., dramatic reduction in the early stage of CRC) have been demonstrated in tissue samples. Specific investigations are warranted in order to add further noninvasive markers to the panel of CRC screening tools.

  14. 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.

  15. Refined exercise testing can aid DNA-based diagnosis in muscle channelopathies.

    PubMed

    Tan, S Veronica; Matthews, Emma; Barber, Melissa; Burge, James A; Rajakulendran, Sanjeev; Fialho, Doreen; Sud, Richa; Haworth, Andrea; Koltzenburg, Martin; Hanna, Michael G

    2011-02-01

    To improve the accuracy of genotype prediction and guide genetic testing in patients with muscle channelopathies we applied and refined specialized electrophysiological exercise test parameters. We studied 56 genetically confirmed patients and 65 controls using needle electromyography, the long exercise test, and short exercise tests at room temperature, after cooling, and rewarming. Concordant amplitude-and-area decrements were more reliable than amplitude-only measurements when interpreting patterns of change during the short exercise tests. Concordant amplitude-and-area pattern I and pattern II decrements of >20% were 100% specific for paramyotonia congenita and myotonia congenita, respectively. When decrements at room temperature and after cooling were <20%, a repeat short exercise test after rewarming was useful in patients with myotonia congenita. Area measurements and rewarming distinguished true temperature sensitivity from amplitude reduction due to cold-induced slowing of muscle fiber conduction. In patients with negative short exercise tests, symptomatic eye closure myotonia predicted sodium channel myotonia over myotonia congenita. Distinctive "tornado-shaped" neuromyotonia-like discharges may be seen in patients with paramyotonia congenita. In the long exercise test, area decrements from pre-exercise baseline were more sensitive than amplitude decrements-from-maximum-compound muscle action potential (CMAP) in patients with Andersen-Tawil syndrome. Possible ethnic differences in the normative data of the long exercise test argue for the use of appropriate ethnically-matched controls. Concordant CMAP amplitude-and-area decrements of >20% allow more reliable interpretation of the short exercise tests and aid accurate DNA-based diagnosis. In patients with negative exercise tests, specific clinical features are helpful in differentiating sodium from chloride channel myotonia. A modified algorithm is suggested. Copyright © 2011 American Neurological

  16. Refined Exercise testing can aid DNA-based Diagnosis in Muscle Channelopathies

    PubMed Central

    Tan, S. Veronica; Matthews, Emma; Barber, Melissa; Burge, James A; Rajakulendran, Sanjeev; Fialho, Doreen; Sud, Richa; Haworth, Andrea; Koltzenburg, Martin; Hanna, Michael G

    2010-01-01

    Objective To improve the accuracy of genotype prediction and guide genetic testing in patients with muscle channelopathies we applied and refined specialised electrophysiological exercise test parameters. Methods We studied 56 genetically confirmed patients and 65 controls using needle electromyography, the long exercise test, and short exercise tests at room temperature, after cooling, and rewarming. Results Concordant amplitude-and-area decrements were more reliable than amplitude-only measurements when interpreting patterns of change during the short exercise tests. Concordant amplitude-and-area pattern I and pattern II decrements of >20% were 100% specific for PMC and MC respectively. When decrements at room temperature and after cooling were <20%, a repeat short exercise test after rewarming was useful in patients with myotonia congenita. Area measurements and rewarming distinguished true temperature sensitivity from amplitude reduction due to cold-induced slowing of muscle fibre conduction. In patients with negative short exercise tests, symptomatic eye closure myotonia predicted sodium channel myotonia over myotonia congenita. Distinctive ‘tornado-shaped’ neuromyotonia-like discharges may be seen in patients with paramyotonia congenita. In the long exercise test, area decrements from pre-exercise baseline were more sensitive than amplitude decrements-from-maximum-CMAP in patients with Andersen-Tawil syndrome. Possible ethnic differences in the normative data of the long exercise test argue for the use of appropriate ethnically-matched controls. Interpretation Concordant CMAP amplitude-and-area decrements of >20% allow more reliable interpretation of the short exercise tests and aid accurate DNA-based diagnosis. In patients with negative exercise tests, specific clinical features are helpful in differentiating sodium from chloride channel myotonia. A modified algorithm is suggested.. PMID:21387378

  17. Assessing the impact of common forensic presumptive tests on the ability to obtain results using a novel rapid DNA platform.

    PubMed

    Donachie, Gillian E; Dawnay, Nick; Ahmed, Romana; Naif, Sarah; Duxbury, Nicola J; Tribble, Nicholas D

    2015-07-01

    The rise of DNA evidence to the forefront of forensic science has led to high sample numbers being submitted for profiling by investigators to casework laboratories: bottleneck effects are often seen resulting in slow turnaround times and sample backlog. The ParaDNA(®) Screening and Intelligence Tests have been designed to guide investigators on the viability of potential sources of DNA allowing them to determine which samples should be sent for full DNA analysis. Both tests are designed to augment the arsenal of available forensic tests for end users and be used concurrently to those commonly available. Therefore, assessing the impact that common forensic tests have on such novel technology is important to measure. The systems were tested against various potential inhibitors to which samples may be exposed as part of the investigative process. Presumptive test agents for biological materials (blood, semen and saliva) and those used as fingerprint enhancement agents were both used. The Screening Test showed a drop in performance following application of aluminium powder and cyanoacrylate (CNA) on fingerprints samples; however this drop in performance was not replicated with high template DNA. No significant effect was observed for any agent using the Intelligence Test. Therefore, both tests stand up well to the chemical agents applied and can be used by investigators with confidence that system performance will be maintained. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Plasmid-mediated genomic recombination at the pilin gene locus enhances the N-acetyl-D-galactosamine-specific haemagglutination activity and the growth rate of Eikenella corrodens.

    PubMed

    Azakami, Hiroyuki; Akimichi, Hiromi; Noiri, Yuichiro; Ebisu, Shigeyuki; Kato, Akio

    2006-03-01

    Eikenella corrodens belongs to a group of periodontopathogenic bacteria and forms unique corroding colonies on solid medium due to twitching motility. It is believed that an N-acetyl-D-galactosamine (GalNAc)-specific lectin on the cell surface contributes significantly to its pathogenicity and can be estimated by its haemagglutination (HA) activity. Recently, a plasmid, pMU1, from strain 1073 has been found; this plasmid affects pilus formation and colony morphology. To identify the gene involved in these phenomena, ORF 4 and ORFs 5-6 on pMU1 were separately subcloned into a shuttle vector, and the resultant plasmids were introduced into E. corrodens 23834. Transformants with the ORF 4 gene, which is identified to be a homologous gene of the type IV pilin gene-specific recombinase, lost their pilus structure and formed non-corroding colonies on a solid medium, whereas transformants with ORFs 5-6 exhibited the same phenotype as the host strain 23834. Southern analysis showed that the introduction of the ORF 4 gene into strain 23834 resulted in genomic recombination at the type IV pilin gene locus. The hybridization pattern of these transformants was similar to that of strain 1073. These results suggest that ORF 4 on pMU1 encodes a site-specific recombinase and causes genomic recombination of the type IV pilin gene locus. Furthermore, the introduction of ORF 4 into strain 23834 increased GalNAc-specific HA activity to a level equivalent to that of strain 1073. Although the morphological colony changes and loss of pilus structure are also observed in phase variation, genomic recombination of the type IV pilin gene locus did not occur in these variants. Moreover, an increase was not observed in the GalNAc-specific HA activity of these variants. These results suggested that the loss of pilus structure, the morphological change in colonies and the increase in HA activity due to plasmid pMU1 might be caused by a mechanism that differs from phase variation, such as a

  19. Evaluation of chlorite and chlorate genotoxicity using plant bioassays and in vitro DNA damage tests.

    PubMed

    Feretti, D; Zerbini, I; Ceretti, E; Villarini, M; Zani, C; Moretti, M; Fatigoni, C; Orizio, G; Donato, F; Monarca, S

    2008-09-01

    In the last few years chlorine dioxide has been increasingly used for disinfecting drinking water in many countries. Although it does not react with humic substances, chlorine dioxide added to water is reduced primarily to chlorite and chlorate ions, compounds that are under investigation for their potential adverse effects on human health. The aim of this research was to study the genotoxicity of chlorite and chlorate and their mixtures. The end-points included two plant tests (chromosomal aberration test in Allium cepa and micronucleus assay in Tradescantia, carried out at different times of exposure) and two genotoxicity tests in human HepG2 cells (comet assay and cytokinesis-blocked micronucleus test). Preliminary toxicity tests were carried out for both plant and HepG2 assays. The results showed that chlorite and chlorate are able to induce chromosomal damage to plant systems, particularly chromosomal aberrations in A. cepa root tip cells, even at concentrations lower than the limit established by Italian normative law and WHO guidelines. In HepG2 cells increased DNA damage was only observed for chlorate at the lowest concentration. No increase in micronuclei frequency was detected in any of the samples tested in human HepG2 cells.

  20. 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

  1. 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).

  2. Sturgeon nucleo-cytoplasmic large DNA virus phylogeny and PCR tests.

    PubMed

    Clouthier, Sharon C; VanWalleghem, Elissa; Anderson, Eric D

    2015-12-09

    Sturgeon epitheliotropic nucleo-cytoplasmic large DNA viruses (NCLDVs) can cause a lethal disease of the integumentary system. These viruses have not been assigned to any currently recognized family or genus. In this study, phylogenetic analyses using the major capsid protein (MCP) showed that the sturgeon NCLDVs formed a cohesive taxonomic group, could be identified to the species or possibly sub-species level and formed a distinct evolutionary lineage within the Megavirales. The genetic relatedness of the sturgeon virus MCP allowed design of 3 PCR diagnostic tests with analytical specificity (ASp) inclusive of this group of viruses. The conventional PCR test, C1, had broader ASp than the 2 quantitative PCR tests, Q1 and Q2, and was inclusive of the sturgeon viruses as well as some viruses belonging to the families Mimi-, Phycodna-, or Iridoviridae. Q2 had broader specificity than Q1 but both tests recognized the sturgeon NCLDVs and did not cross-react with co-localizing sturgeon herpesviruses. Analytical test performance characteristics evaluated for Q1 and Q2 revealed sensitive assays with observed 50% limits of detection between 3 and 6.25 plasmid copies and high intra- and inter-assay repeatability. Q1 was used to test for sturgeon viruses in endangered populations of lake sturgeon Acipenser fulvescens within the Winnipeg River or Nelson River drainage systems of Manitoba, Canada. Test results indicated that namao virus is endemic in the Nelson River water basin. These tests meet the analytical requirements for diagnostic testing in Canada and are useful tools for disease management in sturgeon conservation stocking programs in North America.

  3. 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

  4. Clinical implementation of routine screening for fetal trisomies in the UK NHS: cell-free DNA test contingent on results from first-trimester combined test.

    PubMed

    Gil, M M; Revello, R; Poon, L C; Akolekar, R; Nicolaides, K H

    2016-01-01

    Cell-free DNA (cfDNA) analysis of maternal blood for detection of trisomies 21, 18 and 13 is superior to other methods of screening but is expensive. One strategy to maximize performance at reduced cost is to offer cfDNA testing contingent on the results of the first-trimester combined test that is used currently. The objectives of this study were to report the feasibility of implementing such screening, to examine the factors affecting patient decisions concerning their options for screening and decisions on the management of affected pregnancies and to report the prenatal diagnosis of fetal trisomies and outcome of affected pregnancies following the introduction of contingent screening. We examined routine clinical implementation of contingent screening in 11,692 singleton pregnancies in two National Health Service (NHS) hospitals in the UK. Women with a risk ≥ 1 in 100 (high-risk group) were offered options of invasive testing, cfDNA testing or no further testing, and those with a risk between 1 in 101 and 1 in 2500 (intermediate-risk group) were offered cfDNA testing or no further testing. The trisomic status of the pregnancies was determined by prenatal or postnatal karyotyping or by examination of the neonates. In the study population of 11,692 pregnancies, there were 47 cases of trisomy 21 and 28 of trisomies 18 or 13. Screening with the combined test followed by invasive testing for all patients in the high-risk group potentially could have detected 87% of trisomy 21 and 93% of trisomies 18 or 13, at a false-positive rate of 3.4%; the respective values for cfDNA testing in the high- and intermediate-risk groups were 98%, 82% and 0.25%. However, in the high-risk group, 38% of women chose invasive testing and 60% chose cfDNA testing; in the intermediate-risk group 92% opted for cfDNA testing. A prenatal diagnosis was made in 43 (91.5%) pregnancies with trisomy 21 and all pregnancies with trisomies 18 or 13. In many affected pregnancies the parents chose

  5. Diagnosis of canine Echinococcus multilocularis infections by copro-DNA tests: comparison of DNA extraction techniques and evaluation of diagnostic deworming.

    PubMed

    Irie, Takao; Ito, Takuya; Kouguchi, Hirokazu; Yamano, Kimiaki; Uraguchi, Kohji; Yagi, Kinpei; Nonaka, Nariaki

    2017-08-01

    The use of copro-DNA detection methods for the diagnosis of canine Echinococcus multilocularis infection was evaluated with a focus on DNA extraction techniques: two commercial kits and a modified alkaline-sodium dodecyl sulfate (SDS) technique. Dog feces (0.2 g) mixed with a protoscolex or with 1 or 10 eggs of E. multilocularis were subjected to DNA detection following extraction by these methods. DNA was extracted from all protoscolex samples by all methods, but success for samples with eggs depended on extraction technique with the modified technique showing success on all samples. Following experimental infection of dogs, copro-DNA was successfully extracted from fecal samples (0.2 g) of dogs in the patent period by all methods. In the prepatent period, PCR testing of feces subsamples (0.2 g) extracted by each technique was positive at a rate of 79.6-94.4%. Extraction by the modified technique with fecal samples of over 1 g showed detection of copro-DNA in all samples in both the patent and prepatent periods, and it produced reproducible detection in the addition recovery test using feces from 72 different domestic dogs. As copro-DNA was detected for at least 1 day following deworming with administration of anthelmintic drugs in experimentally infected dogs, diagnostic deworming might be useful for clinical examination. Using the present detection method can provide quick and accurate diagnosis of canine E. multilocularis infection, which with prompt management and treatment of infected dogs can prevent pet owners from becoming infected and prevent echinococcosis from spreading into non-endemic areas.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. Validation of Testing and Interpretation Protocols for Low Template DNA Samples Using AmpFℓSTR® Identifiler®

    PubMed Central

    Caragine, Theresa; Mikulasovich, Rebecca; Tamariz, Jeannie; Bajda, Ewelina; Sebestyen, James; Baum, Howard; Prinz, Mechthild

    2009-01-01

    Aim To test the reliability, robustness, and reproducibility of short tandem repeat (STR) profiling of low template DNA (LT-DNA) when employing a defined set of testing and interpretation parameters. Methods DNA from known donors was measured with a quantitative real time polymerase chain reaction (PCR) assay that consistently detects less than 1 pg/µL of DNA within a factor of 0.3. Extracts were amplified in triplicate with AmpFℓSTR® Identifiler® reagents under enhanced PCR conditions. Replicates were examined independently and alleles confirmed using a consensus approach. Considering observed stochastic effects inherent to LT-DNA samples, interpretation protocols were developed and their accuracy verified through examination of over 800 samples. Results Amplification of 100 pg or less of DNA generated reproducible results with anticipated stochastic effects. Down to 25 pg of DNA, 92% or more of the expected alleles were consistently detected while lower amounts yielded concordant partial profiles. Although spurious alleles were sometimes observed within sample replicates, they did not repeat. To account for allelic dropout, interpretation guidelines were made especially stringent for determining homozygous alleles. Due to increased heterozygote imbalance, stutter filters were set conservatively and minor components of mixtures could not be resolved. Applying the resultant interpretation protocols, 100% accurate allelic assignments for over 107 non-probative casework samples, and subsequently 319 forensic casework samples, were generated. Conclusion Using the protocols and interpretation guidelines described here, LT-DNA testing is reliable and robust. Implementation of this method, or one that is suitably verified, in conjunction with an appropriate quality control program ensures that LT-DNA testing is suitable for forensic purposes. PMID:19480021

  11. Value of HPV-DNA test in women with cytological diagnosis of atypical glandular cells (AGC).

    PubMed

    Zeferino, Luiz Carlos; Rabelo-Santos, Silvia Helena; Villa, Luísa Lina; Sarian, Luis Otávio; Costa, Maria Cecília; do Amaral Westin, Maria Cristina; de Ângelo-Andrade, Liliana Aparecida Lucci; Derchain, Sophie

    2011-11-01

    This study analyzed whether HPV (human papillomavirus) testing contributes towards defining histological abnormalities in women with atypical glandular cells (AGC) diagnosed at cervical cytology. One hundred and eight women with conventional cervical cancer screening smears suggestive of AGC not otherwise specified (AGC-NOS) and favor neoplastic (AGC-FN) were consecutively enrolled. All women underwent colposcopic examinations and biopsy was performed according to the cytopathologic and/or colposcopic abnormalities present. All specimens were tested for high risk HPV genotypes by Roche's polymerase chain reaction reverse line blot assay. The chi-square test was used to evaluate the association between HPV findings and a diagnosis of high-grade pre-invasive or invasive disease (CIN 2 or worse) taking negative tests or CIN 1 as a reference. Odds ratios (OR) with their respective 95% confidence intervals (95%CI) were used to evaluate the magnitude of the association between HPV testing and CIN 2 or worse. Sensitivity, specificity and their respective 95% confidence intervals (95%CI), positive predictive values (PPV) and negative predictive values (NPV) were also calculated. Final diagnosis revealed a negative outcome in 80 cases (74%), cervical epithelial neoplasia 1 (CIN 1) in 13 cases (12%), CIN 2 or worse in 12 cases (11%) and glandular neoplasia in 3 (3%) cases. The overall detection rate of HPV was 21% (23/108). Neoplasia was significantly associated with positive HPV-DNA in women with AGC-NOS (OR=15.21; 95%CI: 2.64-87.50); however, there was no significant association between a histological diagnosis of neoplasia and HPV positivity in women with AGC-FN (OR=3.00; 95%CI: 0.36-24.92). The sensitivity, specificity, positive predictive value and negative predictive value of HPV-DNA testing for the detection of CIN 2 or worse in women with AGC-NOS were 71%, 86%, 29% and 97%, respectively. In women with AGC-FN, these values were 50%, 75%, 66% and 60%, respectively. HPV

  12. North-American survey on HPV-DNA and p16 testing for head and neck squamous cell carcinoma.

    PubMed

    Maniakas, Anastasios; Moubayed, Sami P; Ayad, Tareck; Guertin, Louis; Nguyen-Tan, Phuc Felix; Gologan, Olga; Soulieres, Denis; Christopoulos, Apostolos

    2014-10-01

    Human papillomavirus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) have been shown to have a significantly better prognosis and response to current treatment modalities. Current guidelines recommend systematic HPV-DNA and/or p16 testing on HNSCCs, although treatment approach should not be directed by test results. The objectives of this study were to (1) assess whether HPV-DNA and/or p16 status are systematically evaluated across North American otolaryngologists-head and neck surgeons and (2) whether the status is used to direct treatment approach. A 15-question online survey was sent to three associations: the Association of Oto-rhino-laryngology-Head and Neck Surgery of Quebec, the Canadian Society of Otolaryngology-Head and Neck Surgery, and the American Head and Neck Society. Sixty-seven percent of respondents systematically test for HPV-DNA and/or p16 on HNSCC sites, while 58.3% report using test results to direct treatment for oropharyngeal cancers. A lack of official guidelines was the primary reason (81.8%) physicians did not use test results to direct treatment. Academic centre physicians (83.3%) and physicians with ⩾50% oncologic practice (87.6%) were more likely to test for HPV-DNA and/or p16 in HNSCC compared to non-academic centre physicians (39.7%) and physicians with <50% oncologic practices (51.4%) (p<0.001). Cost of the tests (69.2%), lack of relevance (46.1%) and time constraints (30.8%) were the primary reasons HPV-DNA and/or p16 were not tested. The majority of North American respondents in this survey systematically test for HPV-DNA and/or p16 in HNSCC sites, and most indicate that test results influence their treatment approach for oropharyngeal cancers. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. 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

  14. 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

  15. 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

  16. 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

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

    2011-07-01

    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

  17. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing.

    PubMed

    Frampton, Garrett M; Fichtenholtz, Alex; Otto, Geoff A; Wang, Kai; Downing, Sean R; He, Jie; Schnall-Levin, Michael; White, Jared; Sanford, Eric M; An, Peter; Sun, James; Juhn, Frank; Brennan, Kristina; Iwanik, Kiel; Maillet, Ashley; Buell, Jamie; White, Emily; Zhao, Mandy; Balasubramanian, Sohail; Terzic, Selmira; Richards, Tina; Banning, Vera; Garcia, Lazaro; Mahoney, Kristen; Zwirko, Zac; Donahue, Amy; Beltran, Himisha; Mosquera, Juan Miguel; Rubin, Mark A; Dogan, Snjezana; Hedvat, Cyrus V; Berger, Michael F; Pusztai, Lajos; Lechner, Matthias; Boshoff, Chris; Jarosz, Mirna; Vietz, Christine; Parker, Alex; Miller, Vincent A; Ross, Jeffrey S; Curran, John; Cronin, Maureen T; Stephens, Philip J; Lipson, Doron; Yelensky, Roman

    2013-11-01

    As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95-99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.

  18. Alternative microbial testing: a novel DNA-based detection system for specified microorganisms in pharmaceutical preparations.

    PubMed

    Merker, P; Grohmann, L; Petersen, R; Ladewig, J; Gerbling, K P; Lauter, F R

    2000-01-01

    Fluorescence-coupled PCR technology was employed to quantify DNA segments specific for Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacteriaceae. The PCR procedure is put forward as an alternative method for detecting microbial contaminations in pharmaceutical preparations and is compared to the tests for specified microorganisms described in European Pharmacopoeia (EP) 2, 2.6.13 and the USP, chapter 61. Data presented here describe the validation of this analytical method when used for proof of absence of specified microorganisms. The detection systems were specific for the microorganisms analyzed, and led to linear results over a wide range (more than 6-7 log intervals). The correlation coefficients lay above 0.99. The precision of replicate determinations within a single test was observed to be high, the relative standard deviation being between 0.39% and 1.53%. The precision between different tests was also high, with a relative standard deviation between 0.76% and 1.91%. The sensitivity without pre-enrichment amounted to 1-10 CFU. Since determination of the specified bacteria was performed following pre-enrichment, the limit of detection amounted to 1 CFU. Equivalent results were obtained in a study on nine batches of a milky hydrophilic cream (SH-No. M 440 A) with the conventional test for microbial contamination and the PCR procedure. The data presented here strongly indicate that the use of fluorescence-coupled PCR techniques can prove the absence of specified bacteria faster and more efficiently than conventional methods.

  19. Integrating stakeholder perspectives into the translation of cell-free fetal DNA testing for aneuploidy

    PubMed Central

    2012-01-01

    Background The translation of novel genomic technologies from bench to bedside enjoins the comprehensive consideration of the perspectives of all stakeholders who stand to influence, or be influenced by, the translational course. Non-invasive prenatal aneuploidy testing that utilizes cell-free fetal DNA (cffDNA) circulating in maternal blood is one example of an innovative technology that promises significant benefits for its intended end users; however, it is currently uncertain whether it will achieve widespread clinical implementation. We conducted qualitative interviews with 18 diverse stakeholders in this domain, including prospective users of the technology and healthcare personnel, researchers and developers, and experts in social, legal, and regulatory aspects of genetic technology, and a pilot survey of 62 obstetric healthcare providers. Analysis of interview and survey data was combined with a review of the proceedings of a full-day, multidisciplinary conference on the topic and published scientific and ethics literature surrounding this and other relevant technologies. Discussion We constructed potential pathways for technological implementation, identified broad stakeholder classes party to these translational processes, and performed a preliminary assessment of the viewpoints and interrelations among these diverse stakeholders. Some of the stakeholders whose priorities are critical to understand and integrate into translation include pregnant women and their families; healthcare providers; scientists, their institutions or companies, and the funding agencies that support them; regulatory and judicial bodies; third-party payers; professional societies; educational systems; disability rights communities; and other representatives from civil society. Stakeholder interviews, survey findings, and conference proceedings add complexity to these envisioned pathways and also demonstrate a paramount need to incorporate an iterative stakeholder analysis early and

  20. Stool-based DNA testing, a new noninvasive method for colorectal cancer screening, the first report from Iran

    PubMed Central

    Abbaszadegan, Mohammad Reza; Tavasoli, Alireza; Velayati, Arash; Sima, Hamid Reza; Vosooghinia, Hassan; Farzadnia, Mehdi; Asadzedeh, Hamid; Gholamin, Mehran; Dadkhah, Ezzat; Aarabi, Azadeh

    2007-01-01

    AIM: To detect tumor-associated DNA changes in stool samples among Iranian patients with colorectal cancer (CRC) compared to healthy individuals using BAT-26, p16 hypermethylation and long DNA markers. METHODS: Stool DNA was isolated from 45 subjects including 25 CRC patients and 20 healthy individuals using a new, fast and easy extraction method. Long DNA associated with tumor was detected using polymerase chain reaction method. Microsatellite studies were performed utilizing denaturating polyacrylamide gel to determine the instability of BAT-26. Methylation status of p16 promoter was analyzed using methylation-specific PCR (MSP). RESULTS: The results showed a significant difference in existence of long DNA (16 in patients vs 1 in controls, P < 0.001) and p16 (5 in patients vs none in controls, P = 0.043) in the stool samples of two groups. Long DNA was detected in 64% of CRC patients; whereas just one of the healthy individuals was positive for Long DNA. p16 methylation was found in 20% of patients and in none of healthy individuals. Instability of BAT-26 was not detected in any of stool samples. CONCLUSION: We could detect colorectal cancer related genetic alterations by analyzing stool DNA with a sensitivity of 64% and 20% and a specificity of 95% and 100% for Long DNA and p16 respectively. A non-invasive molecular stool-based DNA testing can provide a screening strategy in high-risk individuals. However, additional testing on more samples is necessary from Iranian subjects to determine the exact specificity and sensitivity of these markers. PMID:17461444

  1. DNA testing of Klinefelter's syndrome in a criminal case using XY chromosomal STR multiplex-PCR.

    PubMed

    Honda, K; Tun, Z; Matoba, R

    2001-09-01

    We report genetic typing of Klinefelter's syndrome applied to casework in forensic DNA testing. In this case, by using extracted DNA from body samples (muscle and bones), we could identify two distinct X alleles in two out of three X-STR loci (HPRTB and ARA), in addition to Y alleles (DYS390, DYS393). The extra X was found to have originated from father, and the victim turned out to have 47XXY Klinefelter's syndrome. The victim was a 30-year-old male, born from relatively elderly parents as a second child. His father was a severe alcoholic and had been malnourished for more than 20 years at the moment of his birth. He exhibited slight mental retardation as a child, and belonged to a criminal group as an adult. The method presented here was useful to accurately diagnose sex chromosomal abnormality instead of conventional chromosomal analysis and Xg blood group typing. A subtype of this syndrome, 48 XXXY or mosaic, for example, could be identified if the intensity of the overlapped X bands were calculated.

  2. Italian mitochondrial DNA database: results of a collaborative exercise and proficiency testing.

    PubMed

    Turchi, Chiara; Buscemi, Loredana; Previderè, Carlo; Grignani, Pierangela; Brandstätter, Anita; Achilli, Alessandro; Parson, Walther; Tagliabracci, Adriano

    2008-05-01

    This work is a review of a collaborative exercise on mtDNA analysis undertaken by the Italian working group (Ge.F.I.). A total of 593 samples from 11 forensic genetic laboratories were subjected to hypervariable region (HVS-I/HVS-II) sequence analysis. The raw lane data were sent to MtDNA Population Database (EMPOP) for an independent evaluation. For the inclusion of data for the Italian database, quality assurance procedures were applied to the control region profiles. Only eight laboratories with a final population sample of 395 subjects passed the quality conformance test. Control region haplogroup (hg) assignments were confirmed by restriction fragment length polymorphism (RFLP) typing of the most common European hg-diagnostic sites. A total of 306 unique haplotypes derived from the combined analysis of control and coding region polymorphisms were found; the most common haplotype--CRS, 263, 309.1C, 315.1C/ not7025 AluI--was shared by 20 subjects. The majority of mtDNAs detected in the Italian population fell into the most common west Eurasian hgs: R0a (0.76%), HV (4.81%), H (38.99%), HV0 (3.55%), J (7.85%), T (13.42%), U (11.65%), K (10.13%), I (1.52%), X (2.78%), and W (1.01%).

  3. An exonic splicing silencer in the testes-specific DNA ligase III β exon

    PubMed Central

    Chew, Shern L.; Baginsky, Lysa; Eperon, Ian C.

    2000-01-01

    Alternative pre-mRNA splicing of two terminal exons (α and β) regulates the expression of the human DNA ligase III gene. In most tissues, the α exon is expressed. In testes and during spermatogenesis, the β exon is used instead. The α exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the β exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the β exon and extending into the downstream intron derepressed splicing to the β exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the β exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the polyadenylation signal was deleted or replaced by a 5′ splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceosomal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation. PMID:10606636

  4. Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case.

    PubMed

    Seifert, Keith A; Samson, Robert A; Dewaard, Jeremy R; Houbraken, Jos; Lévesque, C André; Moncalvo, Jean-Marc; Louis-Seize, Gerry; Hebert, Paul D N

    2007-03-06

    DNA barcoding systems employ a short, standardized gene region to identify species. A 648-bp segment of mitochondrial cytochrome c oxidase 1 (CO1) is the core barcode region for animals, but its utility has not been tested in fungi. This study began with an examination of patterns of sequence divergences in this gene region for 38 fungal taxa with full CO1 sequences. Because these results suggested that CO1 could be effective in species recognition, we designed primers for a 545-bp fragment of CO1 and generated sequences for multiple strains from 58 species of Penicillium subgenus Penicillium and 12 allied species. Despite the frequent literature reports of introns in fungal mitochondrial genomes, we detected introns in only 2 of 370 Penicillium strains. Representatives from 38 of 58 species formed cohesive assemblages with distinct CO1 sequences, and all cases of sequence sharing involved known species complexes. CO1 sequence divergences averaged 0.06% within species, less than for internal transcribed spacer nrDNA or beta-tubulin sequences (BenA). CO1 divergences between species averaged 5.6%, comparable to internal transcribed spacer, but less than values for BenA (14.4%). Although the latter gene delivered higher taxonomic resolution, the amplification and alignment of CO1 was simpler. The development of a barcoding system for fungi that shares a common gene target with other kingdoms would be a significant advance.

  5. Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case

    PubMed Central

    Seifert, Keith A.; Samson, Robert A.; deWaard, Jeremy R.; Houbraken, Jos; Lévesque, C. André; Moncalvo, Jean-Marc; Louis-Seize, Gerry; Hebert, Paul D. N.

    2007-01-01

    DNA barcoding systems employ a short, standardized gene region to identify species. A 648-bp segment of mitochondrial cytochrome c oxidase 1 (CO1) is the core barcode region for animals, but its utility has not been tested in fungi. This study began with an examination of patterns of sequence divergences in this gene region for 38 fungal taxa with full CO1 sequences. Because these results suggested that CO1 could be effective in species recognition, we designed primers for a 545-bp fragment of CO1 and generated sequences for multiple strains from 58 species of Penicillium subgenus Penicillium and 12 allied species. Despite the frequent literature reports of introns in fungal mitochondrial genomes, we detected introns in only 2 of 370 Penicillium strains. Representatives from 38 of 58 species formed cohesive assemblages with distinct CO1 sequences, and all cases of sequence sharing involved known species complexes. CO1 sequence divergences averaged 0.06% within species, less than for internal transcribed spacer nrDNA or β-tubulin sequences (BenA). CO1 divergences between species averaged 5.6%, comparable to internal transcribed spacer, but less than values for BenA (14.4%). Although the latter gene delivered higher taxonomic resolution, the amplification and alignment of CO1 was simpler. The development of a barcoding system for fungi that shares a common gene target with other kingdoms would be a significant advance. PMID:17360450

  6. Perceptions of genetic testing for personalized nutrition: a randomized trial of DNA-based dietary advice.

    PubMed

    Nielsen, Daiva E; Shih, Sarah; El-Sohemy, Ahmed

    2014-01-01

    Direct-to-consumer (DTC) genetic tests have facilitated easy access to personal genetic information related to health and nutrition; however, consumer perceptions of the nutritional information provided by these tests have not been evaluated. The objectives of this study were to assess individual perceptions of personalized nutrition and genetic testing and to determine whether a personalized nutrition intervention modifies perceptions. A double-blind, parallel-group, randomized controlled trial was conducted among healthy men and women aged 20-35 years (n = 138). Participants in the intervention group (n = 92) were given a report of DNA-based dietary advice and those in the control group (n = 46) were given a general dietary advice report. A survey was completed at baseline and 3 and 12 months after distributing the reports to assess perceptions between the two groups. No significant differences in perceptions of personalized nutrition and genetic testing were observed between the intervention and control group, so responses of both groups were combined. As compared to baseline, participant responses increased significantly toward the positive end of a Likert scale at 3 months for the statement 'I am interested in the relationship between diet and genetics' (mean change ± SD: 0.28 ± 0.99, p = 0.0002). The majority of participants indicated that a university research lab (47%) or health care professional (41%) were the best sources for obtaining accurate personal genetic information, while a DTC genetic testing company received the fewest selections (12%). Most participants (56%) considered dietitians to be the best source of personalized nutrition followed by medical doctors (27%), naturopaths (8%) and nurses (6%). These results suggest that perceptions of personalized nutrition changed over the course of the intervention. Individuals view a research lab or health care professional as better providers of genetic information than a DTC genetic testing company

  7. 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

  8. DNA methylation analysis in self-sampled brush material as a triage test in hrHPV-positive women.

    PubMed

    Boers, A; Bosgraaf, R P; van Leeuwen, R W; Schuuring, E; Heideman, D A M; Massuger, L F A G; Verhoef, V M J; Bulten, J; Melchers, W J G; van der Zee, A G J; Bekkers, R L M; Wisman, G B A

    2014-09-09

    Primary high-risk human papillomavirus (hrHPV) testing in cervical cancer screening shows relatively low specificity, which makes triage testing necessary. In this study, DNA methylation analysis was compared with cytology for triage testing in hrHPV-positive women. Moreover, feasibility of DNA methylation analysis directly on brush-based self-sampled specimens was assessed. Non-responding women from population-based screening were invited to self-collect a cervico-vaginal specimen for hrHPV testing; hrHPV-positive women were referred to a physician for triage liquid-based cytology. DNA methylation analysis was performed on 128 hrHPV-positive physician-collected triage samples and 50 matched brush self-samples with QMSP for C13ORF18, EPB41L3, JAM3 and TERT. In physician-taken triage material, DNA methylation analysis of JAM3 showed the highest combined specificity (88%) and sensitivity (82%) for detection of CIN3+, whereas cytology showed a specificity of 48% and a sensitivity of 91%. Out of 39 women with abnormal cytology and normal histology (false-positive by cytology), 87% were negative for JAM3 and 90% for C13ORF18 methylation. Agreement between DNA methylation analysis performed directly on the matched self-sampled material and physician-taken samples was 88% for JAM3 (κ=0.75, P<0.001) and 90% for C13ORF18 (κ=0.77; P<0.001). DNA methylation analysis as a triage test in hrHPV-positive women is an attractive alternative to cytology. Furthermore, DNA methylation is feasible directly on brush-based self-samplers and showed good correlation with matched physician-taken samples. Direct molecular triage on self-collected specimens could optimise the screening program, especially for non-responders, as this would eliminate the need for an additional physician-taken scraping for triage testing.

  9. Does behavior reflect phylogeny in swiftlets (Aves: Apodidae)? A test using cytochrome b mitochondrial DNA sequences.

    PubMed Central

    Lee, P L; Clayton, D H; Griffiths, R; Page, R D

    1996-01-01

    Swiftlets are small insectivorous birds, many of which nest in caves and are known to echolocate. Due to a lack of distinguishing morphological characters, the taxonomy of swiftlets is primarily based on the presence or absence of echolocating ability, together with nest characters. To test the reliability of these behavioral characters, we constructed an independent phylogeny using cytochrome b mitochondrial DNA sequences from swiftlets and their relatives. This phylogeny is broadly consistent with the higher classification of swifts but does not support the monophyly of swiftlets. Echolocating swiftlets (Aerodramus) and the nonecholocating "giant swiftlet" (Hydrochous gigas) group together, but the remaining nonecholocating swiftlets belonging to Collocalia are not sister taxa to these swiftlets. While echolocation may be a synapomorphy of Aerodramus (perhaps secondarily lost in Hydrochous), no character of Aerodramus nests showed a statistically significant fit to the molecular phylogeny, indicating that nest characters are not phylogenetically reliable in this group. Images Fig. 1 PMID:8692950

  10. An extremely sensitive species-specific ARMS PCR test for the presence of tiger bone DNA.

    PubMed

    Wetton, Jon H; Tsang, Carol S F; Roney, Chris A; Spriggs, Adrian C

    2002-04-18

    The survival of the tiger (Panthera tigris) is seriously threatened by poaching to provide raw materials for traditional Chinese medicines (TCMs). Most highly prized are the tiger's bones, which are used in combination with other animal and plant derivatives in pills and plasters for the treatment of rheumatism and other ailments. Hundreds of patent remedies have been produced which claim to contain tiger bone, but proof of its presence is needed if legislation prohibiting the trade in endangered species is to be enforced.A highly sensitive tiger-specific real-time PCR assay has been developed to address this problem. Using primers specific to the tiger mitochondrial cytochrome b gene, successful amplification has been reliably achieved from blood, hair and bone as well as from a range of TCMs spiked with 0.5% tiger bone. Although capable of detecting fewer than 10 substrate molecules, the seven varieties of TCM pills and plasters tested showed no detectable trace of tiger DNA before spiking. Furthermore, sequencing several "tiger bone" fragments seized from TCM shops has shown that they actually originated from cattle and pigs. The potential effects of traditional bone preparation methods, evidence that much lower concentrations are used than alleged on TCM packaging, and substitution of bones from other species all suggest a low likelihood of detecting tiger DNA in patent medicines. Despite this, the basic methods have been thoroughly proven and can be readily applied to derivatives from other Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) protected species, providing a rapid and highly sensitive forensic test for species of origin. Potential applications to the monitoring of wild populations are demonstrated by the successful identification of shed hairs and faecal samples.

  11. 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

  12. 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

  13. Fetal sex chromosome testing by maternal plasma DNA sequencing: clinical laboratory experience and biology.

    PubMed

    Bianchi, Diana W; Parsa, Saba; Bhatt, Sucheta; Halks-Miller, Meredith; Kurtzman, Kathryn; Sehnert, Amy J; Swanson, Amy

    2015-02-01

    To describe the clinical experience with noninvasive prenatal testing for fetal sex chromosomes using sequencing of maternal plasma cell-free DNA in a commercial laboratory. A noninvasive prenatal testing laboratory data set was examined for samples in which fetal sex chromosomes were reported. Available clinical outcomes were reviewed. Of 18,161 samples with sex chromosome results, no sex chromosome aneuploidy was detected in 98.9% and the fetal sex was reported as XY (9,236) or XX (8,721). In 4 of 32 cases in which the fetal sex was reportedly discordant between noninvasive prenatal testing and karyotype or ultrasonogram, a potential biological reason for the discordance exists, including two cases of documented co-twin demise, one case of a maternal kidney transplant from a male donor, and one case of fetal ambiguous genitalia. In the remaining 204 samples (1.1%), one of four sex chromosome aneuploidies (monosomy X, XXX, XXY, or XYY) was detected. The frequency of false positive results for sex chromosome aneuploidies is a minimum of 0.26% and a maximum of 1.05%. All but one of the discordant sex chromosome aneuploidy results involved the X chromosome. In two putative false-positive XXX cases, maternal XXX was confirmed by karyotype. For the false-positive cases, mean maternal age was significantly higher in monosomy X (P<.001) and lower in XXX (P=.008). Noninvasive prenatal testing results for sex chromosome aneuploidy can be confounded by maternal or fetal biological phenomena. When a discordant noninvasive prenatal testing result is encountered, resolution requires additional maternal history, detailed fetal ultrasonography, and determination of fetal and possibly maternal karyotypes.

  14. Development of a genomic DNA reference material panel for Rett syndrome (MECP2-related disorders) genetic testing.

    PubMed

    Kalman, Lisa V; Tarleton, Jack C; Percy, Alan K; Aradhya, Swaroop; Bale, Sherri; Barker, Shannon D; Bayrak-Toydemir, Pinar; Bridges, Christina; Buller-Burckle, Arlene M; Das, Soma; Iyer, Ramaswamy K; Vo, Timothy D; Zvereff, Val V; Toji, Lorraine H

    2014-03-01

    Rett syndrome is a dominant X-linked disorder caused by point mutations (approximately 80%) or by deletions or insertions (approximately 15% to 18%) in the MECP2 gene. It is most common in females but lethal in males, with a distinctly different phenotype. Rett syndrome patients have severe neurological and behavioral problems. Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process; however, none are commercially available for MECP2 genetic testing. The Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with the genetic testing community and the Coriell Cell Repositories, established 27 new cell lines and characterized the MECP2 mutations in these and in 8 previously available cell lines. DNA samples from the 35 cell lines were tested by eight clinical genetic testing laboratories using DNA sequence analysis and methods to assess copy number (multiplex ligation-dependent probe amplification, semiquantitative PCR, or array-based comparative genomic hybridization). The eight common point mutations known to cause approximately 60% of Rett syndrome cases were identified, as were other MECP2 variants, including deletions, duplications, and frame shift and splice-site mutations. Two of the 35 samples were from males with MECP2 duplications. These MECP2 and other characterized genomic DNA samples are publicly available from the NIGMS Repository at the Coriell Cell Repositories.

  15. Stool DNA Test of Methylated Syndecan-2 for the Early Detection of Colorectal Neoplasia.

    PubMed

    Niu, Feng; Wen, Jialing; Fu, Xinhui; Li, Chujun; Zhao, Rongsong; Wu, Shan; Yu, Hao; Liu, Xianglin; Zhao, Xia; Liu, Side; Wang, Xinying; Wang, Jianping; Zou, Hongzhi

    2017-09-01

    Background: Although the incidence of colorectal cancer is steadily increasing, screening for colorectal cancer with conventional approaches is not routinely performed in China. Noninvasive screening methods are attractive options to resolve this issue. Syndecan-2 (SDC2) is frequently methylated in colorectal cancer. However, the value of a stool test of methylated SDC2 for the detection of colorectal cancer is unknown.Methods: Methylation status of SDC2 was tested in cell lines and 398 colorectal tissue samples and further evaluated with 497 stool samples, including 196 from colorectal cancer patients, 122 from adenoma patients, and 179 from normal individuals, using real-time methylation-specific PCR. The impacts of one quantitative partial stool sampling device and 17 potentially interfering substances on the performance of fecal methylated SDC2 were also analyzed. SDC2 expression was also measured.Results:SDC2 methylation level was higher in 96.8% (120/124) of colorectal cancer tissues compared with paired adjacent normal epithelia. Stool test of methylated SDC2 detected 81.1% (159/196) of colorectal cancer and 58.2% (71/122) of adenomas at a specificity of 93.3% (167/179). No significant difference was found between partial and whole stool collection on colorectal cancer detection (P > 0.05, R(2) = 0.80). Among 17 interfering substances, only berberine at high concentrations inhibited fecal detection of methylated SDC2SDC2 was overexpressed in colorectal cancer tissues compared with normal epithelia.Conclusions: Fecal methylated SDC2 is a valuable biomarker for the noninvasive detection of colorectal neoplasms.Impact: Stool DNA test of methylated SDC2 would serve as an alternative method for screening colorectal neoplasms. Cancer Epidemiol Biomarkers Prev; 26(9); 1411-9. ©2017 AACR. ©2017 American Association for Cancer Research.

  16. Mitochondrial DNA in somatic cells: a promising target of routine clinical tests.

    PubMed

    Kang, Dongchon; Hamasaki, Naotaka

    2005-08-01

    Alterations of mitochondrial DNA have long been considered only from a point of view of rare genetic disorders causing neuromyopathy. Recently, alterations of mitochondrial DNA have been found in so-called common diseases such as heart failure, diabetes, and cancer; some of these alterations are inherited, and some are generated and/or accumulated in somatic cells with age. Mitochondrial DNA is more vulnerable to alteration than is nuclear DNA. For example, mitochondria produce a large amount of reactive oxygen species as an inevitable byproduct of oxidative phosphorylation. Therefore, mitochondrial DNA is under much stronger oxidative stress than is nuclear DNA. In spite of the importance, it is much less elucidated in the mitochondrial genome than in the nuclear genome how the genome is maintained. In this review, we focus on maintenance of mitochondrial DNA in somatic cells and its clinical importance.

  17. The clinical research of Thinprep Cytology Test (TCT) combined with HPV-DNA detection in screening cervical cancer.

    PubMed

    Liu, Y; Zhang, L; Zhao, G; Che, L; Zhang, H; Fang, J

    2017-02-28

    Our objective is to explore the clinical value of thinprep cytologic test (TCT) combined with HPV-DNA detection in screening cervical cancer. 420 cervical cancer patients admitted in our hospital between April, 2011-April, 2014 were selected. All patients received TCT and HPV-DNA detection, and cervical tissue biopsy was used to confirm the diagnosis. TCT screening results showed that there were 175 patients were >ASCUS and the positive rate was 41.7%, histopathological screening showed that there were 199 patients were ≥cervical intraepithelial neoplasia (CIN) I and the positive rate was 47.4%. HPV-DNA detection showed 180 patients were positive which was 42.9%, and the positive rate of HPV-DNA detection was increased as the disease severity increased. The sensitivity of TCT combined with HPV-DNA detection was higher than single TCT or HPV-DNA, however the specificity was relatively low, and the positive predictive value and negative predictive value were higher which were similar to pathological results. TCT combined with HPV-DNA detection has high sensitivity and accuracy in screening cervical cancer, which is worthy of clinical application.

  18. A Simple Sperm DNA Toroid Integrity Test and Risk of Miscarriage

    PubMed Central

    Chan, Philip J.; Orzylowska, Eliza M.; Corselli, Johannah U.; Jacobson, John D.; Wei, Albert K.

    2015-01-01

    Current methods of analyzing sperm chromatin competency overlook the inner sperm compartment which is inaccessible to probes and reagents. By breaking the molecular protamine disulfide bridges, the DNA toroids are exposed to integrity analysis. The aim was to develop a simple nuclear toroid test and determine its association with fertilization, pregnancy, and miscarriage. The approach involved treating washed sperm remaining after ICSI procedures (N = 35 cases) with acidified Triton X-100 and dithiothreitol (DTT) before Diff-Quik staining. Percentages of sperm with normal chromatin indicated by light-colored nuclei were assessed. The toroid integrity test showed more sperm with normal chromatin in the pregnant group (73.6 ± 1.7%, mean ± SEM) when compared with the miscarriage (51.2 ± 6.6%) or nonpregnant groups (60.9 ± 4.8%). Furthermore, the toroid results were correlated with ICSI fertilization (R = 0.32, P = 0.04) and pregnancy outcome (pregnant cases 73.6 ± 1.7% versus nonpregnant 58.0 ± 3.9%, P = 0.001). ROC calculated cut-off was >70.0% for normal toroid integrity (sensitivity 0.98, specificity 0.33, and diagnostic accuracy 78.3%). An association between normal sperm toroid integrity and miscarriage was evident when the staining procedure included acidified detergent DTT pretreatment. PMID:25649376

  19. 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.

  20. Sperm DNA integrity testing: big halo is a good predictor of embryo quality and pregnancy after conventional IVF.

    PubMed

    Tandara, M; Bajić, A; Tandara, L; Bilić-Zulle, L; Šunj, M; Kozina, V; Goluža, T; Jukić, M

    2014-09-01

    Sperm DNA integrity is a sperm functional parameter of male fertility evaluation. Two parameters of sperm DNA integrity were observed: DNA damage expressed as DNA fragmentation index (DFI) and percentage of the DNA undamaged spermatozoa expressed as big halo. Halosperm test was used for sperm DNA integrity determination. The aim of this study was to evaluate which DNA integrity parameter is better as an embryo quality and pregnancy prognostic parameter after the conventional IVF. We evaluated two embryo groups (positive and negative group) according to the 3rd day cumulative embryo score. Big halo and DFI, as we expected, showed good correlation (r = -0.69; p < 0.001). Receiver operating characteristic (ROC) analyses show that DFI and big halo are significant (p < 0.001) as prognostic parameters of embryo quality. ROC curves comparison of DFI and big halo revealed the AUC value for big halo to be significantly higher (DFI AUC = 0.71 vs. big halo AUC = 0.83; p = 0.025) than for DFI. Big halo was found to be the only independent predictor of embryo quality. Sperm DNA integrity both parameters are good prognostic parameters of embryo quality after the conventional IVF where big halo seems to be better. ROC analyses show DFI and big halo as significant prognostic parameters for achieved pregnancy (AUC ± SE for DFI was 0.67 ± 0.06 and 0.75 ± 0.06 for big halo). To our knowledge, this is the first study demonstrating the correlation between sperm DNA undamaged rate expressed as big halo parameter and semen characteristics as well as the influence on fertilization rate, embryo quality and pregnancy in conventional IVF. © 2014 American Society of Andrology and European Academy of Andrology.

  1. The Relationship between the Methylated Septin-9 DNA Blood Test and Stool Occult Blood Test for Diagnosing Colorectal Cancer in Taiwanese People.

    PubMed

    Chen, Chung-Hung; Yan, Sheng-Lei; Yang, Tsung-Hsun; Chen, Shih-Feng; Yeh, Yung-Hsiang; Ou, Jing-Jim; Lin, Chien-Hua; Lee, Yueh-Tsung; Chen, Chien-Hua

    2017-01-01

    Colorectal cancer (CRC) is a common and lethal disease in the world. There is an increasing number of cases in Taiwan and a higher rate at advanced stages. The immune fecal occult blood test (iFOBT) has been used as a screening method in Taiwan for years. A new novel diagnostic tool, the Methylated Septin-9 (MS-9) DNA blood test, had been reported to have high sensitivity and specificity for CRC detection. There are no available data in Taiwan, so we conducted this prospective randomized trial to investigate the relationship among the MS-9 DNA blood test, iFOBT, and a combination of the two tests for diagnosing CRC in Taiwanese people. From July 1, 2012 to December 31, 2013, we prospectively selected 60 plasma samples from patients who were diagnosed with CRC and otherwise, the healthy group by colonoscopy in our hospital. Patients were divided into the CRC group and healthy group. CRC stages 0, I, II and stages III and IV were separately analyzed. We calculated the sensitivity and specificity of each group to determine the relationship among the MS-9 DNA blood test, iFOBT, and a combination of the two tests for diagnosing CRC in Taiwanese people. The results of the MS-9 DNA blood test for the 60 samples were divided into three groups, and the sensitivity as well as the specificity of the MS-9 DNA blood test to detect CRC were 47% and 89%, respectively. The results of iFOBT were also divided into three groups, and had higher sensitivity (84%) but lower specificity (55%) using iFOBT to detect CRC. Higher rates could be predicted to detect CRC if both the tests were positive. A combined MS-9 DNA blood test and iFOBT may help in a higher detection rate of CRC. It could be offered to individuals who are unwilling or unable to undergo colonoscopy. Further large prospective, randomized studies are needed in the future. © 2016 Wiley Periodicals, Inc.

  2. Prenatal reflex DNA screening for Down syndrome: enhancing the screening performance of the initial first trimester test.

    PubMed

    Wald, Nicholas J; Bestwick, Jonathan P

    2016-04-01

    To estimate the effect of adding three biochemical markers (alphafetoprotein, inhibin-A, and placental growth factor) and two ultrasound markers (ductus venosus pulsatility index and nasal bone examination) to enhance the initial Combined test in prenatal reflex DNA screening for Down syndrome. Published data were used to estimate screening performance [detection rates (DRs) and false-positive rates (FPRs)] of reflex DNA screening according to the additional markers used, the proportion of women with the highest initial test risks reflexed to DNA testing and the gestational age at the time of blood collection. If 10% of women are reflexed, the addition of the three biochemical markers to the Combined test increases the DR from 90.8% to 92.3% (FPR 0.025% to 0.027%) with markers measured at 11 completed weeks' gestation. With markers measured at 13 completed weeks' gestation the DR increases from 87.7% to 95.2% (FPRs both 0.027%). The further addition of the two ultrasound markers increases the DR to 96.8% and 97.5% at 11 and 13 weeks' respectively (FPR to 0.024% and 0.022% respectively). Adding the specified markers to the Combined test can maintain or improve screening performance with a lower proportion of women reflexed. Our results can be used to determine the most cost-effective reflex DNA screening policy. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

  3. Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification

    PubMed Central

    Brewer, Bonita J.; Payen, Celia; Di Rienzi, Sara C.; Higgins, Megan M.; Ong, Giang; Dunham, Maitreya J.; Raghuraman, M. K.

    2015-01-01

    DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create “closed” forks—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of

  4. Should We Perform Semen Analysis, DNA Fragmentation, and Hypo-osmotic Swelling Tests together?

    PubMed Central

    Hasanzadeh Keshteli, Samaneh; Farsi, Mir Mehrdad; Khafri, Soraya

    2016-01-01

    Semen analysis, sperm DNA fragmentation (SDF) and hypo-osmotic swelling test (HOST) are usually performed for the evaluation of sperm fertilizing ability. There are some debates over the necessity of SDF and HOST incorporation in male infertility work-up.Semen of 77 men was evaluated by SDF and HOST through three semen analyses. Sperm parameters were arranged into different categories: <5%, 5-15%, >15% for normal morphology; <50%, 50-70%, >70 % for motility; and <10, 10-20, 21-34, 35-50, >50 million/ml for concentration. SDF analysis was performed and values under 30% were assumed to be normal. Normal range of HOST was considered to be >60%.Only normal sperm morphology had significant relationship with DF rate (P<0.001). Normal morphology, motility, and concentration of sperms had significant relationship with HOST (P<0.001, 0.05, and <0.003,respectively). There was a significant negative correlation between sperm morphology and DF rate. The correlations between sperm parameters and percentage of HOST were significantly positive (r: 0.44, 0.19, and 0.32 for morphology, motility, and concentration, respectively). Receiver operating characteristic curve (ROC) showed that sperm morphology is a strong predictor of the rate of DF and HOST (accuracy: 0.74‚ and 0.81, respectively). The best sperm morphology cut off point for DF and HOST rate prediction was 4.5% and 5.5%, respectively.Sperm morphology had significant correlation with DF rate and HOST and is supposed to be a predictor for these tests. Performing these three tests collectively for evaluation of semen samples would not be necessarily required in all cases.

  5. 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

  6. [MODELS OF CLINICAL IMPLEMENTATION OF CELL FREE FETAL DNA IN THE MATERNAL SERUM SCREENING TEST-ANALYSIS].

    PubMed

    Yankova, M; Chaveeva, P; Stratieva, V

    2015-01-01

    Prenatal screening by definition is a way of identifying pregnancies, with a high enough risk to specific fetal damage as to justify the subsequent invasive diagnosis among the seemingly normal pregnancies. [1] The aim of the prenatal screening test is to reach the high diagnostic frequency (DR > 95%), with low false-positive rate (FPR < 1%). Several non-invasive prenatal tests (NIPT) are widely adopted and use in clinical practice: 1st Trimester Combined screening (First trimester Combined Screening) and 2nd trimester biochemical screening (Second trimester biochemical screening) and in the last few years through screening Fetal DNA in Maternal serum (cfDNA screening). Since the introduction of the sfDNA test were examined and discussed the results of several ways of application: (1) as a primary screening method without preceding the result of 1st trimester combined screening for chromosomal abnormalities, (2) as a contingent test after 1st trimester combined screening in high risk pregnancies (> 1:100) (3) as a contingent test after 1st trimester combined screening, when the calculated risk is between ( 1:10 to 1:2500). The purpose of the study: to compare the results of different ways of application screening through cfDNA: detection rate (DR) for Tri21, Tri18 and Tri13, procentage of invasive diagnostics and cost-effectiveness ratio of cfDNA test in comparison with the 1st trimester combined screening. To establish the most suitable algorithm for application of cfDNA test. Analyzed were the results of several randomized multi-center clinical studies whose data are processed through a meta-analysis. cfDNA-test has a higher DR for Tri21 for lower FPR, compared to the combined screening in 1st trimester (cfDNA-DR 99%, 1st trimester screening-DR 96% and 0.4%FPR, respectively FPR 5%), but although it is with better results and reduces the incidence of invasive tests, does not justify the significant difference in price-performance ratio. On the other hand cfDNA-test

  7. 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.

  8. Rapid genotyping of carcinogenic human papillomavirus by loop-mediated isothermal amplification using a new automated DNA test (Clinichip HPV™).

    PubMed

    Satoh, Toyomi; Matsumoto, Koji; Fujii, Takuma; Sato, Osamu; Gemma, Nobuhiro; Onuki, Mamiko; Saito, Hiroshi; Aoki, Daisuke; Hirai, Yasuo; Yoshikawa, Hiroyuki

    2013-03-01

    This study was designed to evaluate the Clinichip HPV test, a new DNA test that detects carcinogenic human papillomavirus (HPV) rapidly by loop-mediated isothermal amplification and performs genotyping of all 13 carcinogenic types using automated DNA chip technology with an assay time 2.5h. Using this test, 247 Japanese women (109 with normal cytology, 43 with cervical intraepithelial neoplasia grade 1, 60 with cervical intraepithelial neoplasia grade 2/3 and 35 with invasive cervical cancer) were tested for carcinogenic HPV genotypes. The results were compared to those obtained by the polymerase chain reaction-amplified DNA sequencing using 13 type-specific primers. Overall, there was very good agreement for the detection of carcinogenic HPV between the Clinichip test and direct sequencing, with 95.5% total agreement and a kappa value of 0.91. Comparison of the detection of individual HPV types shows that the overall agreement was also high (range: 96.8-100%). In women with cervical intraepithelial neoplasia grade 2 or worse, the detection rate of carcinogenic HPV was 95.7% by both the Clinichip test and the direct-sequencing method, indicating complete agreement between the two methods. In conclusion, it was found that the Clinichip test is a promising new laboratory method for genotyping of carcinogenic HPV.

  9. Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis

    PubMed Central

    Seol, Yeonee; Strub, Marie-Paule; Neuman, Keir C.

    2016-01-01

    Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis based on the Student’s t-test that accommodates continuous transitions in addition to the discrete transitions assumed in most widely employed analysis routines. To illustrate the single-molecule unwinding assay and the analysis routine, we provide DNA unwinding measurements of Escherichia coli RecQ helicase under a variety of conditions (Na+, ATP, temperature, and DNA substrate geometry). These examples reveal that DNA unwinding measurements under various conditions can aid in elucidating the unwinding mechanism of DNA helicase but also emphasize that environmental effects on DNA helicase activity must be considered in relation to in vivo activity and mechanism. PMID:27131595

  10. Horses for courses: a DNA-based test for race distance aptitude in thoroughbred racehorses.

    PubMed

    Hill, Emmeline W; Ryan, Donal P; MacHugh, David E

    2012-12-01

    Variation at the myostatin (MSTN) gene locus has been shown to influence racing phenotypes in Thoroughbred horses, and in particular, early skeletal muscle development and the aptitude for racing at short distances. Specifically, a single nucleotide polymorphism (SNP) in the first intron of MSTN (g.66493737C/T) is highly predictive of best race distance among Flat racing Thoroughbreds: homozygous C/C horses are best suited to short distance races, heterozygous C/T horses are best suited to middle distance races, and homozygous T/T horses are best suited to longer distance races. Patent applications for this gene marker association, and other linked markers, have been filed. The information contained within the patent applications is exclusively licensed to the commercial biotechnology company Equinome Ltd, which provides a DNA-based test to the international Thoroughbred horse racing and breeding industry. The application of this information in the industry enables informed decision making in breeding and racing and can be used to assist selection to accelerate the rate of change of genetic types among distinct populations (Case Study 1) and within individual breeding operations (Case Study 2).

  11. An ancient DNA test of a founder effect in Native American ABO blood group frequencies.

    PubMed

    Halverson, Melissa S; Bolnick, Deborah A

    2008-11-01

    Anthropologists have assumed that reduced genetic diversity in extant Native Americans is due to a founder effect that occurred during the initial peopling of the Americas. However, low diversity could also be the result of subsequent historical events, such as the population decline following European contact. In this study, we show that autosomal DNA from ancient Native American skeletal remains can be used to investigate the low level of ABO blood group diversity in the Americas. Extant Native Americans exhibit a high frequency of blood type O, which may reflect a founder effect, genetic drift associated with the historical population decline, or natural selection in response to the smallpox epidemics that occurred following European contact. To help distinguish between these possibilities, we determined the ABO genotypes of 15 precontact individuals from eastern North America. The precontact ABO frequencies were not significantly different from those observed in extant Native Americans from the same region, but they did differ significantly from the ABO frequencies in extant Siberian populations. Studies of other precontact populations are needed to better test the three hypotheses for low ABO blood group diversity in the Americas, but our findings are most consistent with the hypothesis of a founder effect during the initial settlement of this continent.

  12. Identification of irradiated wheat by germination test, DNA comet assay and electron spin resonance

    NASA Astrophysics Data System (ADS)

    Barros, Adilson C.; Freund, Maria Teresa L.; Villavicencio, Ana Lúcia C. H.; Delincée, Henry; Arthur, Valter

    2002-03-01

    In several countries, there has been an increase in the use of radiation for food processing thus improving the quality and sanitary conditions, inhibiting pathogenic microorganisms, delaying the natural aging process and so extending product lifetime. The need to develop analytical methods to detect these irradiated products is also increasing. The goal of this research was to identify wheat irradiated using different radiation doses. Seeds were irradiated with a gamma 60Co source (Gammacell 220 GC) in the Centro de Energia Nuclear na Agricultura and the Instituto de Pesquisas Energéticas e Nucleares. Dose rate used were 1.6 and 5.8kGy/h. Applied doses were 0.0, 0.10, 0.25, 0.50, 0.75, 1.0, and 2.0kGy. After irradiation, seeds were analysed over a 6 month period. Three different detection methods were employed to determine how irradiation had modified the samples. Screening methods consisted of a germination test measuring the inhibition of shooting and rooting and analysis of DNA fragmentation. The method of electron spin resonance spectroscopy allowed a better dosimetric evaluation. These techniques make the identification of irradiated wheat with different doses possible.

  13. Rapid method for testing susceptibility of Mycobacterium tuberculosis by using DNA probes.

    PubMed Central

    Martin-Casabona, N; Xairó Mimó, D; González, T; Rossello, J; Arcalis, L

    1997-01-01

    The increasing number of multidrug-resistant Mycobacterium tuberculosis strains has stimulated the interest of investigators in finding a rapid method for susceptibility testing. We used commercially available rRNA DNA-bioluminescence-labelled probes (Accu-Probe, Gen Probe, Inc. San Diego, Calif.) for this purpose. The study was performed in three chronological steps. (i) We studied the correlation between the photometric light units (PLUs) given by the hybridization method, the numbers of CFU per milliliter, and turbidity as nephelometric units for six different inocula of an M. tuberculosis strain over 14 days. A good correlation (c > 0.9; P < 0.05) was found from the third day for all concentrations used. (ii) Over a period of 14 days we studied the evolution of the PLUs for 20 strains growing in medium with 0.2 microl of isoniazid (H) per ml and 18 strains in medium with 1 microl of rifampin (R) per ml to standardize the method. Susceptible and resistant strains were used according to the reference proportions method in Middlebrook 7H10, and the MICs were determined in solid and liquid media. The final inoculum of a 10(-2) dilution from a McFarland no. 1 standard and reading at 3 and 5 days provided the best results. A quotient was established to find a cutoff point between resistant and susceptible strains. (iii) We used the standardized parameters in 117 tests with H and R. On day 3, the sensitivity, specificity, positive predictive value, and negative predictive value for detecting resistant strains were 86.8, 100, 100, and 90.1%, respectively, and on day 5 they were 96.2, 100, 100, and 94%, respectively. We concluded that the method is readily available, is easy to perform, and could be useful for screening resistant M. tuberculosis strains. PMID:9316900

  14. Deep-Sequencing Technologies and Potential Applications in Forensic DNA Testing.

    PubMed

    Zascavage, R R; Shewale, S J; Planz, J V

    2013-03-01

    Development of second- and third-generation DNA sequencing technologies have enabled an increasing number of applications in different areas such as molecular diagnostics, gene therapy, monitoring food and pharmaceutical products, biosecurity, and forensics. These technologies are based on different biochemical principles such as monitoring released pyrophosphate upon incorporation of a base (pyrosequencing), fluorescence detection subsequent to reversible incorporation of a fluorescently labeled terminator base, ligation based approach wherein fluorescence of cleaved nucleotide after ligation is measured, measuring the proton released after incorporation of a base (semiconductor-based sequencing), monitoring incorporation of a nucleotide by measuring the fluorescence of the fluorophore attached to the phosphate chain of the nucleotide, and by detecting the altered charge in a protein nanopore due to released nucleotide by exonuclease cleavage of a DNA strand. Analysis of multiple DNA fragments in parallel increases the depth of coverage while decreasing labor, cost, and time, highlighting some major advantages of deep-sequencing technologies. DNA sequencing has been routinely used in the forensic laboratories for mitochondrial DNA analysis. Fragment analysis, however, is the preferred method for Short Tandem Repeat genotyping due to the cumbersome and costly nature of fi rst-generation DNA sequencing methodologies. Deep-sequencing technologies have brought a new perspective to forensic DNA analysis. Studies include STR analysis to reveal hidden variation in the repeat regions, mtDNA sequencing, Single Nucleotide Polymorphism analysis, mixture resolution, and body fluid identification. Recent publications reveal that attempts are being made to expand the capability.

  15. 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.

  16. 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

  17. Arrested Spermatogenesis and Evidence for DNA Damage in PTIP Mutant Testes

    PubMed Central

    Schwab, Kristopher R.; Smith, Gary D.; Dressler, Gregory R.

    2012-01-01

    The differentiation of mature sperm from male germ cells requires both chromatin remodeling and compaction as well as DNA double stranded break repair of sister chromatids. We examined the function of PTIP, a protein implicated in both DNA repair and histone methylation, during spermatogenesis by using a conditional, inducible mutation in adult male mice. Loss of PTIP led to the developmental arrest of spermatocytes, testicular atrophy, and infertility. By immunostaining with specific markers for different stages of spermatogenesis and for proteins involved in DNA damage and repair mechanisms, we conclude that the lack of PTIP results in genomic instability and DNA damage resulting in the cessation of spermatogenesis in meiosis I. These data underscore the importance of PTIP in the DNA repair process associated with the development of mature spermatozoa. PMID:23063797

  18. Comparison of Human Papillomavirus Detection in Urine and Cervical Samples Using High-Risk HPV DNA Testing in Northern Thailand

    PubMed Central

    Settakorn, Jongkolnee; Sukpan, Kornkanok; Lekawanvijit, Suree; Katruang, Narisara; Siriaunkgul, Sumalee

    2016-01-01

    Objective. To evaluate the performance of high-risk human papillomavirus (HPV) DNA testing in urine samples compared to that of cervical sample testing in Northern Thailand. Methods. Paired urine and cervical samples were collected during the follow-up of women with a previous positive HPV test. HPV testing was performed using the Cobas 4800 HPV Test. Linear Array assay was used for genotyping in selected cases. Results. Paired urine and cervical samples were obtained from 168 women. Of 123 paired samples with valid results, agreement in the detection of high-risk HPV DNA was present in 106 cases (86.2%), with a kappa statistic of 0.65 (substantial agreement). Using the cervical HPV results as a reference, the sensitivity of urine HPV testing was 68.6% (24/35) and the specificity 93.2% (82/88). For the detection of histologic high-grade squamous intraepithelial lesion or worse (HSIL+), the sensitivity of urine HPV testing was 80.0% (4/5) and the specificity 78.0% (92/118). Conclusion. Although urine HPV testing had a rather low sensitivity for HPV detection, its sensitivity for histologic HSIL+ detection was high. For clinical use of urine HPV testing, standardization of specimen collection and processing techniques or application of a more sensitive test, especially in the detection of HPV52 and HPV58, is necessary. PMID:28101107

  19. Multilocus phylogeny of alligator lizards (Elgaria, Anguidae): Testing mtDNA introgression as the source of discordant molecular phylogenetic hypotheses.

    PubMed

    Leavitt, Dean H; Marion, Angela B; Hollingsworth, Bradford D; Reeder, Tod W

    2017-05-01

    The increased availability of nuclear DNA sequence data has led to a better appreciation of the role and frequency of introgressive hybridization and subsequent mitochondrial capture in misleading phylogenetic hypotheses based on mtDNA sequence data alone. Relationships among members of the alligator lizard genus Elgaria have been addressed with morphology, allozyme and mtDNA sequence data with discordant results. In this study, we use seven nuclear loci (total of 5.9kb) and ∼3kb of mtDNA to infer the phylogenetic relationships among Elgaria species and test whether the discordance among previous phylogenetic hypotheses is due to introgression and mtDNA capture. While gene tree topologies varied among the different loci, we recovered a well-resolved coalescent-based species tree. Contrary to our expectations, the nDNA-only species tree does not support the sister relationship between E. kingii and E. panamintina inferred from the previous allozyme study. Nevertheless, we found evidence for possible mitochondrial capture in two unexpected situations. The first instance of mtDNA capture involves E. paucicarinata from the Cape Region of Baja California. MtDNA recovered a clade comprising E. paucicarinata and the other two peninsular endemics, while the nDNA-only species tree recovered E. paucicarinata as sister to the continental E. kingii. We hypothesize that this discordance is the result of ancient mitochondrial capture rather than incomplete lineage sorting. Additionally, analyses of nDNA recovered E. panamintina as sister to an E. multicarinata North lineage, whereas the mtDNA gene tree recovers E. panamintina nested within a southern E. multicarinata clade. We hypothesize that this discordance also may be due to mitochondrial capture. Additionally, hybridization between these two lineages may have resulted in geographically limited nuclear introgression. Divergence dating analyses suggest that oviparous Elgaria species diverged within a relatively narrow

  20. Highly sensitive faecal DNA testing of TWIST1 methylation in combination with faecal immunochemical test for haemoglobin is a promising marker for detection of colorectal neoplasia.

    PubMed

    Suehiro, Yutaka; Zhang, Yibo; Hashimoto, Shinichi; Takami, Taro; Higaki, Shingo; Shindo, Yoshitaro; Suzuki, Nobuaki; Hazama, Shoichi; Oka, Masaaki; Nagano, Hiroaki; Sakaida, Isao; Yamasaki, Takahiro

    2017-01-01

    Background As TWIST1 methylation is specific to colorectal neoplasia, detection of TWIST1 methylation from faeces samples might be useful for colorectal neoplasia screening. However, because the content of human DNA in faeces is very small, it is very difficult to detect TWIST1 methylation by conventional bisulphite-based methylation assays. Therefore, we developed a new methylation assay without bisulphite treatment, the combined restriction digital PCR assay, and evaluated its sensitivity and specificity in combination with and without the faecal immunochemical test for haemoglobin for colorectal neoplasia detection from faeces samples. Methods For the combined restriction digital PCR assay, DNA was treated with three methylation-sensitive restriction enzymes and an exonuclease, followed by measurement of TWIST1 methylation level by droplet digital PCR. Faecal DNA testing and faecal immunochemical test for haemoglobin were performed on 109 patients with colorectal neoplasia and 10 control individuals. Results Basic performance testing showed that the combined restriction digital PCR assay enabled detection of 0.14% of the TWIST1 methylation level for the lymphocyte DNA. The combined restriction digital PCR assay from faeces samples had a sensitivity of 22.2% (95% confidence interval, 2.8-60.0%) for non-advanced adenoma, 47.1% (95% confidence interval, 23.0-72.2%) for advanced adenoma, and 33.7% (95% confidence interval, 23.7-45.0%) for colorectal cancer, and a specificity of 100.0%. Combination of faecal immunochemical test for haemoglobin and faecal combined restriction digital PCR assay increased sensitivity to 82.4% (95% confidence interval, 56.6-96.2%) for the detection of advanced adenoma. Conclusions We developed the combined restriction digital PCR assay, a possible highly sensitive methylation assay. Combination of faecal combined restriction digital PCR assay with faecal immunochemical test for haemoglobin may provide an alternative screening strategy

  1. The prevalence and significance of high-risk human papillomavirus DNA test in southern Malaysia and Singapore.

    PubMed

    Tay, Sun-Kuie; Tay, Yeng-Kwang

    2009-06-01

    To investigate the prevalence of high-risk human papillomavirus (HPV) and its associated cytological abnormalities among women attending cervical screening clinics in southern Malaysia and Singapore. Laboratory results of Hybrid Capture-II (Digene) HPV DNA and liquid-based cytology tests of consecutive women who had screening performed between January 2004 and December 2006 were studied retrospectively. Of 2364 women studied, the overall prevalence of high-risk HPV DNA detection rate was 25.6%. The prevalence peaked at 49.1% for women between 20 and 24 years old and declined to 23% among women between the age of 30 and 49 years. A small second peak of prevalence rate of 30% was observed among women above the age of 50 years old. 76.1% of the high-risk HPV infection regressed within the study period. An incidence infection rate of 16% was noted among a small group of women who had a second HPV DNA test. A total of 1153 women had both the HPV DNA and the cytology tests. Cytological abnormality (ASCUS or more) was detected in 8.9% in HPV DNA-positive group and in 3.1% in HPV DNA-negative group (P < 0.001). The risk ratio for HSIL was 9.8 for HPV-positive women compared to HPV-negative women. The prevalence of cytological abnormalities increased with increasing age of the women. The epidemiology and clinical impact of high-risk HPV infection for women in Southern Malaysia and Singapore were indistinguishable from experience elsewhere. The apparent moderately high incidence of cervical cancer was explainable by suboptimal screening program.

  2. Utility of Filter Paper for Preserving Insects, Bacteria, and Host Reservoir DNA for Molecular Testing

    PubMed Central

    Karimian, F; Sedaghat, MM; Oshaghi, MA; Mohtarami, F; Dehkordi, A Sanei; Koosha, M; Akbari, S; Hashemi-Aghdam, SS

    2011-01-01

    Background: Appropriate methodology for storage biological materials, extraction of DNA, and proper DNA preservation is vital for studies involving genetic analysis of insects, bacteria, and reservoir hosts as well as for molecular diagnostics of pathogens carried by vectors and reservoirs. Here we tried to evaluate the utility of a simple filter paper-based for storage of insects, bacteria, rodent, and human DNAs using PCR assays. Methods: Total body or haemolymph of individual mosquitoes, sand flies or cockroaches squashed or placed on the paper respectively. Extracted DNA of five different bacteria species as well as blood specimens of human and great gerbil Rhombomys opimus was pipetted directly onto filter paper. The papers were stored in room temperature up to 12 months during 2009 until 2011. At monthly intervals, PCR was conducted using a 1-mm disk from the DNA impregnated filter paper as target DNA. PCR amplification was performed against different target genes of the organisms including the ITS2-rDNA of mosquitoes, mtDNA-COI of the sand flies and cockroaches, 16SrRNA gene of the bacteria, and the mtDNA-CytB of the vertebrates. Results: Successful PCR amplification was observed for all of the specimens regardless of the loci, taxon, or time of storage. The PCR amplification were ranged from 462 to 1500 bp and worked well for the specified target gene/s. Time of storage did not affect the amplification up to one year. Conclusion: The filter paper method is a simple and economical way to store, to preserve, and to distribute DNA samples for PCR analysis. PMID:22808417

  3. False-negative post-18-month confirmatory HIV tests in HIV DNA PCR-positive children: a retrospective analysis.

    PubMed

    Garcia-Prats, Anthony J; Draper, Heather R; Sanders, Jill E; Agrawal, Anurag K; Mohapi, Edith Q; Schutze, Gordon E

    2012-09-24

    The WHO guidelines for children less than 18 months old diagnosed with HIV based on presumptive clinical diagnosis or one virologic test recommend confirmatory HIV antibody testing after 18 months of age. This study describes post-18-month HIV test results following this WHO-recommended confirmatory testing strategy. Case series and retrospective review of routine program data. Children enrolled at the Baylor Children's Clinical Center of Excellence, a pediatric and family HIV clinic in Maseru, Lesotho from December 2005 through January 2009 with a positive HIV DNA PCR at less than 18 months of age and HIV rapid test results after 18 months of age were included. Post-18-month confirmatory HIV test results are described. Factors associated with non-positive confirmatory rapid tests were determined using binary logistic regression. Of the 109 children meeting inclusion criteria, 22 (20.2%) had negative and 27 (24.8%) discordant confirmatory rapid tests. Forty-six of these 49 were on antiretroviral therapy (ART). Among these 49, 11 of 24 post-18-month HIV DNA PCRs were negative, whereas nine of 10 post-18-month HIV ELISAs were positive; 29 were definitively and 17 probably HIV-infected, two were uninfected, and one had undetermined status. Only age less than 9 months at ART initiation (odds ratio 4.25, P = 0.002) was associated with non-positive rapid tests. False-negative post-18-month confirmatory rapid tests and HIV DNA PCRs in children on ART are common, associated with early ART initiation, and may lead to inappropriate ART discontinuation and discharge from care of truly HIV-infected children.

  4. 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

  5. Development and Testing of a DNA Macroarray To Assess Nitrogenase (nifH) Gene Diversity

    PubMed Central

    Steward, Grieg F.; Jenkins, Bethany D.; Ward, Bess B.; Zehr, Jonathan P.

    2004-01-01

    A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml−1, a half-saturation of signal at 0.26 ng ml−1, and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment. PMID:15006766

  6. Stool DNA Testing for the Detection of Pancreatic Cancer: Assessment of Methylation Marker Candidates

    PubMed Central

    Kisiel, John B.; Yab, Tracy C.; Taylor, William R.; Chari, Suresh T.; Petersen, Gloria M.; Mahoney, Douglas W.; Ahlquist, David A.

    2011-01-01

    BACKGROUND Pancreatic cancer (PanC) presents at late stage with high mortality. Effective early detection methods are needed. Aberrantly methylated genes are unexplored as markers for noninvasive detection by stool testing. We aimed to select discriminant methylated genes and to assess accuracy of these and mutant KRAS in stool to detect PanC. METHODS Nine target genes were assayed by real-time methylation-specific PCR (MSP) in bisulfite-treated DNA from microdissected frozen specimens of 24 PanC cases and 30 normal colon controls. Archived stools from 58 PanC cases and 65 controls matched on sex, age, and smoking were analyzed. Target genes from fecal supernatants were enriched by hybrid capture, bisulfite-treated, and assayed by MSP. KRAS mutations were assayed using the QuARTS technique. RESULTS Areas under the receiver operating characteristics curves (AUCs) for tissue BMP3, NDRG4, EYA4, UCHL1, MDFI, Vimentin, CNTNAP2, SFRP2 and TFPI2 were 0.90, 0.79, 0.78, 0.78, 0.77, 0.77, 0.69, 0.67, and 0.66, respectively. The top 4 markers and mutant KRAS were evaluated in stool. BMP3 was the most discriminant methylation marker in stool. At 90% specificity: methylated BMP3 alone detected 51% of PanCs, mutant KRAS detected 50%, and combination detected 67%. AUCs for methylated BMP3, mutant KRAS, and combination in stool were 0.73, 0.75, and 0.85, respectively. CONCLUSIONS This study demonstrates that stool assay of a methylated gene marker can detect PanC. Among candidate methylated markers discriminant in tissue, BMP3 alone performed well in stool. Combining methylated BMP3 and mutant KRAS increased stool detection over either marker alone. PMID:22083596

  7. Paternity testing using microsatellite DNA markers in captive Adélie penguins (Pygoscelis adeliae).

    PubMed

    Sakaoka, Ken; Suzuki, Isao; Kasugai, Naeko; Fukumoto, Yohei

    2014-01-01

    We investigated the paternity of 39 Adélie penguins (Pygoscelis adeliae) hatched at the Port of Nagoya Public Aquarium between 1995 and 2005 breeding seasons using microsatellite DNA markers. Among the 13 microsatellite marker loci tested in this study, eight markers amplified and were found to be polymorphic in the colony's founders of the captive population (n = 26). Multiple marker analysis confirmed that all the hatchlings shared alleles with their social fathers and that none of them were sired by any male (all males ≥4 years old in the exhibit tank during each reproductive season; n = 9-15) other than the one carrying out parental duties, except in the case of two inbred hatchlings whose half-sibling parents shared the same father. These results demonstrated that extra-pair paternity (EPP) did not occur in this captive population and that even if EPP has been detected among them, the probability of excluding all other possible fathers in the exhibit tank is extremely high based on paternity exclusion probabilities across the investigated loci. The paternity exclusion probabilities were almost the same between 1994 and 2005. The probability of identity across the investigated loci declined between the two time points, but was still high. These results are reflected in a very short history of breeding in this captive population. In other words, the parentage analyses using a suite of microsatellite markers will be less effective as generations change in small closed populations, such as zoo and aquarium populations. © 2014 Wiley Periodicals, Inc.

  8. Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability.

    PubMed

    Morris, Ulrika; Aydin-Schmidt, Berit; Shakely, Delér; Mårtensson, Andreas; Jörnhagen, Louise; Ali, Abdullah S; Msellem, Mwinyi I; Petzold, Max; Gil, José P; Ferreira, Pedro E; Björkman, Anders

    2013-03-19

    The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations. DNA was extracted from two RDT devices (Paracheck-Pf® and SD Bioline Malaria Pf/Pan®), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman® 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance. The P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples. The results support RDTs as a valuable source of parasite DNA and

  9. Performance of genotypic tropism testing on proviral DNA in clinical practice: results from the DIVA study group.

    PubMed

    Svicher, Valentina; Alteri, Claudia; Montano, Marco; D'Arrigo, Roberta; Andreoni, Massimo; Angarano, Gioacchino; Antinori, Andrea; Antonelli, Guido; Allice, Tiziano; Bagnarelli, Patrizia; Baldanti, Fausto; Bertoli, Ada; Borderi, Marco; Boeri, Enzo; Bon, Isabella; Bruzzone, Bianca; Callegaro, Anna Paola; Capobianchi, Maria Rosaria; Carosi, Giampiero; Cauda, Roberto; Ceccherini-Silberstein, Francesca; Clementi, Massimo; Chirianni, Antonio; Colafigli, Manuela; D'Arminio Monforte, Antonella; De Luca, Andrea; Di Biagio, Antonio; Di Nicuolo, Giuseppe; Di Perri, Giovanni; Di Pietro, Massimo; Di Santo, Fabiola; Fabeni, Lavinia; Fadda, Giovanni; Galli, Massimo; Gennari, William; Ghisetti, Valeria; Giacometti, Andrea; Gori, Caterina; Gori, Andrea; Gulminetti, Roberto; Leoncini, Francesco; Maffongelli, Gaetano; Maggiolo, Franco; Manca, Giuseppe; Gargiulo, Franco; Martinelli, Canio; Maserati, Renato; Mazzotta, Francesco; Meini, Genny; Micheli, Valeria; Monno, Laura; Mussini, Cristina; Narciso, Pasquale; Nozza, Silvia; Paolucci, Stefania; Pal, Giorgio; Parisi, Saverio; Parruti, Giustino; Pignataro, Angela Rosa; Pollicita, Michela; Quirino, Tiziana; Re, Maria Carla; Rizzardini, Giuliano; Santangelo, Rosaria; Scaggiante, Renzo; Sterrantino, Gaetana; Turriziani, Ombretta; Vatteroni, Maria Linda; Vecchi, Laura; Viscoli, Claudio; Vullo, Vincenzo; Zazzi, Maurizio; Lazzarini, Adriano; Perno, Carlo Federico

    2012-01-01

    The DIVA study is aimed at setting up a standardized genotypic tropism-testing on proviral-DNA for the routine clinical diagnostic-laboratory. Twelve local centres and 5 reference centres (previously cross-validated) were identified. For inter-center validation-procedure, 60 peripheral-blood mononuclear cells (PBMCs) aliquots from 45 HAART-treated patients were randomly chosen for population V3 sequencing on proviral-DNA at local HIV centre and at reference-laboratory. Viral tropism was predicted by Geno2Pheno algorithm (False Positive Rate [FPR] = 20%) as proposed by the European-Guidelines. Quantification of total HIV-1 DNA was based on a method described by Viard (2004). Quantification of HIV-1 DNA was available for 35/45 (77.8%) samples, and gave a median value of 598 (IQR:252- 1,203) copies/10 PBMCs. A total of 56/60 (93.3%) samples were successfully amplified by both the reference and the local virological centers. The overall concordance of tropism prediction between local and reference centers was 54/56 (96.4%). Results of tropism prediction by local centers were: 33/54 (61.1%) R5 and 21/54 (38.9%) X4/DM. There was high concordance in the genotypic tropism prediction based on proviral DNA among different virological centers throughout Italy. Our results are in line with other European studies, and support the use of genotypic tropism testing on proviral DNA in patients with suppressed plasma HIV-1 RNA candidate to CCR5-antagonist treatment.

  10. A significantly lower potency observed for the 3rd WHO International Standard for Parvovirus B19V DNA with the cobas TaqScreen DPX test.

    PubMed

    Pisani, G; Cristiano, K; Fabi, S; Simeoni, M; Marino, F; Gaggioli, A

    2016-08-01

    In the context of the Official Medicines Control Laboratories plasma pool testing for Parvovirus B19 DNA, we use the cobas TaqScreen DPX test. When we re-evaluated this method using the 3rd B19 DNA WHO IS at the final concentration of 4 log IU/mL, we observed a titre lower than expected, i.e. 3.79 log IU/mL. Therefore, we further investigated the accuracy of the DPX test. The following B19V DNA materials were tested by using both the DPX test and an in-house real-time PCR: The 1st, 2nd and 3rd WHO ISs for B19V DNA The Non WHO B19V DNA Reference Material for NAT The Biological Reference Preparation B19 virus DNA for NAT testing, batch 1 . The DPX test showed a good accuracy for all B19V DNA materials with the exception of the 3rd WHO IS for B19V DNA. In fact, an underestimation of about 38% was observed for all dilutions of this standard with respect to the nominal titre. With the B19V in-house real-time PCR, all four materials proved to be well calibrated against the 1(st) WHO IS for B19V DNA, used as external standard curve. In this study, we demonstrated that the DPX test underestimates the B19V DNA content of the 3rd WHO IS for B19V DNA and that this is not due to an incorrect potency assigned to the standard but, most probably, to a mismatch between the primers/probe and the sequence of the target region in the 3rd WHO IS for B19V DNA. © 2016 International Society of Blood Transfusion.

  11. Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

    PubMed Central

    Lovell, C R; Hui, Y

    1991-01-01

    The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera. A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism. No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway. DNA purified from cells extracted from horse manure was also screened with the acetogen probe. Six hybrids, indicating at least six detectable acetogen "strains," were observed. Images PMID:1768134

  12. Quantitative field testing Rotylenchulus reniformis DNA from metagenomic samples isolated directly from soil.

    PubMed

    Showmaker, Kurt; Lawrence, Gary W; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P

    2011-01-01

    A quantitative PCR procedure targeting the β-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil.

  13. Speciation and DNA barcodes: testing the effects of dispersal on the formation of discrete sequence clusters.

    PubMed

    Papadopoulou, Anna; Bergsten, Johannes; Fujisawa, Tomochika; Monaghan, Michael T; Barraclough, Timothy G; Vogler, Alfried P

    2008-09-27

    Large-scale sequencing of short mtDNA fragments for biodiversity inventories ('DNA barcoding') indicates that sequence variation in animal mtDNA is highly structured and partitioned into discrete genetic clusters that correspond broadly to species-level entities. Here we explore how the migration rate, an important demographic parameter that is directly related to population isolation, might affect variation in the strength of mtDNA clustering among taxa. Patterns of mtDNA variation were investigated in two groups of beetles that both contain lineages occupying habitats predicted to select for different dispersal abilities: predacious diving beetles (Dytiscidae) in the genus Bidessus from lotic and lentic habitats across Europe and darkling beetles (Tenebrionidae) in the genus Eutagenia from sand and other soil types in the Aegean Islands. The degree of genetic clustering was determined using the recently developed 'mixed Yule coalescent' (MYC) model that detects the transition from between-species to within-population branching patterns. Lineages from presumed stable habitats, and therefore displaying lower dispersal ability and migration rates, showed greater levels of mtDNA clustering and geographical subdivision than their close relatives inhabiting ephemeral habitats. Simulations of expected patterns of mtDNA variation under island models showed that MYC clusters are only detected when the migration rates are much lower than the value of Nm=1 typically used to define the threshold for neutral genetic divergence. Therefore, discrete mtDNA clusters provide strong evidence for independently evolving populations or species, but their formation is suppressed even under very low levels of dispersal.

  14. Randomized trial evaluating self-sampling for HPV DNA based tests for cervical cancer screening in Nigeria.

    PubMed

    Modibbo, Fatima; Iregbu, K C; Okuma, James; Leeman, Annemiek; Kasius, Annemieke; de Koning, Maurits; Quint, Wim; Adebamowo, Clement

    2017-01-01

    Cervical cancer incidence and mortality rates in Sub-Saharan Africa (SSA) remain high due to several factors including low levels of uptake of cervical cancer screening. Self-collection of cervicovaginal samples for HPV DNA testing may be an effective modality that can increase uptake of cervical cancer screening in SSA and hard to reach populations in developed countries. We investigated whether self-collection of cervicovaginal samples for HPV DNA tests would be associated with increased uptake of screening compared with clinic based collection of samples. Furthermore, we compared the quality of samples collected by both approaches for use in HPV genotyping. We conducted a community based randomized trial in a semi-urban district of Abuja, Nigeria with 400 women, aged 30 to 65 years randomized to either hospital-collection or self-collection of cervicovaginal samples. We compared cervical cancer screening uptake among the 2 groups and evaluated the concentration of human DNA in the samples by measuring RNase P gene levels using qPCR. High-risk HPV DNA detection and typing was done using the GP5+/6+ Luminex system. Most participants in the self-collection arm (93%, 185/200) submitted their samples while only 56% (113/200) of those invited to the hospital for sample collection attended and were screened during the study period (p value < 0.001). Human genomic DNA was detected in all but five (1.7%) participants, all of whom were in the self-collection arm. The prevalence of high-risk HPV in the study population was 10% with types 35, 52 and 18 being the commonest. Our study shows that self-sampling significantly increased uptake of HPV DNA based test for cervical cancer screening in this population and the samples collected were adequate for HPV detection and genotyping. Cervical cancer screening programs that incorporate self-sampling and HPV DNA tests are feasible and may significantly improve uptake of cervical cancer screening in SSA.

  15. Using the Cancer Risk Management Model to evaluate the health and economic impacts of cytology compared with human papillomavirus DNA testing for primary cervical cancer screening in Canada

    PubMed Central

    Popadiuk, C.; Gauvreau, C.L.; Bhavsar, M.; Nadeau, C.; Asakawa, K.; Flanagan, W.M.; Wolfson, M.C.; Coldman, A.J.; Memon, S.; Fitzgerald, N.; Lacombe, J.; Miller, A.B.

    2016-01-01

    Background In Canada, discussion about changing from cytology to human papillomavirus (hpv) dna testing for primary screening in cervical cancer is ongoing. However, the Canadian Task Force on Preventive Health Care has not yet made a recommendation, concluding that the evidence is insufficient. Methods We used the cervical cancer and hpv transmission models of the Cancer Risk Management Model to study the health and economic outcomes of primary cytology compared with hpv dna testing in 14 screening scenarios with varying screening modalities and intervals. Projected cervical cancer cases, deaths, colposcopies, screens, costs, and incremental cost-effectiveness were evaluated. We performed sensitivity analyses for hpv dna test costs. Results Compared with triennial cytology from age 25, 5-yearly hpv dna screening alone from age 30 resulted in equivalent incident cases and deaths, but 55% (82,000) fewer colposcopies and 43% (1,195,000) fewer screens. At hpv dna screening intervals of 3 years, whether alone or in an age-based sequence with cytology, screening costs are greater, but at intervals of more than 5 years, they are lower. Scenarios on the cost-effectiveness frontier were hpv dna testing alone every 10, 7.5, 5, or 3 years, and triennial cytology starting at age 21 or 25 when combined with hpv dna testing every 3 years. Conclusions Changing from cytology to hpv dna testing as the primary screening test for cervical cancer would be an acceptable strategy in Canada with respect to incidence, mortality, screening and diagnostic test volumes. PMID:26985148

  16. Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods.

    PubMed

    Dunlop, J; Thompson, C K; Godfrey, S S; Thompson, R C A

    2014-11-01

    Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results.

  17. Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species

    PubMed Central

    Loureiro, João; Rodriguez, Eleazar; Doležel, Jaroslav; Santos, Conceição

    2007-01-01

    Background and Aims After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry. Methods GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of particles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the buffer that performed better for a given species. Key Results In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms in comparison with previously published buffers was achieved in most of the species analysed. Conclusions WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species. Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB or WPB is suitable for preparation of high

  18. Impediments to DNA testing and cascade screening for hypertrophic cardiomyopathy and Long QT syndrome: a qualitative study of patient experiences.

    PubMed

    Smart, Andrew

    2010-12-01

    This paper reports data from a qualitative study of patient experiences of DNA testing and cascade screening for hypertrophic cardiomyopathy and long QT syndrome, cardiac conditions that place sufferers at risk of sudden death. The paper particularly focuses on potential impediments to testing and screening. Semi-structured interviews were undertaken with a purposive sample of 27 people in the UK who had undergone testing. In the context of the uncertainties that can characterize experiences of these disorders, the majority of participants in this sample embraced testing and screening as a way of providing health information for themselves or their relatives (particularly children). There was nevertheless evidence of ambivalence about the value and impact of the DNA test information which could influence participants' dispositions toward testing, and play into dilemmas about family communication. Other concerns arose in relation to communicating about these disorders, decisions to involve elderly relatives and pressures relating to family responsibility. The evidence of ambivalence provides insight into why some people may be resistant to testing, screening and sharing information. The findings about communication processes indicate potential areas of concern for the cascading process.

  19. Dipstick test for DNA-based food authentication. Application to coffee authenticity assessment.

    PubMed

    Trantakis, Ioannis A; Spaniolas, Stelios; Kalaitzis, Panagiotis; Ioannou, Penelope C; Tucker, Gregory A; Christopoulos, Theodore K

    2012-01-25

    This paper reports DNA-based food authenticity assays, in which species identification is accomplished by the naked eye without the need of specialized instruments. Strongly colored nanoparticles (gold nanoparticles) are employed as reporters that enable visual detection. Furthermore, detection is performed in a low-cost, disposable, dipstick-type device that incorporates the required reagents in dry form, thereby avoiding multiple pipetting and incubation steps. Due to its simplicity, the method does not require highly qualified personnel. The procedure comprises the following steps: (i) PCR amplification of the DNA segment that flanks the unique SNP (species marker); (ii) a 15 min extension reaction in which DNA polymerase extends an allele-specific primer only if it is perfectly complementary with the target sequence; (iii) detection of the products of the extension reaction within a few minutes by the naked eye employing the dipstick. No purification is required prior to application of the extension products to the dipstick. The method is general and requires only a unique DNA sequence for species discrimination. The only instrument needed is a conventional thermocycler for PCR, which is common equipment in every DNA laboratory. As a model, the method was applied to the discrimination of Coffea robusta and arabica species in coffee authenticity assessment. As low as 5% of Robusta coffee can be detected in the presence of Arabica coffee.

  20. [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

  1. 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.

  2. Genotypic Tropism Testing in HIV-1 Proviral DNA Can Provide Useful Information at Low-Level Viremia

    PubMed Central

    Fabeni, Lavinia; Berno, Giulia; Svicher, Valentina; Ceccherini-Silberstein, Francesca; Gori, Caterina; Bertoli, Ada; Mussini, Cristina; Lichtner, Miriam; Zaccarelli, Mauro; Ammassari, Adriana; Pinnetti, Carmela; Cicalini, Stefania; Mastroianni, Claudio Maria; Andreoni, Massimo; Antinori, Andrea; Perno, Carlo Federico

    2015-01-01

    The possibility of performing genotypic tropism testing (GTT) with proviral DNA (pvDNA) even during suppressed viremia would facilitate the use of CCR5 inhibitors as part of switching, simplification, or intensification strategies. Thus, we aimed to evaluate the tropism concordance between plasma RNA and pvDNA samples and to assess which factors could affect possible discrepancies between the two compartments. GTT was performed using both plasma RNA and pvDNA from 55 sample pairs from drug-experienced patients. Potential differences between the two compartments were evaluated by analyzing coreceptor usage and genetic variability. Paired samples were also stratified in three levels of viremia (<50, 51 to 500, and >500 copies/ml). Overall, Geno2Pheno comparisons of false-positive rates in the two compartments showed good correlation (r = 0.72). A high level of concordance in tropism predictions for the two compartments was found (46/55 sample pairs [83.6%]). Among the 9 sample pairs with discordant tropisms, a larger proportion of pvDNA samples harboring CXCR4/dual-mixed-tropic viruses was found, in comparison with plasma RNA samples (88.9% versus 11.1%; P = 0.0034). Discordant samples were characterized by greater genetic variability than were concordant samples. With stratification of the paired samples according to viremia levels, the prevalence of discordant samples decreased with increasing viremia (<50 copies/ml, 21.4%; 51 to 500 copies/ml, 15.4%; >500 copies/ml, 6.7%; P = 0.2). Our findings confirm that prediction of viral tropism using pvDNA is feasible even in low-level viremia and provides useful information for therapy optimization for patients with low or suppressed viremia. PMID:26135872

  3. 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.

  4. An integrated system of ABO typing and multiplex STR testing for forensic DNA analysis.

    PubMed

    Jiang, Xianhua; He, Juan; Jia, Fei; Shen, Hongying; Zhao, Jinling; Chen, Chuguang; Bai, Liping; Liu, Feng; Hou, Guangwei; Guo, Faye

    2012-12-01

    A new amplification system for ABO and STR genotyping in a single reaction has been successfully developed. Two types of information can be obtained from a biological sample at one time. One is the classical information of ABO blood group typing for screening suspects and the other is STR information for individual identification. The system allows for the simultaneous detection of 15 autosomal STR loci (containing all CODIS STR loci as well as Penta D and Penta E), six ABO genotypes (O/O, B/B, A/A, A/O, A/B, and B/O) and the gender-determining locus Amelogenin. Primers are designed so that the amplicons are distributed ranging from 75bp to 500bp within a four-dye fluorescent design, leaving a fourth dye for the internal size standard. With 30 cycles, the results showed that the optimal amount of DNA template for this multiplex ranges from 250pg to 2ng and the lowest detection threshold is 125pg (as low as 63pg for ABO loci). For the DNA template outside the optimal detection range, we could adjust the number of cycles to obtain the robust profiles. Mixture studies showed that over 83% of minor alleles were detected at 1:9 ratios. The full profiles were still observed when 4ng of degraded DNA was digested by DNase I and 1ng undegraded DNA was added to 40μM haematin. Polymerase chain reaction (PCR)-based conditions including the concentrations of primers, magnesium and the Taq polymerase as well as volume, cycle numbers and annealing temperature were examined and optimised. In addition, the system was validated by 364 bloodstain samples and 32 common casework samples. According to the Chinese National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, our system demonstrates good detection performance and is an ideal tool for forensic DNA typing with potential application.

  5. Assessment of sperm DNA fragmentation in stallion (Equus caballus) and donkey (Equus asinus) using the sperm chromatin dispersion test.

    PubMed

    Cortés-Gutiérrez, E I; Crespo, F; Serres-Dalmau, C; Gutiérrez de las Rozas, A L; Dávila-Rodríguez, M I; López-Fernández, C; Gósalvez, J

    2009-10-01

    Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys.

  6. 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

  7. Phylogeography of Douglas-fir based on mitochondrial and chloroplast DNA sequences: testing hypotheses from the fossil record.

    PubMed

    Gugger, Paul F; Sugita, Shinya; Cavender-Bares, Jeannine

    2010-05-01

    The integration of fossil and molecular data can provide a synthetic understanding of the ecological and evolutionary history of an organism. We analysed range-wide maternally inherited mitochondrial DNA and paternally inherited chloroplast DNA sequence data with coalescent simulations and traditional population genetic methods to test hypotheses of population divergence generated from the fossil record of Douglas-fir (Pseudotsuga menziesii), an ecologically and economically important western North American conifer. Specifically, we tested (i) the hypothesis that the Pliocene orogeny of the Cascades and Sierra Nevada caused the divergence of coastal and Rocky Mountain Douglas-fir varieties; and (ii) the hypothesis that multiple glacial refugia existed on the coast and in the Rocky Mountains. We found that Douglas-fir varieties diverged about 2.11 Ma (4.37 Ma-755 ka), which could be consistent with a Pliocene divergence. Rocky Mountain Douglas-fir probably resided in three or more glacial refugia. More variable molecular markers would be required to detect the two coastal refugia suggested in the fossil record. Comparison of mitochondrial DNA and chloroplast DNA variation revealed that gene flow via pollen linked populations isolated from seed exchange. Postglacial colonization of Canada from coastal and Rocky Mountain refugia near the ice margin at the Last Glacial Maximum produced a wide hybrid zone among varieties that formed almost exclusively by pollen exchange and chloroplast DNA introgression, not seed exchange. Postglacial migration rates were 50-165 m/year, insufficient to track projected 21st century warming in some regions. Although fossil and genetic data largely agree, each provides unique insights.

  8. 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

  9. Quantitative Field Testing Heterodera glycines from Metagenomic DNA Samples Isolated Directly from Soil under Agronomic Production

    PubMed Central

    Li, Yan; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

    2014-01-01

    A quantitative PCR procedure targeting the Heterodera glycines ortholog of the Caenorhabditis elegans uncoordinated-78 gene was developed. The procedure estimated the quantity of H. glycines from metagenomic DNA samples isolated directly from field soil under agronomic production. The estimation of H. glycines quantity was determined in soil samples having other soil dwelling plant parasitic nematodes including Hoplolaimus, predatory nematodes including Mononchus, free-living nematodes and biomass. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from field soil. PMID:24587100

  10. Quantitative field testing Heterodera glycines from metagenomic DNA samples isolated directly from soil under agronomic production.

    PubMed

    Li, Yan; Lawrence, Gary W; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P

    2014-01-01

    A quantitative PCR procedure targeting the Heterodera glycines ortholog of the Caenorhabditis elegans uncoordinated-78 gene was developed. The procedure estimated the quantity of H. glycines from metagenomic DNA samples isolated directly from field soil under agronomic production. The estimation of H. glycines quantity was determined in soil samples having other soil dwelling plant parasitic nematodes including Hoplolaimus, predatory nematodes including Mononchus, free-living nematodes and biomass. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from field soil.

  11. 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.

  12. High-risk human papillomavirus DNA test: could it be useful in low-grade cervical lesion triage? Five-year follow-up.

    PubMed

    Saccardi, Carlo; Gizzo, Salvatore; Noventa, Marco; Anis, Omar; Di Gangi, Stefania; Patrelli, Tito Silvio; D'Antona, Donato; Nardelli, Giovanni Battista

    2014-02-01

    We conducted a retrospective, observational study in order to evaluate the role of high-risk human papillomavirus (hrHPV)-DNA test in patients with first diagnosis of low-grade squamous intraepithelial lesions (L-SILs).Patients were divided into group A, annual Papanicolaou test and hrHPV-DNA tests (167 patients) and group B, immediate colposcopy, followed by annual papanicolaou test and hrHPV-DNA tests (164 patients). We assessed sensitivity, specificity, negative predictive value (NPV), positive predictive value, positive-negative likelihood ratio of hrHPV-DNA test, and 5-year relative risk of cervical intraepithelial neoplasia grade 2 in hrHPV-DNA+. Colposcopy is still considered the best choice for women with L-SIL and hrHPV-DNA+ test. High sensitivity and NPV of hrHPV-DNA test permit to use it in the follow-up of L-SIL with a HPV-negative status, without necessity of referring to colposcopy.

  13. 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

  14. 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.

  15. The cytology and DNA detection by the PapilloCheck® test in the diagnosis of human papillomavirus infection

    PubMed Central

    Vieira, L.

    2013-01-01

    The aim of this study was to make a comparison between the results obtained by cytologies and by the detection and genotyping of human papillomavirus (HPV) DNA in the screening of cervical cancer. In this study, there were 994 samples used from human females. These were obtained from liquid-based preparations. The samples were analyzed by cytological technique and by the detection of HPV DNA using the PapilloCheck® Test. The HPV was detected in 28% of the samples. Most of the cytology lesions appeared in HPV positive samples and, within these, the most serious injuries occurred mostly in samples with multiple HPV infections. The results indicate that, in general, there is a correlation between the detection of HPV DNA and cytology. However, there were some cases that emphasize the limitations of both diagnosis methods (27% cases with viral HPV DNA positive and normal cytologies and about 2% of cytological lesions detected in samples HPV negatives). It is possible to conclude that none of the two techniques is enough by itself and should be applied together in order to increase the accuracy of cervical cancer screening. PMID:24265920

  16. Testing the utility of matK and ITS DNA regions for discrimination of Allium species

    USDA-ARS?s Scientific Manuscript database

    Molecular phylogenetic analysis of the genus Allium L. has been mainly based on the nucleotide sequences of ITS region. In 2009 matK and rbcL were accepted as a two-locus DNA barcode to classify plant species by the Consortium for the Barcode of Life (CBOL) Plant Working Group. MatK region has been ...

  17. PCR detection of cytomegalovirus DNA in serum as a diagnostic test for congenital cytomegalovirus infection.

    PubMed Central

    Nelson, C T; Istas, A S; Wilkerson, M K; Demmler, G J

    1995-01-01

    PCR detected cytomegalovirus (CMV) DNA in the serum of 18 of 18 infants with symptomatic congenital CMV infection, 1 of 2 infants with asymptomatic congenital CMV infection, and 0 of 32 controls. Serum CMV PCR provided a rapid, sensitive, and specific method for diagnosis of congenital CMV infection in infants who were symptomatic at birth. PMID:8586726

  18. DNA analysis of first-trimester chorionic villous biopsies: test for maternal contamination.

    PubMed Central

    de Martinville, B; Blakemore, K J; Mahoney, M J; Francke, U

    1984-01-01

    We investigated the reliability of chorionic villous biopsy as a method to obtain tissues reflecting the genetic constitution of the embryo. In 12 pregnancies before elective termination, we searched for detectable maternal tissue after careful dissection of villi from small 2-5-mg specimens that yielded 7 micrograms of DNA per mg tissue. In Southern blotting experiments (1-2 micrograms DNA per lane), restriction fragment length polymorphisms (RFLPs) at an autosomal (D14S1) and a sex chromosomal (DXYS1) locus allowed recognition of maternally and embryonically derived alleles. Pure villi were obtained in six of the seven informative cases. One biopsy was not dissected satisfactorily; a mixture of embryonic and maternal DNA was found. Nonvillous tissues were mostly maternally derived in eight informative cases. Sex determination by molecular analysis (alleles at the DXYS1 locus) agreed with the karyotypes of uncultured or cultured villi. In one continuing pregnancy, distinct RFLPs indicated maternal inheritance of the alpha-thalassemia 1 trait in a female embryo without detectable maternal contamination. Reliable prenatal diagnosis depends on the specimen's purity. Maternal contamination can be evaluated by DNA analyses. Images p1360-a p1364-a PMID:6517057

  19. Identifying Insects with Incomplete DNA Barcode Libraries, African Fruit Flies (Diptera: Tephritidae) as a Test Case

    PubMed Central

    Virgilio, Massimiliano; Jordaens, Kurt; Breman, Floris C.; Backeljau, Thierry; De Meyer, Marc

    2012-01-01

    We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods. PMID:22359600

  20. Identifying insects with incomplete DNA barcode libraries, African fruit flies (Diptera: Tephritidae) as a test case.

    PubMed

    Virgilio, Massimiliano; Jordaens, Kurt; Breman, Floris C; Backeljau, Thierry; De Meyer, Marc

    2012-01-01

    We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods.

  1. Usefulness of Integrase resistance testing in proviral HIV-1 DNA in patients with Raltegravir prior failure.

    PubMed

    Fernández-Caballero, Jose Ángel; Chueca, Natalia; Álvarez, Marta; Mérida, María Dolores; López, Josefa; Sánchez, José Antonio; Vinuesa, David; Martínez, María Ángeles; Hernández, José; García, Federico

    2016-05-13

    In our study, we have hypothesized that proviral DNA may show the history of mutations that emerged at previous failures to a Raltegravir containing regimen, in patients who are currently undetectable and candidates to simplification to a Dolutegravir containing regimen, in order to decide on once a day or twice a day dosing. We have performed a pilot, observational, retrospective, non interventional study, including 7 patients infected by HIV-1, all with a history of previous failure to a RAL containing regimen, that were successfully salvaged and had reached viral suppression. A genotypic viral Integrase region study was available for each patient at the moment of RAL failure. After an average (IQR) time of 48 months (29-53) Integrase resistance mutations in proviral DNA were studied. All the patients were infected by HIV-1 B subtypes, with a mean age of 55 (range 43 to 56), originating from Spain, and 4 were women. Median viral load (log) and CD4 count at the moment of the study on proviral DNA was of 1.3 log cp/ml (range 0-1.47) and 765.5 cells/μL (range; 436.75-1023.75). The median time (IQR) between previous failure to RAL and the study on proviral DNA was 48 (29-53) months. At Raltegravir failure, N155H was detected in four patients, and other secondary mutations were detected in five patients (71.4 %). In proviral DNA, N155H was detected by population sequencing in three patients (42.8 %), and UDS demonstrated a 9.77 % relative abundance of N155H in the remaining patient. Sanger sequencing correctly identified all the secondary mutations. This is a pilot study that demonstrates the possibility of properly identifying N155H and some secondary mutations 29-53 months after failure.

  2. Developmental Validation of the ParaDNA® Screening System - A presumptive test for the detection of DNA on forensic evidence items.

    PubMed

    Dawnay, Nick; Stafford-Allen, Beccy; Moore, Dave; Blackman, Stephen; Rendell, Paul; Hanson, Erin K; Ballantyne, Jack; Kallifatidis, Beatrice; Mendel, Julian; Mills, DeEtta K; Nagy, Randy; Wells, Simon

    2014-07-01

    Current assessment of whether a forensic evidence item should be submitted for STR profiling is largely based on the personal experience of the Crime Scene Investigator (CSI) and the submissions policy of the law enforcement authority involved. While there are chemical tests that can infer the presence of DNA through the detection of biological stains, the process remains mostly subjective and leads to many samples being submitted that give no profile or not being submitted although DNA is present. The ParaDNA(®) Screening System was developed to address this issue. It consists of a sampling device, pre-loaded reaction plates and detection instrument. The test uses direct PCR with fluorescent HyBeacon™ detection of PCR amplicons to identify the presence and relative amount of DNA on an evidence item and also provides a gender identification result in approximately 75 minutes. This simple-to-use design allows objective data to be acquired by both DNA analyst and non-specialist personnel, to enable a more informed submission decision to be made. The developmental validation study described here tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Screening System on a range of mock evidence items. The data collected demonstrates that the ParaDNA Screening System identifies the presence of DNA on a variety of evidence items including blood, saliva and touch DNA items.

  3. Experimental Test of Connector Rotation during DNA Packaging into Bacteriophage ϕ29 Capsids

    PubMed Central

    Jardine, Paul J; Grimes, Shelley; Walter, Jessica M; Falk, Wayne; Anderson, Dwight L; Bustamante, Carlos

    2007-01-01

    The bacteriophage ϕ29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question. PMID:17311473

  4. Injection molded nanofluidic chips: fabrication method and functional tests using single-molecule DNA experiments.

    PubMed

    Utko, Pawel; Persson, Fredrik; Kristensen, Anders; Larsen, Niels B

    2011-01-21

    We demonstrate that fabrication of well-defined nanofluidic systems can be greatly simplified by injection molding of thermoplastic polymers. Chips featuring nanochannel arrays, microchannels and integrated interconnects are produced in a single processing step by injection molding. The resulting open channel structures are subsequently sealed by facile plasma-enhanced thermal bonding of a polymer film. This fast, inexpensive and industry-compatible method thus provides a single-use all-polymer platform for nanofluidic lab-on-a-chip applications. Its applicability for nanofluidics is demonstrated by DNA stretching experiments performed on individual double-stranded DNA molecules confined in the injection molded nanochannels. The obtained results are consistent with measurements performed in costly state-of-the-art silica nanochannels, for both straight and tapered channel geometries.

  5. Fluorescent studies on the interaction of DNA and ternary lanthanide complexes with cinnamic acid-phenanthroline and antibacterial activities testing.

    PubMed

    Sun, Hui-Juan; Wang, Ai-Ling; Chu, Hai-Bin; Zhao, Yong-Liang

    2015-03-01

    Twelve lanthanide complexes with cinnamate (cin(-) ) and 1,10-phenanthroline (phen) were synthesized and characterized. Their compositions were assumed to be RE(cin)3 phen (RE(3+)  = La(3+) , Pr(3+) , Nd(3+) , Sm(3+) , Eu(3+) , Gd(3+) , Tb(3+) , Dy(3+) , Ho(3+) , Tm(3+) , Yb(3+) , Lu(3+) ). The interaction mode between the complexes and DNA was investigated by fluorescence quenching experiment. The results indicated the complexes could bind to DNA and the main binding mode is intercalative binding. The fluorescence quenching constants of the complexes increased from La(cin)3 phen to Lu(cin)3 phen. Additionally, the antibacterial activity testing showed that the complexes exhibited excellent antibacterial ability against Escherichia coli, and the changes of antibacterial ability are in agreement with that of the fluorescence quenching constants.

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

    DTIC Science & Technology

    2012-12-12

    nogenicity in hamsters, and the results indicated that there was no antibody response, even after five vaccinations delivered by gene gun (data not...infective doses [ID50]) (data not shown). In an earlier study , it was determined that the ANDV M gene-based DNA vaccine failed to elicit a de...data indicated that improve- ments in the quality of the immune response, i.e., the neutralizing antibody response, were needed. Synthetic, mammalian

  7. Single cells for forensic DNA analysis--from evidence material to test tube.

    PubMed

    Brück, Simon; Evers, Heidrun; Heidorn, Frank; Müller, Ute; Kilper, Roland; Verhoff, Marcel A

    2011-01-01

    The purpose of this project was to develop a method that, while providing morphological quality control, allows single cells to be obtained from the surfaces of various evidence materials and be made available for DNA analysis in cases where only small amounts of cell material are present or where only mixed traces are found. With the SteREO Lumar.V12 stereomicroscope and UV unit from Zeiss, it was possible to detect and assess single epithelial cells on the surfaces of various objects (e.g., glass, plastic, metal). A digitally operated micromanipulator developed by aura optik was used to lift a single cell from the surface of evidence material and to transfer it to a conventional PCR tube or to an AmpliGrid(®) from Advalytix. The actual lifting of the cells was performed with microglobes that acted as carriers. The microglobes were held with microtweezers and were transferred to the DNA analysis receptacles along with the adhering cells. In a next step, the PCR can be carried out in this receptacle without removing the microglobe. Our method allows a single cell to be isolated directly from evidence material and be made available for forensic DNA analysis.

  8. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

    PubMed Central

    Elbrecht, Vasco; Taberlet, Pierre; Dejean, Tony; Valentini, Alice; Usseglio-Polatera, Philippe; Beisel, Jean-Nicolas; Coissac, Eric; Boyer, Frederic

    2016-01-01

    Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate. PMID:27114891

  9. Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China.

    PubMed

    Chen, Juan; Zhao, Jietang; Erickson, David L; Xia, Nianhe; Kress, W John

    2015-03-01

    The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear-cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH-psbA and trnL-F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra- and inter-specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH-psbA (100%), trnL-F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH-psbA and trnL-F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers.

  10. Analysis of first-trimester combined test results in preparation for a cell-free fetal DNA era.

    PubMed

    Kose, Semir; Cımrın, Dilek; Yıldırım, Nuri; Aksel, Ozge; Keskinoglu, Pembe; Bora, Elcin; Cankaya, Tufan; Altunyurt, Sabahattin

    2016-11-01

    To survey experience with the first-trimester combined test (FCT) for trisomy 21 (T21) in different risk score groups to determine the most useful clinical application of cell-free fetal DNA (cffDNA) screening. In a retrospective study, the records of FCT results obtained at a center in Turkey between January 2009 and January 2014 were reviewed. The FCT results and rates of uptake of invasive diagnostic testing were compared among different risk score groups. FCT results were available for 4804 pregnancies; 276 (5.7%) had IDT results. Ten (72.7%) of 11 cases of T21 had a risk score of 1:300 or more. The IDT uptake rates were 54.5%, 51.9%, and 47.4% at risk scores of 1:100 or more, 1:200 or more, and 1:300 or more, respectively. In the group at intermediate risk (1:1001-1:3000), no pregnancy had an FCT result of both low pregnancy-associated plasma protein A and high free β-human chorionic gonadotropin, but 30 (3.9%) of 766 pregnancies had both advanced maternal age and high β-human chorionic gonadotropin. cffDNA screening should be used to optimize IDT uptake in pregnancies with a risk score of 1:101-1:1000. The selective power of the FCT diminishes beyond the 1:1001 score and cffDNA screening cannot yet be recommended routinely. Copyright © 2016 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

  11. DNA barcoding in the cycadales: testing the potential of proposed barcoding markers for species identification of cycads.

    PubMed

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

    2007-11-07

    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.

  12. Relevance of the Pap Test: A Report of HPV-DNA Test-Negative High-Grade Squamous Intraepithelial Lesions of the Female Lower Genital Tract.

    PubMed

    Hui, Yiang; Hansen, Katrine; Murthy, Jayasimha; Chau, Danielle; Sung, C James; Quddus, M Ruhul

    2016-01-01

    A vast majority of cervicovaginal intraepithelial lesions are caused by high-risk human papillomaviruses (HPVs). The Pap test has been the sole method used for the screening of cervicovaginal squamous intraepithelial lesions (SIL). Recently, the FDA approved an HPV-DNA assay as a method of primary screening. We report on a series of FDA-approved HPV-DNA test-negative SIL with HPV genotyping, using an alternative method on the corresponding surgical biopsy specimens. A retrospective review identified cytology-positive HPV-negative cases over a 15-month period at a tertiary care gynecologic oncology institution. Corresponding biopsies were reviewed and genotyped for high-risk HPVs. Of the 18,200 total cases, 17 patients meeting the study criteria were selected with 27 surgical specimens corresponding to their cytologic diagnoses. Four patients with high-grade lesions were identified, 3 of whom (75%) were positive for HPV. One of these 4 patients (25%) showed high-grade SIL on biopsies from 4 separate sites in the cervix and vagina. Multiviral HPV infections were frequent. We discuss the relevance of cotesting for screening cervical SILs and emphasize that false-negative results are possible with the FDA-approved HPV screening assay, also in patients with high-grade SIL. These cases may be detectable by cytologic examination and this suggests that the Pap test remains an important diagnostic tool. © 2016 S. Karger AG, Basel.

  13. Cancer worries, risk perceptions and associations with interest in DNA testing and clinic satisfaction in a familial colorectal cancer clinic.

    PubMed

    Collins, V; Halliday, J; Warren, R; Williamson, R

    2000-12-01

    Multi-disciplinary familial cancer clinics are becoming an integral part of cancer services. It is, therefore, important to assess how attendance at these clinics impacts on cancer-related concerns, risk perceptions and behavioural intentions, and how the clinic services are being received by those using them. This study has assessed a familial colorectal cancer clinic with respect to cancer-related worries and risk perceptions and their impact on interest in DNA testing and overall satisfaction with the clinic. Pre- and post-clinic questionnaires were completed by 127 patients and relatives attending the clinic. After attending the clinic, the proportion of people 'very' or 'extremely' worried about developing bowel cancer reduced from 49 (pre-clinic) to 34% (p = 0.002). Worry about bowel cancer was positively associated with younger age, higher education level and higher perceived risk of developing cancer. A reduction in level of risk perception correlated with a lower likelihood of feeling 'very worried' about developing bowel cancer. Of those intending to go ahead with DNA testing, 58% were 'very worried' about bowel cancer compared with 15% of those not intending to proceed with testing, suggesting that worry was a motivation for interest in DNA testing. One-third of participants indicated another session of genetic counselling would be helpful. Within this group, a higher proportion was very worried about bowel cancer (43%) than for those who did not want another session (17%). Attendance at this familial colorectal cancer clinic alleviated worry for many individuals, partly due to improved information about risk of colorectal cancer.

  14. Comparison of post-thaw DNA integrity of boar spermatozoa assessed with the neutral comet assay and Sperm-Sus Halomax test kit.

    PubMed

    Fraser, L; Parda, A; Filipowicz, K; Strzeżek, J

    2010-10-01

    In this study, we tested the hypothesis whether the neutral Comet assay (NCA) and the Sperm-Sus-Halomax (SSH) test kit could provide similar measurements of post-thaw DNA fragmentation of boar spermatozoa. Whole ejaculates or sperm-rich fractions of boar semen were frozen in an extender containing lactose, lipoprotein fractions isolated from ostrich egg yolk (LPFo), glycerol (lactose-LPFo-G) or in a standard boar semen extender (K3), without the addition of cryoprotective substances. In all boars, both the NCA and SSH test showed similar levels of post-thaw sperm DNA fragmentation in samples of the same ejaculates, regardless of the ejaculate collection procedure and extender. Yet, the levels of post-thaw sperm DNA damage, detected by the NCA and SSH test, were more accentuated in spermatozoa frozen in the absence of cryoprotective substances. Both the NCA and SSH detected variations among individual boars in terms of post-thaw sperm DNA fragmentation. Agreement between the measurements of the NCA and SSH was confirmed by scatter plots of differences, suggesting that the DNA integrity tests could detect the same sperm populations, which were susceptible to cryo-induced DNA damage. The findings of this study indicate that the NCA and the SSH test are effective in detecting similar levels of sperm DNA fragmentation and reinforce their importance in the assessment of frozen-thawed boar semen quality.

  15. Cell-free fetal DNA analysis in maternal plasma as a screening test for trisomy 21, 18 and 13 in twin pregnancies.

    PubMed

    Le Conte, Grégoire; Letourneau, Alexandra; Jani, Jacques; Kleinfinger, Pascale; Lohmann, Laurence; Costa, Jean-Marc; Benachi, Alexandra

    2017-08-18

    To evaluate the utility of noninvasive prenatal testing using cell-free circulating fetal DNA (cfDNA) in screening for the three main autosomal fetal trisomies in twin pregnancies. CfDNA testing was offered to 492 patients with twin pregnancies without ultrasound anomalies as a first-line screening test or after serum screening in clinical practice. Data were collected prospectively and a retrospective analysis was performed. CfDNA analysis was performed by massively parallel sequencing. The fetal fraction threshold for test evaluation was 8%. Regression analysis was performed to investigate the effect on the test failure rate of various variables. Performance of the test is also reported. The test was performed first line (after first-trimester scan) in 377 patients and following serum screening in 115. 78.8% of pregnancies were dichorionic-diamniotic. The test failed at the first attempt in 12 (2.9%) of 420 pregnancies with available outcomes, and regression analysis demonstrated that only maternal weight was a significant independent predictor of test failure. After redraw, a result was achieved in 10 cases. CfDNA identified all 3 cases of trisomy 21, and the only case of trisomy 18. For trisomy 21, the specificity was 99.8% (95% CI [98.7% - 100.0%]). When considering the spontaneous or ART origin of pregnancies, there were no significant differences in terms of maternal weight or of no-result rate for cfDNA screening in the two groups. In twin pregnancies without fetal ultrasound abnormalities, cfDNA had a high success rate and performance. Therefore, in routine practice, cfDNA could be considered as a first- or second-line screening test. This article is protected by copyright. All rights reserved.

  16. Accuracy of bacterial DNA testing for central venous catheter-associated bloodstream infection in children with cancer.

    PubMed

    Millar, M; Zhou, W; Skinner, R; Pizer, B; Hennessy, E; Wilks, M; Gilbert, R E

    2011-02-01

    Central venous catheters (CVCs) are widely used for children with cancer and are a major risk factor for bloodstream infection. Early and specific diagnosis of CVC-associated bloodstream infection allows early targeted treatment, reducing the risk of CVC removal and avoiding the operative risks and trauma of reinsertion, but peripheral vein sampling, as used in adults, improves specificity but is not usually acceptable in children. To improve the detection and treatment of CVC-associated bloodstream infection in children (aged 0-18 years) with cancer admitted with fever. There were four main studies: (1) evaluation of the diagnostic accuracy of a quantitative molecular method for the detection of bacterial deoxyribonucleic acid (DNA), based solely on blood samples drawn through the CVC; (2) analysis of the prognostic risk of CVC removal and duration of intravenous (i.v.) antibiotic treatment days in relation to presenting clinical features, blood culture results and bacterial DNA test results; (3) systematic reviews of treatment options for CVC-associated infection and a questionnaire survey of current practice in paediatric oncology centres; (4) evaluation of the clinical effectiveness of different test-treatment strategies to reduce i.v. antibiotic treatment days and unnecessary CVC removals. (1) The bacterial DNA test detected two-thirds [95% confidence interval (CI) 44% to 83%] of children classified with probable CVC-associated infection - specificity was 88% (95% CI 84% to 92%). Although high bacterial DNA concentrations were associated with subsequent CVC removal and long duration of i.v. antibiotic treatment, the test did not improve the prediction of these outcomes over and above clinical signs of CVC-associated infection combined with blood culture results. (2) High DNA load was predictive of CVC removal and i.v. treatment duration, before blood culture results became available at 48 hours after sampling. (3) There was limited evidence that antibiotic

  17. Epigenetic DNA Methylation Profiling with MSRE: A Quantitative NGS Approach Using a Parkinson's Disease Test Case.

    PubMed

    Marsh, Adam G; Cottrell, Matthew T; Goldman, Morton F

    2016-01-01

    Epigenetics is a rapidly developing field focused on deciphering chemical fingerprints that accumulate on human genomes over time. As the nascent idea of precision medicine expands to encompass epigenetic signatures of diagnostic and prognostic relevance, there is a need for methodologies that provide high-throughput DNA methylation profiling measurements. Here we report a novel quantification methodology for computationally reconstructing site-specific CpG methylation status from next generation sequencing (NGS) data using methyl-sensitive restriction endonucleases (MSRE). An integrated pipeline efficiently incorporates raw NGS metrics into a statistical discrimination platform to identify functional linkages between shifts in epigenetic DNA methylation and disease phenotypes in samples being analyzed. In this pilot proof-of-concept study we quantify and compare DNA methylation in blood serum of individuals with Parkinson's Disease relative to matched healthy blood profiles. Even with a small study of only six samples, a high degree of statistical discrimination was achieved based on CpG methylation profiles between groups, with 1008 statistically different CpG sites (p < 0.0025, after false discovery rate correction). A methylation load calculation was used to assess higher order impacts of methylation shifts on genes and pathways and most notably identified FGF3, FGF8, HTT, KMTA5, MIR8073, and YWHAG as differentially methylated genes with high relevance to Parkinson's Disease and neurodegeneration (based on PubMed literature citations). Of these, KMTA5 is a histone methyl-transferase gene and HTT is Huntington Disease Protein or Huntingtin, for which there are well established neurodegenerative impacts. The future need for precision diagnostics now requires more tools for exploring epigenetic processes that may be linked to cellular dysfunction and subsequent disease progression.

  18. Epigenetic DNA Methylation Profiling with MSRE: A Quantitative NGS Approach Using a Parkinson's Disease Test Case

    PubMed Central

    Marsh, Adam G.; Cottrell, Matthew T.; Goldman, Morton F.

    2016-01-01

    Epigenetics is a rapidly developing field focused on deciphering chemical fingerprints that accumulate on human genomes over time. As the nascent idea of precision medicine expands to encompass epigenetic signatures of diagnostic and prognostic relevance, there is a need for methodologies that provide high-throughput DNA methylation profiling measurements. Here we report a novel quantification methodology for computationally reconstructing site-specific CpG methylation status from next generation sequencing (NGS) data using methyl-sensitive restriction endonucleases (MSRE). An integrated pipeline efficiently incorporates raw NGS metrics into a statistical discrimination platform to identify functional linkages between shifts in epigenetic DNA methylation and disease phenotypes in samples being analyzed. In this pilot proof-of-concept study we quantify and compare DNA methylation in blood serum of individuals with Parkinson's Disease relative to matched healthy blood profiles. Even with a small study of only six samples, a high degree of statistical discrimination was achieved based on CpG methylation profiles between groups, with 1008 statistically different CpG sites (p < 0.0025, after false discovery rate correction). A methylation load calculation was used to assess higher order impacts of methylation shifts on genes and pathways and most notably identified FGF3, FGF8, HTT, KMTA5, MIR8073, and YWHAG as differentially methylated genes with high relevance to Parkinson's Disease and neurodegeneration (based on PubMed literature citations). Of these, KMTA5 is a histone methyl-transferase gene and HTT is Huntington Disease Protein or Huntingtin, for which there are well established neurodegenerative impacts. The future need for precision diagnostics now requires more tools for exploring epigenetic processes that may be linked to cellular dysfunction and subsequent disease progression. PMID:27853465

  19. [Genetic counseling and DNA testing in patients with increased risks for malignant melanoma].

    PubMed

    Itin, P H; Fistarol, S K

    2003-08-01

    There are numerous risk factors for the development of malignant melanoma. It has been documented that genetic predisposition exists but exogenous factors are also very important. In familial melanomas it has been well established that mutation in the CDKN2A gene which is located at chromosome 9 leads to a marked risk for malignant melanoma. This tumor-suppressor gene is important for the regulation of the cell cycle and mutation in this gene is associated also with an increased rate of pancreas cancer. The penetrance of this mutation is influenced by UV-energy. In addition it has been shown that a second cluster for the familial atypical nevus syndrome is located at chromosome 1p36. Patients with the rare disease xeroderma pigmentosum have a defect in the DNA-repair mechanism inherited in an autosomal recessive trait and therefore develop within the first 20 years of life numerous malignant skin tumours including malignant melanomas. But also in non-syndromic patients a decrease of DNA-repair ability may occur. It has been shown recently that reduced DNA-repair ability is an independent risk factor for malignant melanoma and may contribute to susceptibility to sunlight-induced melanoma among the general population. Other constitutional risk factors for the development of malignant melanoma are fair skin, red hair and blue eyes. The most important exogenous risk factor is UV-exposition. Extensive and repetitive sunburns before the age of 15 years are especially predisposing to malignant melanoma. The most important preventive measures are continuous sun-protection including avoidance of sun in noon time on tropical and subtropical places, wearing a hut and sunglasses and application of sun-screens with high sun-protection factor. Furthermore a regular check for changing moles is indicated in persons with multiple atypical nevi or a familial melanoma syndrome. Nowadays molecular genetic screenings are available within research projects for members of melanoma

  20. Development of a Genomic DNA Reference Material Panel for Myotonic Dystrophy Type 1 (DM1) Genetic Testing

    PubMed Central

    Kalman, Lisa; Tarleton, Jack; Hitch, Monica; Hegde, Madhuri; Hjelm, Nick; Berry-Kravis, Elizabeth; Zhou, Lili; Hilbert, James E.; Luebbe, Elizabeth A.; Moxley, Richard T.; Toji, Lorraine

    2014-01-01

    Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG triplet repeat in the 3′ untranslated region of the DMPK gene that encodes a serine-threonine kinase. Patients with larger repeats tend to have a more severe phenotype. Clinical laboratories require reference and quality control materials for DM1 diagnostic and carrier genetic testing. Well-characterized reference materials are not available. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community, the National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members, and the Coriell Cell Repositories, has established and characterized cell lines from patients with DM1 to create a reference material panel. The CTG repeats in genomic DNA samples from 10 DM1 cell lines were characterized in three clinical genetic testing laboratories using PCR and Southern blot analysis. DMPK alleles in the samples cover four of five DM1 clinical categories: normal (5 to 34 repeats), mild (50 to 100 repeats), classical (101 to 1000 repeats), and congenital (>1000 repeats). We did not identify or establish Coriell cell lines in the premutation range (35 to 49 repeats). These samples are publicly available for quality control, proficiency testing, test development, and research and should help improve the accuracy of DM1 testing. PMID:23680132

  1. Development of a genomic DNA reference material panel for myotonic dystrophy type 1 (DM1) genetic testing.

    PubMed

    Kalman, Lisa; Tarleton, Jack; Hitch, Monica; Hegde, Madhuri; Hjelm, Nick; Berry-Kravis, Elizabeth; Zhou, Lili; Hilbert, James E; Luebbe, Elizabeth A; Moxley, Richard T; Toji, Lorraine

    2013-07-01

    Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG triplet repeat in the 3' untranslated region of the DMPK gene that encodes a serine-threonine kinase. Patients with larger repeats tend to have a more severe phenotype. Clinical laboratories require reference and quality control materials for DM1 diagnostic and carrier genetic testing. Well-characterized reference materials are not available. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community, the National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members, and the Coriell Cell Repositories, has established and characterized cell lines from patients with DM1 to create a reference material panel. The CTG repeats in genomic DNA samples from 10 DM1 cell lines were characterized in three clinical genetic testing laboratories using PCR and Southern blot analysis. DMPK alleles in the samples cover four of five DM1 clinical categories: normal (5 to 34 repeats), mild (50 to 100 repeats), classical (101 to 1000 repeats), and congenital (>1000 repeats). We did not identify or establish Coriell cell lines in the premutation range (35 to 49 repeats). These samples are publicly available for quality control, proficiency testing, test development, and research and should help improve the accuracy of DM1 testing.

  2. Comparison of the Cobas 4800 HPV and HPV 9G DNA Chip Tests for Detection of High-Risk Human Papillomavirus in Cervical Specimens of Women with Consecutive Positive HPV Tests But Negative Pap Smears.

    PubMed

    Jun, Sun-Young; Park, Eun Su; Kim, Jiyoung; Kang, Jun; Lee, Jae Jun; Bae, Yoonjin; Kim, Sang-Il; Maeng, Lee-So

    2015-01-01

    Detecting high-risk (HR) HPV is important for clinical management of women with persistent HPV-positive and Pap-negative results. The Cobas 4800 HPV test is the first FDA-approved HPV DNA test that can be used alone as a first-line screening tool. The HPV 9G DNA chip test is a PCR-based DNA microarray assay. We evaluated the patients of consecutive HPV-positivity on HPV 9G DNA chip test without cytologic abnormalities. We then compared the performances of HPV 9G DNA chip and the Cobas 4800 HPV tests for detecting HR HPV with each other and confirmed HPV genotyping using direct sequencing. All 214 liquid-based cytology specimens were collected from 100 women with consecutive HPV-positive and Pap-negative results on the HPV 9G DNA chip test between May 2012 and Dec 2013, but only 180 specimens were available for comparing HPV test results. The HPV 9G DNA chip and the Cobas 4800 HPV tests agreed with each other in 81.7% of the samples, and the concordance rate was greater than 97.2% for detecting HPV-16 or -18. For HR genotypes other than HPV types 16 and 18, the two tests agreed for 81.1% of the samples. The sensitivity of both assays for detecting HR HPV was 100%, regardless of HR genotypes. The HPV 9G DNA chip test may be as effective as the Cobas 4800 HPV test in detecting HR HPV, and has a similar ability to identify HPV-16 and -18.

  3. Comparison of the Cobas 4800 HPV and HPV 9G DNA Chip Tests for Detection of High-Risk Human Papillomavirus in Cervical Specimens of Women with Consecutive Positive HPV Tests But Negative Pap Smears

    PubMed Central

    Jun, Sun-Young; Park, Eun Su; Kim, Jiyoung; Kang, Jun; Lee, Jae Jun; Bae, Yoonjin; Kim, Sang-Il; Maeng, Lee-So

    2015-01-01

    Detecting high-risk (HR) HPV is important for clinical management of women with persistent HPV-positive and Pap-negative results. The Cobas 4800 HPV test is the first FDA-approved HPV DNA test that can be used alone as a first-line screening tool. The HPV 9G DNA chip test is a PCR-based DNA microarray assay. We evaluated the patients of consecutive HPV-positivity on HPV 9G DNA chip test without cytologic abnormalities. We then compared the performances of HPV 9G DNA chip and the Cobas 4800 HPV tests for detecting HR HPV with each other and confirmed HPV genotyping using direct sequencing. All 214 liquid-based cytology specimens were collected from 100 women with consecutive HPV-positive and Pap-negative results on the HPV 9G DNA chip test between May 2012 and Dec 2013, but only 180 specimens were available for comparing HPV test results. The HPV 9G DNA chip and the Cobas 4800 HPV tests agreed with each other in 81.7% of the samples, and the concordance rate was greater than 97.2% for detecting HPV-16 or -18. For HR genotypes other than HPV types 16 and 18, the two tests agreed for 81.1% of the samples. The sensitivity of both assays for detecting HR HPV was 100%, regardless of HR genotypes. The HPV 9G DNA chip test may be as effective as the Cobas 4800 HPV test in detecting HR HPV, and has a similar ability to identify HPV-16 and -18. PMID:26469982

  4. Lab-on-a-chip platforms based on highly sensitive nanophotonic Si biosensors for single nucleotide DNA testing

    NASA Astrophysics Data System (ADS)

    Sánchez del Rio, J.; Carrascosa, L. G.; Blanco, F. J.; Moreno, M.; Berganzo, J.; Calle, A.; Dominguez, C.; Lechuga, L. M.

    2007-02-01

    In order to solve the drawbacks of sensitivity and portability in optical biosensors we have developed ultrasensitive and miniaturized photonic silicon sensors able to be integrated in a "lab-on-a-chip" microsystem platform. The sensors are integrated Mach-Zehnder interferometers based on TIR optical waveguides (Si/SiO II/Si 3N 4) of micro/nanodimensions. We have applied this biosensor for DNA testing and for detection of single nucleotide polymorphisms at BRCA-1 gene, involved in breast cancer development, without target labeling. The oligonucleotide probe is immobilized by covalent attachment to the sensor surface through silanization procedures. The hybridization was performed for different DNA target concentrations showing a lowest detection limit at 10 pM. Additionally, we have detected the hybridization of different concentrations of DNA target with two mismatching bases corresponding to a mutation of the BRCA-1 gene. Following the way of the lab-on-a-chip microsystem, integration with the microfluidics has been achieved by using a novel fabrication method of 3-D embedded microchannels using the polymer SU-8 as structural material. The optofluidic chip shows good performances for biosensing.

  5. Cell-free DNA testing of an extended range of chromosomal anomalies: clinical experience with 6,388 consecutive cases

    PubMed Central

    Pescia, Graziano; Guex, Nicolas; Iseli, Christian; Brennan, Liam; Osteras, Magne; Xenarios, Ioannis; Farinelli, Laurent; Conrad, Bernard

    2017-01-01

    Purpose: Cell-free DNA (cfDNA) testing for fetal aneuploidies was broadly implemented for common trisomies and sex-chromosome anomalies (SCAs). However, such an approach identifies only 75 to 85% of clinically relevant aneuploidies. Methods: We present a consecutive series of 6,388 cases, thus uncovering a broader array of aneuploidies, including the rare autosomal trisomies (RATs) and the maternally inherited deletion and duplication copy-number variations (CNVs), with complete and stratified follow-up by amniocentesis. Combined measurements of z-scores and the fetal fraction, in conjunction with fetal cfDNA enrichment, were used to stratify the likelihood of true and false results. Results: We obtained an incremental diagnostic yield of 50%; RATs and CNVs were found to be significant causes of fetal pathology. Scrutinizing z-scores and the fetal fraction made it possible to distinguish the sources of false-negative results; predict the likelihood of false-positive results for major trisomies and SCAs; classify maternal mosaic SCAs and CNVs, preventing false-positive results; and robustly identify maternally inherited CNVs and detect recurrent genomic disorders as a standardized function of the fetal fraction. Conclusion: With the clinical pertinence of this broader detection scheme confirmed, we offer recommendations for its implementation. Genet Med 19 2, 169–175. PMID:27362910

  6. Results of External Quality Assessment for Proviral DNA Testing of HIV Tropism in the Maraviroc Switch Collaborative Study

    PubMed Central

    Land, Sally; Pett, Sarah; Emery, Sean; Marks, Kat; Kelleher, Anthony D.; Kaye, Steve; Kaiser, Rolf; Schuelter, Eugene; Harrigan, Richard

    2013-01-01

    The Maraviroc Switch collaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n = 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination using the bioinformatic algorithm geno2pheno, which generates a false-positive rate (FPR). DNA sequences with low FPRs are more likely to be from CXCR4-using (X4) viruses. Phase 1 of the EQA involved chromatogram interpretation. Phases 2, 2/3, and 3 involved patient and clonal samples. Clinical samples used in these phases were from treatment-experienced HIV-infected volunteers; 18/20 had viral loads of <50 copies/ml, and 10/15 were CXCR4-tropic on prior phenotyping. All samples were tested in triplicate, and any replicate with a geno2pheno FPR of <10% was designated X4. Performance was deemed adequate if ≤2 R5 and ≤1 X4 specimens were miscalled. For several clinical samples in the EQA, triplicate testing revealed marked DNA variability (FPR range, 0 to 96.7%). Therefore, a consensus-based approach was employed for each sample, i.e., a median FPR across laboratories was used to define sample tropism. Further sequencing analysis showed mixed viral populations in the clinical samples, explaining the differences in tropism predictions. All laboratories passed the EQA after achieving predefined competence thresholds in either of the phase 2 rounds. The use of clinical samples from patients resembling those who were likely to be screened in the MARCH, coupled with triplicate testing, revealed inherent DNA variability that might have been missed if single or duplicate testing and/or clonal samples alone were used. These data highlight the importance of intensive EQA of tropism

  7. Results of external quality assessment for proviral DNA testing of HIV tropism in the Maraviroc Switch collaborative study.

    PubMed

    Tu, Elise; Swenson, Luke C; Land, Sally; Pett, Sarah; Emery, Sean; Marks, Kat; Kelleher, Anthony D; Kaye, Steve; Kaiser, Rolf; Schuelter, Eugene; Harrigan, Richard

    2013-07-01

    The Maraviroc Switch collaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n = 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination using the bioinformatic algorithm geno2pheno, which generates a false-positive rate (FPR). DNA sequences with low FPRs are more likely to be from CXCR4-using (X4) viruses. Phase 1 of the EQA involved chromatogram interpretation. Phases 2, 2/3, and 3 involved patient and clonal samples. Clinical samples used in these phases were from treatment-experienced HIV-infected volunteers; 18/20 had viral loads of <50 copies/ml, and 10/15 were CXCR4-tropic on prior phenotyping. All samples were tested in triplicate, and any replicate with a geno2pheno FPR of <10% was designated X4. Performance was deemed adequate if ≤2 R5 and ≤1 X4 specimens were miscalled. For several clinical samples in the EQA, triplicate testing revealed marked DNA variability (FPR range, 0 to 96.7%). Therefore, a consensus-based approach was employed for each sample, i.e., a median FPR across laboratories was used to define sample tropism. Further sequencing analysis showed mixed viral populations in the clinical samples, explaining the differences in tropism predictions. All laboratories passed the EQA after achieving predefined competence thresholds in either of the phase 2 rounds. The use of clinical samples from patients resembling those who were likely to be screened in the MARCH, coupled with triplicate testing, revealed inherent DNA variability that might have been missed if single or duplicate testing and/or clonal samples alone were used. These data highlight the importance of intensive EQA of tropism

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

    PubMed Central

    Brocato, R. L.; Josleyn, M. J.; Wahl-Jensen, V.; Schmaljohn, C. S.

    2013-01-01

    Puumala virus (PUUV) is a causative agent of hemorrhagic fever with renal syndrome (HFRS). Although PUUV-associated HFRS does not result in high case-fatality rates, the social and economic impact is considerable. There is no licensed vaccine or specific therapeutic to prevent or treat HFRS. Here we report the synthesis of a codon-optimized, full-length M segment open reading frame and its cloning into a DNA vaccine vector to produce the plasmid pWRG/PUU-M(s2). pWRG/PUU-M(s2) delivered by gene gun produced high-titer neutralizing antibodies in hamsters and nonhuman primates. Vaccination with pWRG/PUU-M(s2) protected hamsters against infection with PUUV but not against infection by related HFRS-associated hantaviruses. Unexpectedly, vaccination protected hamsters in a lethal disease model of Andes virus (ANDV) in the absence of ANDV cross-neutralizing antibodies. This is the first evidence that an experimental DNA vaccine for HFRS can provide protection in a hantavirus lethal disease model. PMID:23239797

  9. DNA-testing for immigration cases: the risk of erroneous conclusions.

    PubMed

    Karlsson, Andreas O; Holmlund, Gunilla; Egeland, Thore; Mostad, Petter

    2007-10-25

    Making the correct decision based on results from DNA analyses and other information in family reunification cases can be complicated for a number of reasons. These include stratified populations, cultural differences in family constellations, families with different population origin, and complicated family relations giving complex pedigrees. The aim of this study was to analyze the risk of erroneous conclusions in immigration cases and to propose alternative procedures to current methods to reduce the risk of making such errors. A simulation model was used to study different issues. For simplicity, we focus on cases which can be formulated as questions about paternity. We present an overview of error rates (of falsely included men as the true father and of falsely excluded true fathers) for fairly standard computations, and we show how these are affected by different factors. For example, adding more DNA markers to a case will decrease the error rates, as will the inclusion of more children. We found that using inappropriate population frequency databases had just minor effects on the error rates, but the likelihood ratios varied from an underestimation of 100 times up to an overestimation of 100,000 times. To reduce the risk of falsely including a man related to the true father we propose a more refined prior including five hypotheses instead of the two normally used. Simulations showed that this method gave reduced error rates compared with standard computations, even when the prior does not exactly correspond to reality.

  10. [The development of a test-system for the quantitative and qualitative evaluation of DNA content in criminalistic objects by the real-time polymerase chain reaction].

    PubMed

    Lapenkov, M I; Plakhina, N V; Alekseev, Ia I; Varlamov, D A

    2011-01-01

    An original test-system for the preliminary quantitative and qualitative evaluation of isolated DNA is proposed by the polymerase chain reaction in real time (PCR-RT) based on the TaqMan technology. This test-system permits to simultaneously measure the amount of DNA in the sample, identify the genetic gender, and detect PCR inhibitors. The method has been approbated in the practical work of forensic medical experts.

  11. Integration of Noninvasive DNA Testing for Aneuploidy into Prenatal Care: What Has Happened Since the Rubber Met the Road?

    PubMed Central

    Bianchi, Diana W.; Wilkins-Haug, Louise

    2016-01-01

    BACKGROUND Over the past 2 years, noninvasive prenatal testing (NIPT), which uses massively parallel sequencing to align and count DNA fragments floating in the plasma of pregnant women, has become integrated into prenatal care. Professional societies currently recommend offering NIPT as an advanced screen to pregnant women at high risk for fetal aneuploidy, reserving invasive diagnostic procedures for those at the very highest risk. CONTENT In this review, we summarize the available information on autosomal and sex chromosome aneuploidy detection. Clinical performance in CLIA-certified, College of American Pathology–accredited laboratories appears to be equivalent to prior clinical validation studies, with high sensitivities and specificities and very high negative predictive values. The main impact on clinical care has been a reduction in invasive procedures. Test accuracy is affected by the fetal fraction, the percentage of fetal DNA in the total amount of circulating cell-free DNA. Fetal fraction is in turn affected by maternal body mass index, gestational age, type of aneuploidy, singleton vs multiples, and mosaicism. Three studies comparing NIPT to serum or combined screening for autosomal aneuploidy all show that NIPT has significantly lower false-positive rates (approximately 0.1%), even in all-risk populations. A significant number of the discordant positive cases have underlying biological reasons, including confined placental mosaicism, maternal mosaicism, cotwin demise, or maternal malignancy. SUMMARY NIPT performs well as an advanced screen for whole chromosome aneuploidy. Economic considerations will likely dictate whether its use can be expanded to all risk populations and whether it can be applied routinely for the detection of subchromosome abnormalities. PMID:24255077

  12. A series of recommended tests when validating probabilistic DNA profile interpretation software.

    PubMed

    Bright, Jo-Anne; Evett, Ian W; Taylor, Duncan; Curran, James M; Buckleton, John

    2015-01-01

    There has been a recent push from many jurisdictions for the standardisation of forensic DNA interpretation methods. Current research is moving from threshold-based interpretation strategies towards continuous interpretation strategies. However laboratory uptake of software employing probabilistic models is slow. Some of this reluctance could be due to the perceived intimidating calculations to replicate the software answers and the lack of formal internal validation requirements for interpretation software. In this paper we describe a set of experiments which may be used to internally validate in part probabilistic interpretation software. These experiments included both single source and mixed profiles calculated with and without dropout and drop-in and studies to determine the reproducibility of the software with replicate analyses. We do this by way of example using three software packages: STRmix™, LRmix, and Lab Retriever. We outline and demonstrate the profile examples where the expected answer may be calculated and provide all calculations.

  13. Abnormal plasma DNA profiles in early ovarian cancer using a non-invasive prenatal testing platform: implications for cancer screening.

    PubMed

    Cohen, Paul A; Flowers, Nicola; Tong, Stephen; Hannan, Natalie; Pertile, Mark D; Hui, Lisa

    2016-08-24

    Non-invasive prenatal testing (NIPT) identifies fetal aneuploidy by sequencing cell-free DNA in the maternal plasma. Pre-symptomatic maternal malignancies have been incidentally detected during NIPT based on abnormal genomic profiles. This low coverage sequencing approach could have potential for ovarian cancer screening in the non-pregnant population. Our objective was to investigate whether plasma DNA sequencing with a clinical whole genome NIPT platform can detect early- and late-stage high-grade serous ovarian carcinomas (HGSOC). This is a case control study of prospectively-collected biobank samples comprising preoperative plasma from 32 women with HGSOC (16 'early cancer' (FIGO I-II) and 16 'advanced cancer' (FIGO III-IV)) and 32 benign controls. Plasma DNA from cases and controls were sequenced using a commercial NIPT platform and chromosome dosage measured. Sequencing data were blindly analyzed with two methods: (1) Subchromosomal changes were called using an open source algorithm WISECONDOR (WIthin-SamplE COpy Number aberration DetectOR). Genomic gains or losses ≥ 15 Mb were prespecified as "screen positive" calls, and mapped to recurrent copy number variations reported in an ovarian cancer genome atlas. (2) Selected whole chromosome gains or losses were reported using the routine NIPT pipeline for fetal aneuploidy. We detected 13/32 cancer cases using the subchromosomal analysis (sensitivity 40.6 %, 95 % CI, 23.7-59.4 %), including 6/16 early and 7/16 advanced HGSOC cases. Two of 32 benign controls had subchromosomal gains ≥ 15 Mb (specificity 93.8 %, 95 % CI, 79.2-99.2 %). Twelve of the 13 true positive cancer cases exhibited specific recurrent changes reported in HGSOC tumors. The NIPT pipeline resulted in one "monosomy 18" call from the cancer group, and two "monosomy X" calls in the controls. Low coverage plasma DNA sequencing used for prenatal testing detected 40.6 % of all HGSOC, including 38 % of early stage cases. Our

  14. HPV DNA testing in population-based cervical screening (VUSA-Screen study): results and implications

    PubMed Central

    Rijkaart, D C; Berkhof, J; van Kemenade, F J; Coupe, V M H; Rozendaal, L; Heideman, D A M; Verheijen, R H M; Bulk, S; Verweij, W; Snijders, P J F; Meijer, C J L M

    2012-01-01

    Background: Human papillomavirus (HPV) testing is more sensitive than cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We evaluated the performance of high-risk HPV (hrHPV) testing in routine screening. Methods: In all, 25 871 women (29–61) enrolled in our population-based cohort study were offered both cytology and hrHPV testing. High-risk HPV-positive women with normal cytology and an age-matched subcohort of hrHPV-negative women with normal cytology were invited for repeat testing after 1 and/or 2 years and were referred for colposcopy if they presented with abnormal cytology and/or a positive hrHPV test. The hrHPV-positive women with borderline or mild dyskaryosis (BMD) and all women with moderate dyskaryosis or worse (>BMD) were directly referred for colposcopy. Women with BMD and an hrHPV-negative test were advised to repeat cytology at 6 and 18 months and were referred for colposcopy if the repeat cytology test was abnormal. The main outcome measure was CIN grade 3 or worse (CIN3+). Results were adjusted for non-attendance at repeat testing. Results: The hrHPV-positive women with abnormal cytology had a CIN3+ risk of 42.2% (95% confidence interval (CI): 36.4–48.2), whereas the hrHPV-positive women with normal cytology had a much lower risk of 5.22% (95% CI: 3.72–7.91). In hrHPV-positive women with normal cytology, an additional cytology step after 1 year reduced the CIN3+ risk to only 1.6% (95% CI: 0.6–4.9) if the repeat test was normal. The CIN3+ risk in women with hrHPV-positive normal cytology was higher among women invited for the first time (29–33 years of age) (9.1% 95% CI: 5.6–14.3) than among older women (3.0% 95% CI: 1.5–5.5). Conclusion: Primary hrHPV screening with cytology triage in women aged ⩾30 years is an effective way to stratify women on CIN3+ risk and seems a feasible alternative to cytological screening. Repeat cytology after 1 year for hrHPV-positive women with normal cytology is however

  15. Testing three pipelines for 18S rDNA-based metabarcoding of soil faunal diversity.

    PubMed

    Yang, ChenXue; Ji, YingQiu; Wang, XiaoYang; Yang, ChunYang; Yu, Douglas W

    2013-01-01

    A number of basic and applied questions in ecology and environmental management require the characterization of soil and leaf litter faunal diversity. Recent advances in high-throughput sequencing of barcode-gene amplicons ('metabarcoding') have made it possible to survey biodiversity in a robust and efficient way. However, one obstacle to the widespread adoption of this technique is the need to choose amongst many candidates for bioinformatic processing of the raw sequencing data. We compare three candidate pipelines for the processing of 18S small subunit rDNA metabarcode data from solid substrates: (i) USEARCH/CROP, (ii) Denoiser/UCLUST, and (iii) OCTUPUS. The three pipelines produced reassuringly similar and highly correlated assessments of community composition that are dominated by taxa known to characterize the sampled environments. However, OCTUPUS appears to inflate phylogenetic diversity, because of higher sequence noise. We therefore recommend either the USEARCH/CROP or Denoiser/UCLUST pipelines, both of which can be run within the QIIME (Quantitative Insights Into Microbial Ecology) environment.

  16. Applying and testing the conveniently optimized enzyme mismatch cleavage method to clinical DNA diagnosis.

    PubMed

    Niida, Yo; Kuroda, Mondo; Mitani, Yusuke; Okumura, Akiko; Yokoi, Ayano

    2012-11-01

    Establishing a simple and effective mutation screening method is one of the most compelling problems with applying genetic diagnosis to clinical use. Because there is no reliable and inexpensive screening system, amplifying by PCR and performing direct sequencing of every coding exon is the gold standard strategy even today. However, this approach is expensive and time consuming, especially when gene size or sample number is large. Previously, we developed CEL nuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) as an ideal simple mutation screening system constructed with only conventional apparatuses and commercially available reagents. In this study, we evaluated the utility of CHIPS technology for genetic diagnosis in clinical practice by applying this system to screening for the COL2A1, WRN and RPS6KA3 mutations in newly diagnosed patients with Stickler syndrome (autosomal dominant inheritance), Werner syndrome (autosomal recessive inheritance) and Coffin-Lowry syndrome (X-linked inheritance), respectively. In all three genes, CHIPS detected all DNA variations including disease causative mutations within a day. Direct sequencing of all coding exons of these genes confirmed 100% sensitivity and specificity. We demonstrate high sensitivity, high cost performance and reliability of this simple system, with compatibility to all inheritance modes. Because of its low technology, CHIPS is ready to use and potentially disseminate to any laboratories in the world.

  17. DNA from fecal immunochemical test can replace stool for detection of colonic lesions using a microbiota-based model.

    PubMed

    Baxter, Nielson T; Koumpouras, Charles C; Rogers, Mary A M; Ruffin, Mack T; Schloss, Patrick D

    2016-11-14

    There is a significant demand for colorectal cancer (CRC) screening methods that are noninvasive, inexpensive, and capable of accurately detecting early stage tumors. It has been shown that models based on the gut microbiota can complement the fecal occult blood test and fecal immunochemical test (FIT). However, a barrier to microbiota-based screening is the need to collect and store a patient's stool sample. Using stool samples collected from 404 patients, we tested whether the residual buffer containing resuspended feces in FIT cartridges could be used in place of intact stool samples. We found that the bacterial DNA isolated from FIT cartridges largely recapitulated the community structure and membership of patients' stool microbiota and that the abundance of bacteria associated with CRC were conserved. We also found that models for detecting CRC that were generated using bacterial abundances from FIT cartridges were equally predictive as models generated using bacterial abundances from stool. These findings demonstrate the potential for using residual buffer from FIT cartridges in place of stool for microbiota-based screening for CRC. This may reduce the need to collect and process separate stool samples and may facilitate combining FIT and microbiota-based biomarkers into a single test. Additionally, FIT cartridges could constitute a novel data source for studying the role of the microbiome in cancer and other diseases.

  18. Problem-Based Test: Replication of Mitochondrial DNA during the Cell Cycle

    ERIC Educational Resources Information Center

    Setalo, Gyorgy, Jr.

    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,…

  19. Problem-Based Test: Replication of Mitochondrial DNA during the Cell Cycle

    ERIC Educational Resources Information Center

    Setalo, Gyorgy, Jr.

    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,…

  20. Exploring tuberculosis by molecular tests on DNA isolated from smear microscopy slides.

    PubMed

    Rakotosamimanana, Niaina; Rabodoarivelo, Marie Sylvianne; Palomino, Juan Carlos; Martin, Anandi; Razanamparany, Voahangy Rasolofo

    2017-03-01

    Tuberculosis (TB) is an infectious disease of global public health importance caused by Mycobacterium tuberculosis complex. The disease has worsened with the emergence of multidrug-resistant (MDR)-TB strains. The timely diagnosis and treatment of TB remains a key public health priority, and laboratories have a critical role in the rapid and accurate detection of TB and drug resistance. Molecular assays based on nucleic acid amplification techniques have been developed for the rapid, sensitive, and specific diagnosis of TB, with the ability to determine the drug sensitivity status. These molecular techniques are now available or are being implemented in developing countries. However, traditional microscopy and culture methods cannot yet be replaced; the molecular assays can be applied in parallel with these tests for the diagnosis of TB or for drug susceptibility testing. Performing such molecular tests is often restricted by constraints with regard to sputum sample storage and safe transportation from remote health centres to central laboratories. Since smear slides are performed routinely for the diagnosis of TB in most TB diagnostic laboratories, they are readily available and could be the ideal tool to transport sputum for further molecular tests. The aim of this review was to provide a comprehensive survey on the use of smear slides for both TB diagnosis and the molecular test approach. Based on the literature, stained smear microscopy slides can be a safe system for the transportation of sputum specimens from remote health centres to reference TB laboratories for further molecular TB or MDR-TB detection, and could help in the rapid diagnosis and therefore timely management of TB patients. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Tracing the geographic origin of traded leopard body parts in the indian subcontinent with DNA-based assignment tests.

    PubMed

    Mondol, Samrat; Sridhar, Vanjulavalli; Yadav, Prasanjeet; Gubbi, Sanjay; Ramakrishnan, Uma

    2015-04-01

    Illicit trade in wildlife products is rapidly decimating many species across the globe. Such trade is often underestimated for wide-ranging species until it is too late for the survival of their remaining populations. Policing this trade could be vastly improved if one could reliably determine geographic origins of illegal wildlife products and identify areas where greater enforcement is needed. Using DNA-based assignment tests (i.e., samples are assigned to geographic locations), we addressed these factors for leopards (Panthera pardus) on the Indian subcontinent. We created geography-specific allele frequencies from a genetic reference database of 173 leopards across India to infer geographic origins of DNA samples from 40 seized leopard skins. Sensitivity analyses of samples of known geographic origins and assignments of seized skins demonstrated robust assignments for Indian leopards. We found that confiscated pelts seized in small numbers were not necessarily from local leopards. The geographic footprint of large seizures appeared to be bigger than the cumulative footprint of several smaller seizures, indicating widespread leopard poaching across the subcontinent. Our seized samples had male-biased sex ratios, especially the large seizures. From multiple seized sample assignments, we identified central India as a poaching hotspot for leopards. The techniques we applied can be used to identify origins of seized illegal wildlife products and trade routes at the subcontinent scale and beyond. © 2014 Society for Conservation Biology.

  2. Changes occurred in the testes and DNA pattern of males wax moth (Galleria mellonella) first generation as a result of irradiation of their parents.

    PubMed

    Rizk, Salwa Abdo; Abdalla, Ragaa Sayed; Sayed, Rehab Mahmoud

    2017-08-01

    Nowadays, the sterile insect technique is broadly used as a pest control measure. Therefore, the present study was conducted to investigate the alteration occurred in testes and DNA pattern as an effect of inherited sterility. Full grown pupae of the wax moth, Galleria mellonella were irradiated with 80 and 160 Gy of γ irradiation. The size of the testes was decreased by increasing of γ irradiation dose. Also, the size of the testes was decreased in F 1 males comparing with the size of the testes of both the parents and the untreated control. The effects of γ rays on the DNA patterns of adult male parents and F 1 males showed alterations among the controls, the treated parents and F 1 individuals. Exposure to radiation caused very frequently the appearance of some extra bands and the deficiency of others in the arbitrary random amplified polymorphic DNA-polymerase chain reaction amplification patterns of the irradiated insects.

  3. DNA paternity tests in Spain without the mother's consent: the legal responsibility of the laboratories.

    PubMed

    Barrot, C; Sánchez, C; Ortega, M; De Alcaraz-Fossoul, J; Carreras, C; Medallo, J; Bono, N; Royes, A; Gené, M

    2014-01-01

    It is technically feasible to perform paternity diagnosis testing solely involving an alleged father and his descendent. However, there are serious legal and ethical problems for forensic genetics laboratories when it comes to paternity testing cases for investigating the alleged father-child relationship if the biological mother has not given consent to access her genetic information. Based on the Spanish Constitution, the new Code of Ethics of the Spanish Medical Association includes several articles on studies about genetic information and their acceptance by all the individuals involved. This problem is greater when the child is a minor, mentally incapacitated or psychologically incapable, because current Spanish law requires informed consent from legal representatives, but the law does not typify what happens when one parent gives consent (the putative father) and the other parent (the mother) does not agree. The aim of this study is to put forward legal solutions to avoid potential legal problems.

  4. DNA integrity of canine spermatozoa during chill storage assessed by the sperm chromatin dispersion test using bright-field or fluorescence microscopy.

    PubMed

    Hidalgo, M; Urbano, M; Ortiz, I; Demyda-Peyras, S; Murabito, M R; Gálvez, M J; Dorado, J

    2015-08-01

    The objective of this study was to evaluate the effect of chill storage on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test using bright-field microscopy with Wright solution (sDF-B) or fluorescence microscopy with propidium iodide (sDF-F). The relationship and agreement between the results obtained with both staining methods were analyzed. The values of DNA fragmentation indexes (sDF-F and sDF-B) were compared at each time of chill storage (0, 24, 48, 72, and 96 hours). Additionally, the sperm DNA fragmentation rate (slope) was compared between the methods during chill storage. Good agreement and no significant differences between values obtained with both staining procedures were observed. Finally, the effect of chill storage for up to 96 hours was assessed on sperm motility parameters and DNA fragmentation indexes. Significant differences were found after 48 hours of chill storage, obtaining greater values of fragmented DNA. Progressive sperm motility was lower just after 96 hours of chill storage, and no effect was found in total sperm motility. In conclusion, the Sperm-Halomax kit, developed for canine semen and based on the sperm chromatin dispersion test, can be used accurately under bright-field or fluorescence microscopy to assess the sperm DNA integrity of canine semen during chill storage. The sperm DNA fragmentation index increased after 48 hours of chill storage, thereby detecting sperm damage earlier than other routine sperm parameters, such as sperm motility.

  5. DNA demethylation mediated by down-regulated TETs in the testes of rare minnow Gobiocypris rarus under bisphenol A exposure.

    PubMed

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

    2017-03-01

    Inevitable BPA exposure resulted in disturbance of DNA methylation status and our published study suspected that BPA has the potentiality to disturb DNA demethylation and GSH production in Gobiocypris rarus testes. To confirm this conjecture, several experiments were carried out in the present study. Adult male G. rarus was exposed to 1, 15 and 225 μg L(-1) (nominal concentration) BPA for two weeks. The levels of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), glutathione (GSH), and enzyme levels for DNA methylation and GSH synthesis in the testes were detected. Meanwhile, the contents of substrates for GSH synthesis were measured. Furthermore, the transcriptional changes of the studied genes were examined. Results indicated that 1-225 μg L(-1) BPA caused decrease of testicular ten-eleven translocation proteins (TETs) with more obvious effects at low concentrations. Moreover, all concentrations of BPA resulted in decrease of 5hmC levels while only 225 μg L(-1) BPA resulted in significant increase of 5mC. In addition, all treatments resulted in significant decrease of GSH and the replenishment of GSH might be mainly accomplished by circular synthesis. These results indicated that BPA exposure inhibited TETs-mediated DNA demethylation and the declined DNA demethylation mediated by TETs may result in DNA hypermethylation at 225 μg L(-1) BPA. In addition, the changes of DNA methylation status were irrelevant with GSH levels.

  6. The efficacy of individual-donation and minipool testing to detect low-level hepatitis B virus DNA in Taiwan.

    PubMed

    Yang, Meng-Hua; Li, Lei; Hung, Ying-Shen; Hung, Cheng-Shen; Allain, Jean-Pierre; Lin, Kuo-Sin; Tsai, Su-Jen Lin

    2010-01-01

    Financial constraints are the main concern in implementing nucleic acid testing (NAT) as routine blood screening in Taiwan. The PROCLEIX ULTRIO assay (Ultrio) on the TIGRIS System (Novartis Diagnostics) was evaluated for its operational performance both for individual-donation testing (IDT) and in minipools of 4 (MP4) to develop a feasible solution. Analytical sensitivity was determined by testing WHO international standards. We tested 10,290 blood donors, 4210 in IDT and 6080 in MP4. Potential hepatitis B virus (HBV) yield donors (hepatitis B surface antigen [HBsAg] negative/NAT reactive) were evaluated for up to 9 months' follow-up. Discordant results between the Ultrio assay and the HBsAg tests were further analyzed by HBV antibody serology, alternative NATs, HBV DNA quantification, and sequencing. The 95% limits of detection in IU/mL (95% confidence interval) were as follows: human immunodeficiency virus Type 1 (HIV-1), 18 (12-34); hepatitis C virus (HCV), 4.4 (2.8-8.9); and HBV, 6.3 (4.4-11). The retest rates were 0.55% for IDT and 0.33% for MP4. No HIV or HCV yield cases were found, while there were 12 potential HBV yield cases, nine from IDT and three from MP4 testing. Eleven of them were successfully genotyped as B2. Ten of them returned for follow-up and mostly were determined as occult HBV infection (OBI). The IDT yield rate of 9 in 4210 (0.21%) was fourfold greater than the MP4 yield rate of 3 in 6080 (0.05%; p < 0.05). The higher yield rate for IDT versus MP4 demonstrates the benefit to implement a more sensitive NAT strategy in regions having significant OBI carriers such as Taiwan.

  7. [Performance and indications of noninvasive prenatal testing using cell free circulating fetal DNA (cffDNA) for the detection of fetal trisomy 21, 18 and 13 in France].

    PubMed

    Benachi, A; Letourneau, A; Kleinfinger, P; Senat, M-V; Gautier, E; Favre, R; Bidat, L; Houfflin-Debarge, V; Querol, V; Bouyer, J; Costa, J-M

    2016-06-01

    To evaluate de performances of noninvasive prenatal testing using cell free circulating fetal DNA (cffDNA) for the detection of fetal trisomy 21, 18 and 13 in a French population. cffDNA analysis was performed by massive parallel sequencing during a multicenter, non interventional, prospective study and the results were compared with a standard fetal karyotype. Results were available for 886 patients who have been classified as high- or moderate-risk depending on the presence of fetal abnormalities on ultrasound examination. For the high-risk group (n=376), the sensitivity and specificity of the test were 100% and 99.9% for trisomy 21, 88% and 99.9% for trisomy 18 and 100% and 99.9% for trisomy 13. The rate of other pathogenic chromosomal abnormalities with a negative NIPT was 7.9%. In the low-risk group (n=510), the sensitivity was 100% and the specificity 99.8% for trisomy 21, and only 0.4% of pathogenic chromosomal abnormalities were revealed by fetal karyotyping but not detected by cffDNA analysis. Noninvasive prenatal testing using cffDNA for high risk patients without fetal anomalies at ultrasound could be recommended in France after counseling on the possible risk of undiagnosed anomalies. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  8. Sex differences in DNA methylation and expression in zebrafish brain: a test of an extended 'male sex drive' hypothesis.

    PubMed

    Chatterjee, Aniruddha; Lagisz, Malgorzata; Rodger, Euan J; Zhen, Li; Stockwell, Peter A; Duncan, Elizabeth J; Horsfield, Julia A; Jeyakani, Justin; Mathavan, Sinnakaruppan; Ozaki, Yuichi; Nakagawa, Shinichi

    2016-09-30

    The sex drive hypothesis predicts that stronger selection on male traits has resulted in masculinization of the genome. Here we test whether such masculinizing effects can be detected at the level of the transcriptome and methylome in the adult zebrafish brain. Although methylation is globally similar, we identified 914 specific differentially methylated CpGs (DMCs) between males and females (435 were hypermethylated and 479 were hypomethylated in males compared to females). These DMCs were prevalent in gene body, intergenic regions and CpG island shores. We also discovered 15 distinct CpG clusters with striking sex-specific DNA methylation differences. In contrast, at transcriptome level, more female-biased genes than male-biased genes were expressed, giving little support for the male sex drive hypothesis. Our study provides genome-wide methylome and transcriptome assessment and sheds light on sex-specific epigenetic patterns and in zebrafish for the first time.

  9. Chronic carriers of hepatitis B virus in Bangladesh: a comparative analysis of HBV-DNA, HBeAg/anti-HBe, and liver function tests.

    PubMed

    Hasan, K N; Rumi, M A K; Hasanat, M A; Azam, M G; Ahmed, S; Salam, M A; Islam, L N; Hassan, M S

    2002-03-01

    Serological markers of hepatitis B virus (HBV), liver function tests and quantitative estimation of HBV-DNA are important in the assessment of the state of infection and prognosis following treatment for hepatitis B. This study aimed to determine whether low-cost assays, eg hepatitis B e antigen (HBeAg) and liver function tests, could be used for the assessment of infectivity as an alternative to HBV-DNA estimation. We tested 125 hepatitis B carriers for HBeAg, antibody to HBeAg (anti-HBe), and serum HBV-DNA; we also carried out a range of standard liver function tests. Seventy-three subjects were positive and 52 were negative for HBeAg. Of the HBeAg positive cases, 3 were also positive for anti-HBe; of the HBeAg negative cases, 5 were also negative for anti-HBe. Of these 8 cases, 7 had no detectable HBV-DNA. Most of the HBeAg positive but anti-HBe negative subjects were positive for HBV-DNA (74.3%; 52/ 70) whereas most of the HBeAg negative and anti-HBe positive subjects (93.6%; 44/47) were also negative for HBV-DNA. Of 56 HBV-DNA positive individuals, alanine transaminase (ALT) was found to be raised in 69.6% (p=0.066) and aspartate transaminase (AST) was raised in 66.1% (p=0.011), while 67.9% had normal alkaline phosphatase (ALP) (p=0.054). HBeAg (p=0.018) and raised ALT (p=0.008) were found to be independent predictors for HBV-DNA positivity among HBV carriers. This study suggests that HBeAg positive and anti-HBe negative hepatitis B carriers with raised ALT and AST are likely to be positive for HBV-DNA; the combination of routine serology and biochemical tests may be considered as an alternative to HBV-DNA in evaluating the state of chronic HBV infection. However, HBV-DNA should be specifically assessed if discordance is observed between seromarkers and transaminases.

  10. Non-invasive prenatal diagnostic test accuracy for fetal sex using cell-free DNA a review and meta-analysis.

    PubMed

    Wright, Caroline F; Wei, Yinghui; Higgins, Julian P T; Sagoo, Gurdeep S

    2012-09-01

    Cell-free fetal DNA (cffDNA) can be detected in maternal blood during pregnancy, opening the possibility of early non-invasive prenatal diagnosis for a variety of genetic conditions. Since 1997, many studies have examined the accuracy of prenatal fetal sex determination using cffDNA, particularly for pregnancies at risk of an X-linked condition. Here we report a review and meta-analysis of the published literature to evaluate the use of cffDNA for prenatal determination (diagnosis) of fetal sex. We applied a sensitive search of multiple bibliographic databases including PubMed (MEDLINE), EMBASE, the Cochrane library and Web of Science. Ninety studies, incorporating 9,965 pregnancies and 10,587 fetal sex results met our inclusion criteria. Overall mean sensitivity was 96.6% (95% credible interval 95.2% to 97.7%) and mean specificity was 98.9% (95% CI = 98.1% to 99.4%). These results vary very little with trimester or week of testing, indicating that the performance of the test is reliably high. Based on this review and meta-analysis we conclude that fetal sex can be determined with a high level of accuracy by analyzing cffDNA. Using cffDNA in prenatal diagnosis to replace or complement existing invasive methods can remove or reduce the risk of miscarriage. Future work should concentrate on the economic and ethical considerations of implementing an early non-invasive test for fetal sex.

  11. Comparative test of DNA probes for detection of Plasmodium vivax circumsporozoite protein polymorphs VK 247 and VK 210.

    PubMed

    Sattabongkot, J; Suwanabun, N; Rongnoparut, P; Wirtz, R A; Kain, K C; Rosenberg, R

    1994-02-01

    Oligonucleotide probes specific to the characteristic repeat sequences of two alleles of the circumsporozoite protein gene of Plasmodium vivax (VK 210 and VK 247) were selected, synthesized, and tested on matched blood and sporozoite DNA amplified by polymerase chain reaction from 182 cases naturally acquired in Thailand. Probe results were compared to those of circumsporozoite phenotype-specific ELISAs used to evaluate sporozoites from the same cases. There was a 96% agreement between probe results for blood and for sporozoites. Although there was also a nearly complete agreement between probe and ELISA results for cases producing only VK 210 or VK 247 sporozoites, the probes detected 45% more mixed infections than did the ELISAs when used to test specimens from western and southern Thailand; there was no discrepancy when mixed cases from Cambodia were tested. Examination of Southern blots from ambiguous mixed cases demonstrated the presence of both genes, suggesting suppression of VK 247 in some mixed cases to numbers below those detectable by the ELISA.

  12. Amelogenin test abnormalities revealed in Belarusian population during forensic DNA analysis.

    PubMed

    Borovko, Sergey; Shyla, Alena; Korban, Victorya; Borovko, Alexandra

    2015-03-01

    Study of gender markers is a part of routine forensic genetic examination of crime scene and reference samples, paternity testing and personal identification. Amelogenin locus as a gender marker is included in majority of forensic STR kits of different manufacturers. In current study we report 11 cases of amelogenin abnormalities identified in males of Belarusian origin: 9 cases of AMELY dropout and 2 cases of AMELX dropout. Cases were obtained from forensic casework (n=9) and paternity testing (n=2) groups. In 4 out of 9 AMELY-negative cases deletion of AMELY was associated with the loss of DYS458 marker. In addition, we identified 3 males with SRY-positive XX male syndrome. Deletion of the long arm of the Y-chromosome was detected in two XX males. Loss of the major part of the Y-chromosome was identified in the third XX male. The presence of two X-chromosomes in XX males was confirmed with the use of Mentype(®) Argus X-8 PCR Amplification Kit. AMELY null allele observed in 2 out of 9 cases with AMELY dropout can be caused by mutation in the primer-binding site of AMELY allele. Primer-binding site mutations of AMELX can result in AMELX dropout identified in 2 cases with amplification failure of AMELX. Our study represents the first report and molecular genetic investigation of amelogenin abnormalities in the Belarusian population.

  13. 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.

  14. NAD+ administration significantly attenuates synchrotron radiation X-ray-induced DNA damage and structural alterations of rodent testes

    PubMed Central

    Sheng, Caibin; Chen, Heyu; Wang, Ban; Liu, Tengyuan; Hong, Yunyi; Shao, Jiaxiang; He, Xin; Ma, Yingxin; Nie, Hui; Liu, Na; Xia, Weiliang; Ying, Weihai

    2012-01-01

    Synchrotron radiation (SR) X-ray has great potential for its applications in medical imaging and cancer treatment. In order to apply SR X-ray in clinical settings, it is necessary to elucidate the mechanisms underlying the damaging effects of SR X-ray on normal tissues, and to search for the strategies to reduce the detrimental effects of SR X-ray on normal tissues. However, so far there has been little information on these topics. In this study we used the testes of rats as a model to characterize SR X-ray-induced tissue damage, and to test our hypothesis that NAD+ administration can prevent SR X-ray-induced injury of the testes. We first determined the effects of SR X-ray at the doses of 0, 0.5, 1.3, 4 and 40 Gy on the biochemical and structural properties of the testes one day after SR X-ray exposures. We found that 40 Gy of SR X-ray induced a massive increase in double-strand DNA damage, as assessed by both immunostaining and Western blot of phosphorylated H2AX levels, which was significantly decreased by intraperitoneally (i.p.) administered NAD+ at doses of 125 and 625 mg/kg. Forty Gy of SR X-ray can also induce marked increases in abnormal cell nuclei as well as significant decreases in the cell layers of the seminiferous tubules one day after SR X-ray exposures, which were also ameliorated by the NAD+ administration. In summary, our study has shown that SR X-ray can produce both molecular and structural alterations of the testes, which can be significantly attenuated by NAD+ administration. These results have provided not only the first evidence that SR X-ray-induced tissue damage can be ameliorated by certain approaches, but also a valuable basis for elucidating the mechanisms underlying SR X-ray-induced tissue injury. PMID:22518270

  15. Parentage test in broad-snouted caimans (Caiman latirostris, Crocodylidae) using microsatellite DNA

    PubMed Central

    2009-01-01

    In this study, microsatellite markers, developed for Alligator mississipiensis and Caiman latirostris, were used to assess parentage among individuals from the captive colony of Caiman latirostris at the University of São Paulo, in Piracicaba, São Paulo, Brazil. Many of the females in the colony were full siblings, which made maternal identification difficult due to genotypic similarity. Even so, the most likely mother could be identified unambiguously among offspring in most of the clutches studied. Two non-parental females displayed maternal behavior which would have misled managers in assigning maternity based on behavior alone. This set of variable loci demonstrates the utility of parentage testing in captive propagation programs. PMID:21637468

  16. Rapid DNA haplotyping using a multiplex heteroduplex approach: application to Duchenne muscular dystrophy carrier testing.

    PubMed

    Prior, T W; Wenger, G D; Papp, A C; Snyder, P J; Sedra, M S; Bartolo, C; Moore, J W; Highsmith, W E

    1995-01-01

    A new strategy has been developed for rapid haplotype analysis based on an initial multiplex amplification of several polymorphic sites, followed by heteroduplex detection. Heteroduplexes formed between two different alleles are detected because they migrate differently than the corresponding homoduplexes in Hydrolink-MDE gel. This simple, rapid method does not depend on specific sequences such as restriction enzyme sites or CA boxes and does not require the use of isotope. This approach has been tested using commonly occurring polymorphisms spanning the dystrophin gene as a model. We describe the use of the method to assign the carrier status of females in Duchenne muscular dystrophy (DMD) pedigrees. The method may be used for other genetic diseases when mutations are unknown or there are few dinucleotide markers in the gene proximity, and for the identification of haplotype backgrounds of mutant alleles.

  17. Sensitive neutralization test for rubella antibody.

    PubMed Central

    Sato, H; Albrecht, P; Krugman, S; Ennis, F A

    1979-01-01

    A modified rubella virus plaque neutralization test for measuring rubella antibody was developed based on the potentiation of the virus-antibody complex by heterologous anti-immunoglobulin. The test is highly sensitive, yielding titers on the average 50 to 100 times higher than the haemagglutination inhibition test or the conventional plaque neutralization test. The sensitivity of this enhanced neutralization test is somewhat limited by the existence of a prozone phenomenon which precludes testing of low-titered sera below a dilution of 1:16. No prozone effect was observed with cerebrospinal fluids. The specificity of the enhanced neutralization test was determined by seroconversion of individuals receiving rubella vaccine. Although the rubella hemagglutination inhibition test remains the test of choice in routine diagnostic and surveillance work, the enhanced rubella neutralization test is particularly useful in monitoring low-level antibody in the cerebrospinal fluid in patients with neurological disorders and in certain instances of vaccine failure. PMID:107192

  18. 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

  19. The power of DNA double-strand break (DSB) repair testing to predict breast cancer susceptibility.

    PubMed

    Keimling, Marlen; Deniz, Miriam; Varga, Dominic; Stahl, Andreea; Schrezenmeier, Hubert; Kreienberg, Rolf; Hoffmann, Isabell; König, Jochem; Wiesmüller, Lisa

    2012-05-01

    Most presently known breast cancer susceptibility genes have been linked to DSB repair. To identify novel markers that may serve as indicators for breast cancer risk, we performed DSB repair analyses using a case-control design. Thus, we examined 35 women with defined familial history of breast and/or ovarian cancer (first case group), 175 patients with breast cancer (second case group), and 245 healthy women without previous cancer or family history of breast cancer (control group). We analyzed DSB repair in peripheral blood lymphocytes (PBLs) by a GFP-based test system using 3 pathway-specific substrates. We found increases of microhomology-mediated nonhomologous end joining (mmNHEJ) and nonconservative single-strand annealing (SSA) in women with familial risk vs. controls (P=0.0001-0.0022) and patients with breast cancer vs. controls (P=0.0004-0.0042). Young age (<50) at initial diagnosis of breast cancer, which could be indicative of genetic predisposition, was associated with elevated SSA using two different substrates, amounting to similar odds ratios (ORs=2.54-4.46, P=0.0059-0.0095) as for familial risk (ORs=2.61-4.05, P=0.0007-0.0045). These findings and supporting validation data underscore the great potential of detecting distinct DSB repair activities in PBLs as method to estimate breast cancer susceptibility beyond limitations of genotyping and to predict responsiveness to therapeutics targeting DSB repair-dysfunctional tumors.

  20. HPV DNA testing with cytology triage in cervical cancer screening: Influence of revealing HPV infection status.

    PubMed

    Richardson, Lyndsay Ann; El-Zein, Mariam; Ramanakumar, Agnihotram V; Ratnam, Samuel; Sangwa-Lugoma, Ghislain; Longatto-Filho, Adhemar; Cardoso, Marly Augusto; Coutlée, Francois; Franco, Eduardo L

    2015-12-01

    Knowledge of cervical human papillomavirus (HPV) status might influence a cytotechnician's assessment of cellular abnormalities. The authors compared original cytotechnicians' Papanicolaou (Pap) readings for which HPV status was concealed with Pap rereads for which HPV status was revealed separately for 3 screening populations. Previously collected cervical Pap smears and clinical data were obtained from the Canadian Cervical Cancer Screening Trial (study A), the Democratic Republic of Congo Community-Based Screening Study (study B), and the Brazilian Investigation into Nutrition and Cervical Cancer Prevention (study C). Smears were reread with knowledge of HPV status for all HPV-positive women as well as a sample of HPV-negative women. Diagnostic performance of Pap cytology was compared between original readings and rereads. A total of 1767 Pap tests were reread. Among 915 rereads for HPV-positive women, the contrast between "revealed" and "concealed" Pap readings demonstrated revisions from negative to positive results for 109 women (cutoff was atypical squamous cells of undetermined significance or worse) and 124 women (cutoff was low-grade squamous intraepithelial lesions [LSIL] or worse). For a disease threshold of cervical intraepithelial neoplasia of grade 2 or worse, specificity significantly declined at the atypical squamous cells of undetermined significance cutoff for studies A (86.6% to 75.3%) and C (42.5% to 15.5%), and at the LSIL cutoff for study C (61.9% to 37.6%). Sensitivity remained nearly unchanged between readings, except in study C, in which reread performance was superior (91.3% vs 71.9% for the LSIL cutoff). A reduction in the diagnostic accuracy of Pap cytology was observed when revealing patients' cervical HPV status, possibly due to a heightened awareness of potential abnormalities, which led to more false-positive results. © 2015 American Cancer Society.

  1. Introducing a High-Risk HPV DNA Test Into a Public Sector Screening Program in El Salvador.

    PubMed

    Cremer, Miriam L; Maza, Mauricio; Alfaro, Karla M; Kim, Jane J; Ditzian, Lauren R; Villalta, Sofia; Alonzo, Todd A; Felix, Juan C; Castle, Philip E; Gage, Julia C

    2016-04-01

    In a primary human papillomavirus (HPV) screening program, we compared the 6-month follow-up among colposcopy and noncolposcopy-based management strategies for screen-positive women. Women aged 30 to 49 years were screened with HPV DNA tests using both self-collection and provider collection of samples. Women testing positive received either (1) colposcopy management (CM) consisting of colposcopy and management per local guidelines or (2) screen-and-treat (ST) management using visual inspection with acetic acid to determine cryotherapy eligibility, with eligible women undergoing immediate cryotherapy. One thousand women were recruited in each cohort. Of these, 368 (18.4%) of 2000 women were recruited using a more intensive outreach strategy. Demographics, HPV positivity, and treatment compliance were compared across recruitment and management strategies. More women in the ST cohort received treatment within 6 months compared with those in the CM cohort (117/119 [98.3%] vs 64/93 [68.8%]; p < .001). Women recruited through more intensive outreach were more likely to be HPV positive, lived in urban areas, were more educated, and had higher numbers of lifetime sexual partners and fewer children. Women in the CM arm were less likely to complete care than women in the ST arm. Targeted outreach to underscreened women successfully identified women with higher prevalence of HPV and possibly higher disease burden.

  2. Introducing a High-Risk HPV DNA Test Into a Public Sector Screening Program in El Salvador

    PubMed Central

    Cremer, Miriam L.; Maza, Mauricio; Alfaro, Karla M.; Kim, Jane J.; Ditzian, Lauren R.; Villalta, Sofia; Alonzo, Todd A.; Felix, Juan C.; Castle, Philip E.; Gage, Julia C.

    2016-01-01

    Objective In a primary human papillomavirus (HPV) screening program, we compared the 6-month follow-up among colposcopy and noncolposcopy-based management strategies for screen-positive women. Materials and Methods Women aged 30 to 49 years were screened with HPV DNA tests using both self-collection and provider collection of samples. Women testing positive received either (1) colposcopy management (CM) consisting of colposcopy and management per local guidelines or (2) screen-and-treat (ST) management using visual inspection with acetic acid to determine cryotherapy eligibility, with eligible women undergoing immediate cryotherapy. One thousand women were recruited in each cohort. Of these, 368 (18.4%) of 2000 women were recruited using a more intensive outreach strategy. Demographics, HPV positivity, and treatment compliance were compared across recruitment and management strategies. Results More women in the ST cohort received treatment within 6 months compared with those in the CM cohort (117/119 [98.3%] vs 64/93 [68.8%]; p < .001). Women recruited through more intensive outreach were more likely to be HPV positive, lived in urban areas, were more educated, and had higher numbers of lifetime sexual partners and fewer children. Conclusions Women in the CM arm were less likely to complete care than women in the ST arm. Targeted outreach to underscreened women successfully identified women with higher prevalence of HPV and possibly higher disease burden. PMID:26890683

  3. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing.

    PubMed

    Li, Jin; Wang, Lilin; Mamon, Harvey; Kulke, Matthew H; Berbeco, Ross; Makrigiorgos, G Mike

    2008-05-01

    PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.

  4. Evaluation of the impact of genetic linkage in forensic identity and relationship testing for expanded DNA marker sets.

    PubMed

    Tillmar, Andreas O; Phillips, Chris

    2017-01-01

    Advances in massively parallel sequencing technology have enabled the combination of a much-expanded number of DNA markers (notably STRs and SNPs in one or combined multiplexes), with the aim of increasing the weight of evidence in forensic casework. However, when data from multiple loci on the same chromosome are used, genetic linkage can affect the final likelihood calculation. In order to study the effect of linkage for different sets of markers we developed the biostatistical tool ILIR, (Impact of Linkage on forensic markers for Identity and Relationship tests). The ILIR tool can be used to study the overall impact of genetic linkage for an arbitrary set of markers used in forensic testing. Application of ILIR can be useful during marker selection and design of new marker panels, as well as being highly relevant for existing marker sets as a way to properly evaluate the effects of linkage on a case-by-case basis. ILIR, implemented via the open source platform R, includes variation and genomic position reference data for over 40 STRs and 140 SNPs, combined with the ability to include additional forensic markers of interest. The use of the software is demonstrated with examples from several different established marker sets (such as the expanded CODIS core loci) including a review of the interpretation of linked genetic data.

  5. Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA

    PubMed Central

    Yadav, Arun A.; Wu, Xing; Patel, Daywin; Yalowich, Jack C.; Hasinoff, Brian B.

    2014-01-01

    Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds were designed, synthesized and biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topoisomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid had a mean GI50 in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the N-mustard compounds from which they were derived. These results supported the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA. PMID:25282653

  6. Disposable DNA biosensor with the carbon nanotubes-polyethyleneimine interface at a screen-printed carbon electrode for tests of DNA layer damage by quinazolines.

    PubMed

    Galandová, Júlia; Ovádeková, Renáta; Ferancová, Adriana; Labuda, Ján

    2009-06-01

    A screen-printed carbon working electrode within a commercially available screen-printed three-electrode assembly was modified by using a composite of multiwalled carbon nanotubes (MWCNT) dispersed in polyethylenimine (PEI) followed by covering with the calf thymus dsDNA layer. Several electrochemical methods were used to characterize the biosensor and to evaluate damage to the surface-attached DNA: square wave voltammetry of the [Ru(bpy)(3)](2+) redox indicator and mediator of the guanine moiety oxidation, cyclic voltammetry and electrochemical impedance spectroscopy in the presence of the [Fe(CN)(6)](3-/4-) indicator in solution. Due to high electroconductivity and large surface area of MWCNT and positive charge of PEI, the MWCNT-PEI composite is an advantageous platform for the DNA immobilization by the polyelectrolyte complexation and its voltammetric and impedimetric detection. In this respect, the MWCNT-PEI interface exhibited better properties than the MWCNT-chitosan one reported from our laboratory previously. A deep DNA layer damage at incubation of the biosensor in quinazoline solution was found, which depends on the quinazoline concentration and incubation time.

  7. An Independent Evaluation of the Validity of a DNA-Based Prognostic Test for Adolescent Idiopathic Scoliosis.

    PubMed

    Roye, Benjamin D; Wright, Margaret L; Matsumoto, Hiroko; Yorgova, Petya; McCalla, Daren; Hyman, Joshua E; Roye, David P; Shah, Suken A; Vitale, Michael G

    2015-12-16

    ScoliScore is a DNA-based prognostic test, designed and used to help to predict the risk of curve progression in patients with adolescent idiopathic scoliosis. The role of this test in clinical practice remains unclear as the published results of the ScoliScore have not been validated independently. The purpose of this study was to determine if the ScoliScore effectively predicted the risk of curve progression in patients with mild and moderate adolescent idiopathic scoliosis in two urban academic medical centers. One hundred and twenty-six patients with adolescent idiopathic scoliosis who met inclusion criteria at two centers were administered the ScoliScore test. Two groups were created: a progression group (those who had a Cobb angle of >40° or those who had undergone surgical fusion) and a non-progression group (those who had skeletal maturity without curve progression to 40°). ScoliScore values and risk levels were compared between the two groups. The negative predictive value was calculated for low-risk scores and the positive predictive value was calculated for high-risk scores. There was no significant difference (p = 0.706) in the mean ScoliScore (and standard deviation) between patients with curve progression (107 ± 55 points) and those without curve progression (102 ± 62 points). There was also no significant difference (p = 0.399) in curve progression between patients with high-risk scores (26.7%) and those with low-risk scores (12.9%). The positive predictive value of the test was 0.27 (95% confidence interval, 0.09 to 0.55), and the negative predictive value was 0.87 (95% confidence interval, 0.69 to 0.96). ScoliScores and rates of progression were not affected by brace-wear. ScoliScores did not differ between patients with and without curve progression, and the negative and positive predictive values were lower in our study than in the previously published validation study by the developers of the test. This may be due to differences in our test

  8. Genotypic tropism testing in proviral DNA to guide maraviroc initiation in aviremic subjects: 48-week analysis of the PROTEST study

    PubMed Central

    Garcia, Federico; Poveda, Eva; Jesús Pérez-Elías, Maria; Hernández Quero, José; Àngels Ribas, Maria; Martínez-Madrid, Onofre J; Flores, Juan; Crespo, Manel; Gutiérrez, Félix; García-Deltoro, Miguel; Imaz, Arkaitz; Ocampo, Antonio; Artero, Arturo; Blanco, Francisco; Bernal, Enrique; Pasquau, Juan; Mínguez-Gallego, Carlos; Pérez, Núria; Aiestarán, Aintzane; Paredes, Roger

    2014-01-01

    Introduction In a previous interim 24-week virological safety analysis of the PROTEST study [1], initiation of Maraviroc (MVC) plus 2 nucleoside reverse-transcriptase inhibitors (NRTIs) in aviremic subjects based on genotypic tropism testing of proviral HIV-1 DNA was associated with low rates of virological failure. Here we present the final 48-week analysis of the study. Methods PROTEST was a phase 4, prospective, single-arm clinical trial (ID: NCT01378910) carried on in 24 HIV care centres in Spain. Maraviroc-naïve HIV-1-positive adults with HIV-1 RNA (VL) <50 c/mL on stable ART during the previous 6 months, requiring an ART change due to toxicity, with no antiretroviral resistance to the ART started, and R5 HIV by proviral DNA genotypic tropism testing (defined as a G2P FPR >10% in a singleton), initiated MVC with 2 NRTIs and were followed for 48 weeks. Virological failure was defined as two consecutive VL>50 c/mL. Recent adherence was calculated as: (# pills taken/# pills prescribed during the previous week)*100. Results Tropism results were available from 141/175 (80.6%) subjects screened: 87/141 (60%) were R5 and 74/87 (85%) were finally included in the study. Their median age was 48 years, 16% were women, 31% were MSM, 36% had CDC category C at study entry, 62% were HCV+ and 10% were HBV+. Median CD4+ counts were 616 cells/mm3 at screening, and median nadir CD4+ counts were 143 cells/mm3. Previous ART included PIs in 46 (62%) subjects, NNRTIs in 27 (36%) and integrase inhibitors (INIs) in 1 (2%). The main reasons for treatment change were dyslipidemia (42%), gastrointestinal symptoms (22%), and liver toxicity (15%). MVC was given alongside TDF/FTC in 40 (54%) subjects, ABC/3TC in 30 (40%), AZT/3TC in 2 (3%) and ABC/TDF in 2 (3%). Sixty-two (84%) subjects maintained VL<50 c/mL through week 48, whereas 12 (16%) discontinued treatment: two (3%) withdrew informed consent, one (1%) had a R5→X4 shift in HIV tropism between the screening and baseline visits, one

  9. The accuracy of cell-free fetal DNA-based non-invasive prenatal testing in singleton pregnancies: a systematic review and bivariate meta-analysis.

    PubMed

    Mackie, F L; Hemming, K; Allen, S; Morris, R K; Kilby, M D

    2017-01-01

    Cell-free fetal DNA (cffDNA) non-invasive prenatal testing (NIPT) is rapidly expanding, and is being introduced at varying rates depending on country and condition. Determine accuracy of cffDNA-based NIPT for all conditions. Evaluate influence of other factors on test performance. Medline, Embase, CINAHL, Cochrane Library, from 1997 to April 2015. Cohort studies reporting cffDNA-based NIPT performance in singleton pregnancies. Bivariate or univariate meta-analysis and subgroup analysis performed to explore influence of test type and population risk. A total of 117 studies were included that analysed 18 conditions. Bivariate meta-analysis demonstrated sensitivities and specificities, respectively, for: fetal sex, 0.989 (95% CI 0.980-0.994) and 0.996 (95% CI 0.989-0.998), 11 179 tests; rhesus D, 0.993 (95% CI 0.982-0.997) and 0.984 (95% CI 0.964-0.993), 10 290 tests; trisomy 21, 0.994 (95% CI 0.983-0.998) and 0.999 (95% CI 0.999-1.000), 148 344 tests; trisomy 18, 0.977 (95% CI 0.952-0.989) and 0.999 (95% CI 0.998-1.000), 146 940 tests; monosomy X, 0.929 (95% CI 0.741-0.984) and 0.999 (95% CI 0.995-0.999), 6712 tests. Trisomy 13 was analysed by univariate meta-analysis, with a summary sensitivity of 0.906 (95% CI 0.823-0.958) and specificity of 1.00 (95% CI 0.999-0.100), from 134 691 tests. False and inconclusive results were poorly reported across all conditions. Although the test type affected both sensitivity and specificity, there was no evidence that population risk had any effect. Performance of cffDNA-based NIPT is affected by condition under investigation. For fetal sex and rhesus D status, NIPT can be considered diagnostic. For trisomy 21, 18, and 13, the lower sensitivity, specificity, and disease prevalence, combined with the biological influence of confined placental mosaicism, designates it a screening test. These factors must be considered when counselling patients and assessing the cost of introduction into routine care

  10. The introduction of a choice to learn pre-symptomatic DNA test results for BRCA or Lynch syndrome either face-to-face or by letter.

    PubMed

    Voorwinden, J S; Jaspers, J P C; ter Beest, J G; Kievit, Y; Sijmons, R H; Oosterwijk, J C

    2012-05-01

    In predictive DNA testing for hereditary cancer, test results should traditionally be disclosed face-to-face. Increasingly, however, counselees ask to receive their test result at home by letter. To compare the quality of genetic counselling in the traditional way to a procedure in which counselees are offered a choice on how to get their test result. Counselees from families with a known BRCA1/2 or Lynch syndrome mutation were randomised into two groups. The control group was given the DNA test result in a face-to-face consultation. In the intervention group people could choose to learn their test result face-to-face or by letter. The quality of genetic counselling was assessed through questionnaires at three different moments. Data of 198 counselees were analysed. The quality of genetic counselling and psychological functioning were equally good in both groups. The majority of cases chose for disclosure by letter. The counselees with a good test result in the intervention group were the most satisfied. Our results indicate that in predictive DNA testing for BRCA1/2 and Lynch syndrome, a choice protocol is equally safe and more satisfying. Moreover, it is more efficient for both counsellor and counselee.

  11. DNA Damage Potential of Engine Emissions Measured In Vitro by Micronucleus Test in Human Bronchial Epithelial Cells.

    PubMed

    Cervena, Tereza; Rossnerova, Andrea; Sikorova, Jitka; Beranek, Vit; Vojtisek-Lom, Michal; Ciganek, Miroslav; Topinka, Jan; Rossner, Pavel

    2016-10-26

    Internal combustion engine emissions belong among the major anthropogenic sources of air pollution in urban areas. According to the International Agency for Research on Cancer, there is sufficient evidence of the carcinogenicity of diesel exhaust in human beings. Although alternative fuels, mainly biodiesel, have recently become popular, little is still known about the genotoxicity of emissions from these fuels. We analysed DNA damage expressed as the frequency of micronuclei (MN) in human bronchial epithelial cells (BEAS-2B), induced by extractable organic matter (EOM; tested concentrations: 1, 10 and 25 μg/ml) obtained from particle emissions from various blends of biodiesel with diesel fuels (including neat diesel fuel (B0), a blend of 70% B0 and 30% biodiesel (B30) and neat biodiesel (B100)). We also tested the effect of selected diesel exhaust organic/genotoxic components [benzo[a]pyrene (B[a]P) concentrations: 25, 100 and 200 μM; 1-nitropyrene (1-NP) concentrations: 1, 5 and 10 μM; 3-nitrobenzanthrone (3-NBA) concentrations: 1, 5 and 50 μM]. The cells were treated with the compounds for 28 and 48 hr. Our results showed that most of the tested compounds (except for the 25 μM B[a]P, 28-hr treatment) significantly increased MN frequency. The genotoxicity of EOMs from the engine emissions of diesel and biodiesel engines was comparable. Both nitro-PAH compounds demonstrated higher genotoxic potential in comparison with B[a]P. Considering our results and due to increasing popularity of alternative fuels, it is prudent that the potential genotoxic effects of various fuels are investigated across engine technologies and operating conditions in a relevant model system.

  12. 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.

  13. 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

  14. 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.

  15. False negative PCR despite high levels of JC virus DNA in spinal fluid: Implications for diagnostic testing.

    PubMed

    Landry, Marie L; Eid, Tore; Bannykh, Serguei; Major, Eugene

    2008-10-01

    Genome amplification methods such as polymerase chain reaction (PCR) have revolutionized our ability to detect viruses in spinal fluids of patients with neurologic diseases. It is not as well appreciated among clinicians that PCR protocols, quality assurance, and technical expertise vary significantly among laboratories. In a multi-laboratory blinded study of herpes simplex virus PCR, the most widely used and best validated CSF PCR assay, low-level positives were often missed and false positives were not uncommon [Schloss L, van Loon AM, Cinque P, Cleator G, Echevarria JM, Falk KI, et al. An international external quality assessment of nucleic acid amplification of herpes simplex virus. J Clin Virol 2003;28(2):175-85]. In addition, genome variability and mutations, which are increasingly recognized for a number of different viruses, can lead to falsely low or negative results. Both clinicians and laboratories must recognize the limitations of PCR, since misleading results may have serious consequences. We present here a case of a rapidly progressive, fatal neurologic illness in a young mother, whose CSF JCV DNA PCR at a reference laboratory was falsely negative. Ultimately, brain biopsy established the diagnosis of progressive multifocal leukoencephalopathy (PML). Repeat PCR testing of the same CSF targeting a different region of the genome yielded a high positive result.

  16. HPV DNA test

    MedlinePlus

    ... evaluation, and management. Committee Opinion No. 463. American College of Obstetricians and Gynecologists. Obstet Gynecol . 2010;116:469-472. U.S. Preventive Services Task Force. Screening for cervical cancer: U.S. Preventive ...

  17. Mitochondrial DNA copy number augments performance of A1C and oral glucose tolerance testing in the prediction of type 2 diabetes

    PubMed Central

    Cho, Seong Beom; Koh, InSong; Nam, Hye-Young; Jeon, Jae-Pil; Lee, Hong Kyu; Han, Bok-Ghee

    2017-01-01

    Here, we tested the performance of the mitochondrial DNA copy number (mtDNA-CN) in predicting future type 2 diabetes (n = 1108). We used the baseline clinical data (age, sex, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure) and the mtDNA-CN, hemoglobin A1c (A1C) levels and results of oral glucose tolerance test (OGTT) including fasting plasma glucose, 1-hour glucose, and 2-hour glucose levels, to predict future diabetes. We built a prediction model using the baseline data and the diabetes status at biannual follow-up of 8 years. The mean area under curve (AUC) for all follow-ups of the full model including all variables was 0.92 ± 0.04 (mean ± standard deviation), while that of the model excluding the mtDNA-CN was 0.90 ± 0.03. The sensitivity of the f4ull model was much greater than that of the model not including mtDNA-CN: the mean sensitivities of the model with and without mtDNA-CN were 0.60 ± 0.06 and 0.53 ± 0.04, respectively. We found that the mtDNA-CN of peripheral leukocytes is a biomarker that augments the predictive power for future diabetes of A1C and OGTT. We believe that these results could provide invaluable information for developing strategies for the management of diabetes. PMID:28251996

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. Cervical human papilloma virus (HPV) DNA primary screening test: Results of a population-based screening programme in central Italy.

    PubMed

    Passamonti, Basilio; Gustinucci, Daniela; Giorgi Rossi, Paolo; Cesarini, Elena; Bulletti, Simonetta; Carlani, Angela; Martinelli, Nadia; Broccolini, Massimo; D'Angelo, Valentina; D'Amico, Maria Rosaria; Di Dato, Eugenio; Galeazzi, Paola; Malaspina, Morena; Spita, Nicoletta; Tintori, Beatrice; Giaimo, Maria Donata

    2017-09-01

    Objective To present the results of the first and second round human papilloma virus (HPV)-based screening programme in the Umbria region after three years. Methods From August 2010 to November 2011, the entire female population aged 35-64 in a local health district was invited for HPV testing (HPV-DNA cobas4800 on a liquid-based cytology sample). HPV-negative women were re-invited after three years. For HPV-positive women, a slide was prepared and interpreted. Positive cytologies were referred to colposcopy; negatives were referred to repeat HPV after one year. If HPV was persistently positive, women were referred to colposcopy; if negative, to normal screening. Indicators of the first and second round are compared with those of cytology screening in the same area in the preceding three years. Results Participation was 56.5%, the same as cytology (56.6%). HPV-positivity was 6.4% (396/6272), cytology triage positivity was 35.6%; 251 cytology negative women were referred to one-year HPV retesting, 84.1% complied, and 55.5% were positive. Total colposcopy referral was 4.1%, and for cytology 1%. The detection rate for cervical intraepithelial neoplasia grade 2 or more severe was 10‰, compared with 3.7‰ using cytology. After three years, HPV-positivity was 3.4% (129/3831), overall colposcopy referral was 2.3% (most at one-year follow-up), and detection rate was 0.5/1000. Conclusions The first round detection rate was more than twice that of cytology screening, while colposcopy referral increased fourfold. At the second round, the detection rate decreased dramatically, showing that longer interval and more conservative protocols are needed.

  3. Comparison of HPV DNA testing in cervical exfoliated cells and tissue biopsies among HIV-positive women in Kenya.

    PubMed

    De Vuyst, Hugo; Chung, Michael H; Baussano, Iacopo; Mugo, Nelly R; Tenet, Vanessa; van Kemenade, Folkert J; Rana, Farzana S; Sakr, Samah R; Meijer, Chris J L M; Snijders, Peter J F; Franceschi, Silvia

    2013-09-15

    HIV-positive women are infected with human papillomavirus (HPV) (especially with multiple types), and develop cervical intraepithelial neoplasia (CIN) and cervical cancer more frequently than HIV-negative women. We compared HPV DNA prevalence obtained using a GP5+/6+ PCR assay in cervical exfoliated cells to that in biopsies among 468 HIV-positive women from Nairobi, Kenya. HPV prevalence was higher in cells than biopsies and the difference was greatest in 94 women with a combination normal cytology/normal biopsy (prevalence ratio, PR = 3.7; 95% confidence interval, CI: 2.4-5.7). PR diminished with the increase in lesion severity (PR in 58 women with high-grade squamous intraepithelial lesions (HSIL)/CIN2-3 = 1.1; 95% CI: 1.0-1.2). When HPV-positive, cells contained 2.0- to 4.6-fold more multiple infections than biopsies. Complete or partial agreement between cells and biopsies in the detection of individual HPV types was found in 91% of double HPV-positive pairs. The attribution of CIN2/3 to HPV16 and/or 18 would decrease from 37.6%, when the presence of these types in either cells or biopsies was counted, to 20.2% when it was based on the presence of HPV16 and/or 18 (and no other types) in biopsies. In conclusion, testing HPV on biopsies instead of cells results in decreased detection but not elimination of multiple infections in HIV-positive women. The proportion of CIN2/3 attributable to HPV16 and/or 18 among HIV-positive women, which already appeared to be lower than that in HIV-negative, would then further decrease. The meaning of HPV detection in cells and random biopsy from HIV-positive women with no cervical abnormalities remains unclear. Copyright © 2013 UICC.

  4. Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

    PubMed Central

    Leontiou, Chrysanthia A.; Hadjidaniel, Michael D.; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C.

    2015-01-01

    Introduction Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Methods Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. Results The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Conclusion Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed

  5. Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing.

    PubMed

    Leontiou, Chrysanthia A; Hadjidaniel, Michael D; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C

    2015-01-01

    Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite

  6. Frequency of HBV DNA detection in US blood donors testing positive for the presence of anti-HBc: implications for transfusion transmission and donor screening.

    PubMed

    Kleinman, Steven H; Kuhns, Mary C; Todd, Deborah S; Glynn, Simone A; McNamara, Anne; DiMarco, Anthony; Busch, Michael P

    2003-06-01

    An estimate of the rate of HBV DNA-positive, anti-HBc-positive units is important for evaluating the need for anti-HBc donor screening, especially in the context of HBV NAT. HBsAg EIA-nonreactive, anti-HBc-reactive (Corzyme, Abbott Laboratories) specimens were retrieved from a repository and were retested for anti-HBc (with PRISM HBcore, Abbott Laboratories, currently under FDA review) and anti-HBs (with PRISM Ausab, Abbott Laboratories, research assay). HBV DNA testing using a PCR assay with a greater than 95 percent detection rate of less than 50 copies per mL was performed on a subset of specimens that were PRISM HBcore-reactive and were anti-HBs- negative or reactive at less than 100 IU per L. A total of 395 of 1231 specimens eligible by our serologic criteria were tested by PCR. Four anti-HBs-negative specimens were PCR-positive with estimated HBV DNA copy numbers of 10 per 30 copies per mL in two specimens and 50 to 100 copies per mL in two others. The HBV DNA detection rate in anti-HBs-negative specimens was 3.7 percent, and the projected rate among all Corzyme-reactive specimens was 0.24 percent, leading to an estimated yield of 1 HBV DNA-positive, anti-HBc-positive unit in 49,000 units that were otherwise eligible for transfusion (95% CI, 1 in 16,600-1 in 152,600). Anti-HBc screening detects HBsAg EIA-negative, HBV-infected donors at a rate comparable to the estimated residual risk for HBV window-period infections. The low viral load in the HBV DNA-positive samples suggests that minipool NAT will not detect most potentially infectious units from anti-HBc-positive donors.

  7. Validation of a rapid DNA process with the RapidHIT(®) ID system using GlobalFiler(®) Express chemistry, a platform optimized for decentralized testing environments.

    PubMed

    Salceda, Susana; Barican, Arnaldo; Buscaino, Jacklyn; Goldman, Bruce; Klevenberg, Jim; Kuhn, Melissa; Lehto, Dennis; Lin, Frank; Nguyen, Phong; Park, Charles; Pearson, Francesca; Pittaro, Rick; Salodkar, Sayali; Schueren, Robert; Smith, Corey; Troup, Charles; Tsou, Dean; Vangbo, Mattias; Wunderle, Justus; King, David

    2017-05-01

    The RapidHIT(®) ID is a fully automated sample-to-answer system for short tandem repeat (STR)-based human identification. The RapidHIT ID has been optimized for use in decentralized environments and processes presumed single source DNA samples, generating Combined DNA Index System (CODIS)-compatible DNA profiles in less than 90min. The system is easy to use, requiring less than one minute of hands-on time. Profiles are reviewed using centralized linking software, RapidLINK™ (IntegenX, Pleasanton, CA), a software tool designed to collate DNA profiles from single or multiple RapidHIT ID systems at different geographic locations. The RapidHIT ID has been designed to employ GlobalFiler(®) Express and AmpFLSTR(®) NGMSElect™, Thermo Fisher Scientific (Waltham, MA) STR chemistries. The Developmental Validation studies were performed using GlobalFiler(®) Express with single source reference samples according to Scientific Working Group for DNA Analysis Methods guidelines. These results show that multiple RapidHIT ID systems networked with RapidLINK software form a highly reliable system for wide-scale deployment in locations such as police booking stations and border crossings enabling real-time testing of arrestees, potential human trafficking victims, and other instances where rapid turnaround is essential.

  8. Acceptability of self-collection sampling for HPV-DNA testing in low-resource settings: a mixed methods approach.

    PubMed

    Bansil, Pooja; Wittet, Scott; Lim, Jeanette L; Winkler, Jennifer L; Paul, Proma; Jeronimo, Jose

    2014-06-12

    Vaginal self-sampling with HPV-DNA tests is a promising primary screening method for cervical cancer. However, women's experiences, concerns and the acceptability of such tests in low-resource settings remain unknown. In India, Nicaragua, and Uganda, a mixed-method design was used to collect data from surveys (N = 3,863), qualitative interviews (N = 72; 20 providers and 52 women) and focus groups (N = 30 women) on women's and providers' experiences with self-sampling, women's opinions of sampling at home, and their future needs. Among surveyed women, 90% provided a self- collected sample. Of these, 75% reported it was easy, although 52% were initially concerned about hurting themselves and 24% were worried about not getting a good sample. Most surveyed women preferred self-sampling (78%). However it was not clear if they responded to the privacy of self-sampling or the convenience of avoiding a pelvic examination, or both. In follow-up interviews, most women reported that they didn't mind self-sampling, but many preferred to have a provider collect the vaginal sample. Most women also preferred clinic-based screening (as opposed to home-based self-sampling), because the sample could be collected by a provider, women could receive treatment if needed, and the clinic was sanitary and provided privacy. Self-sampling acceptability was higher when providers prepared women through education, allowed women to examine the collection brush, and were present during the self-collection process. Among survey respondents, aids that would facilitate self-sampling in the future were: staff help (53%), additional images in the illustrated instructions (31%), and a chance to practice beforehand with a doll/model (26%). Self-and vaginal-sampling are widely acceptable among women in low-resource settings. Providers have a unique opportunity to educate and prepare women for self-sampling and be flexible in accommodating women's preference for self-sampling.

  9. Acceptability of self-collection sampling for HPV-DNA testing in low-resource settings: a mixed methods approach

    PubMed Central

    2014-01-01

    Background Vaginal self-sampling with HPV-DNA tests is a promising primary screening method for cervical cancer. However, women’s experiences, concerns and the acceptability of such tests in low-resource settings remain unknown. Methods In India, Nicaragua, and Uganda, a mixed-method design was used to collect data from surveys (N = 3,863), qualitative interviews (N = 72; 20 providers and 52 women) and focus groups (N = 30 women) on women’s and providers’ experiences with self-sampling, women’s opinions of sampling at home, and their future needs. Results Among surveyed women, 90% provided a self- collected sample. Of these, 75% reported it was easy, although 52% were initially concerned about hurting themselves and 24% were worried about not getting a good sample. Most surveyed women preferred self-sampling (78%). However it was not clear if they responded to the privacy of self-sampling or the convenience of avoiding a pelvic examination, or both. In follow-up interviews, most women reported that they didn’t mind self-sampling, but many preferred to have a provider collect the vaginal sample. Most women also preferred clinic-based screening (as opposed to home-based self-sampling), because the sample could be collected by a provider, women could receive treatment if needed, and the clinic was sanitary and provided privacy. Self-sampling acceptability was higher when providers prepared women through education, allowed women to examine the collection brush, and were present during the self-collection process. Among survey respondents, aids that would facilitate self-sampling in the future were: staff help (53%), additional images in the illustrated instructions (31%), and a chance to practice beforehand with a doll/model (26%). Conclusion Self-and vaginal-sampling are widely acceptable among women in low-resource settings. Providers have a unique opportunity to educate and prepare women for self-sampling and be flexible in accommodating women

  10. Enhancing capacitive DNA biosensor performance by target overhang with application on screening test of HLA-B*58:01 and HLA-B*57:01 genes.

    PubMed

    Thipmanee, Orawan; Numnuam, Apon; Limbut, Warakorn; Buranachai, Chittanon; Kanatharana, Proespichaya; Vilaivan, Tirayut; Hirankarn, Nattiya; Thavarungkul, Panote

    2016-08-15

    A highly sensitive label-free DNA biosensor based on PNA probes immobilized on a gold electrode was used to detect a hybridization event. The effect of a target DNA overhang on the hybridization efficiency was shown to enhance the detected signal and allowed detection at a very low concentration. The sensors performances were investigated with a complementary target that had the same length as the probe, and the signal was compared to the target DNAs with different lengths and overhangs. A longer target DNA overhang was found to provide a better response. When the overhang was on the electrode side the signal enhancement was greater than when the overhang was on the solution side due to the increased thickness of the sensing surface, hence produced a larger capacitance change. Using conformationally constrained acpcPNA probes, double stranded DNA was detected sensitively and specifically without any denaturing step. When two acpcPNA probes were applied for the screening test for the double stranded HLA-B*58:01 and HLA-B*57:01 genes that are highly similar, the method differentiated the two genes in all samples. Both purified and unpurified PCR products gave comparable results. This method would be potentially useful as a rapid screening test without the need for purification and denaturation of the PCR products.

  11. 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.

  12. Exploring hereditary cancer among dying cancer patients--a cross-sectional study of hereditary risk and perceived awareness of DNA testing and banking.

    PubMed

    Quillin, John Martin; Bodurtha, Joann N; Siminoff, Laura A; Smith, Thomas J

    2010-10-01

    Hereditary cancer assessment at the end of life is a relatively unexplored area, but it could be critical for surviving family members. This study explored the prevalence of hereditary cancer among dying cancer patients and assessed patients' perceived awareness of DNA testing and/or banking in a public access hospital. Palliative care patients with cancer from a single institution (or their medical-decision-making surrogates for patients unable to answer for themselves) completed structured interviews. Information was collected through medical records review and structured interviews for 43 dying cancer patients. Information for 9 patients was collected from surrogates. Nine patients (21%, 95% CI = 8.8% to 33.1%) had strong genetic risk. Currently available genetic tests could have addressed this risk for several patients. None had previous genetic counseling, testing or DNA banking. Among strong-risk patients, about half of patients/surrogates had heard/read "almost nothing" about genetic testing (44%) and DNA banking (67%). Perceived genetic awareness was not associated with genetic risk, and neither were sociodemographic characteristics. The proportion of hereditary cancer may be at least as high in the palliative care population as in other clinical settings, but awareness and uptake among patients are low. These conditions are not being recognized upstream and families are losing valuable information.

  13. Diagnostic accuracy of DNA methylation for head and neck cancer varies by sample type and number of markers tested

    PubMed Central

    Ji, Xu; Guan, Chao; Jiang, Xuejun; Li, Hong

    2016-01-01

    Abnormal methylation of certain cancer related genes strongly predicts a diagnosis of head and neck cancer (HNC), while the predictive power of methylation of other DNA markers for HNC remains unclear. To systemically assess the diagnostic value of DNA methylation patterns for HNC and the effect of methylation platform techniques and sample types, we performed a PubMed search for studies of the correlation between DNA methylation and HNC completed before July 2016, and extracted the sensitivity and specificity for methylated biomarkers. Across these studies, DNA methylation showed high sensitivity for diagnosing HNC in solid tissue (0.57), and high specificity in saliva (0.89). Area under the curve (AUC) from summary receiver operating characteristic (SROC) curves revealed that DNA methylation had more diagnostic power in solid tissue (AUC = 0.82) than saliva (AUC = 0.80) or blood (AUC = 0.77). Combinations of multiple methylated genes were more sensitive diagnostic markers than single methylated genes. Our results suggest that the diagnostic accuracy of methylated biomarkers for HNC varied by sample type and were most accurate when results from multiple sample types were considered. PMID:27683120

  14. Catalytic nanomedicine: a new field in antitumor treatment using supported platinum nanoparticles. In vitro DNA degradation and in vivo tests with C6 animal model on Wistar rats.

    PubMed

    López, T; Figueras, F; Manjarrez, J; Bustos, J; Alvarez, M; Silvestre-Albero, J; Rodríguez-Reinoso, F; Martínez-Ferre, A; Martínez, E

    2010-05-01

    Novel nanostructured TiO2 and SiO2 based biocatalysts, with 3-4 wt. % of Pt have been developed. The obtained materials exhibit a high surface area together with a broad pore size distribution. The method of synthesis allowed obtaining high dispersed platinum metal nanoparticles. In vitro DNA reactivity test of the biocatalysts were carried out by electrophoresis and formation of DNA adducts was observed. The most active biocatalyst was H2PtCl6/SiO2. These biocatalysts were also tested in an experimental model of C6 brain tumours in Wistar rats. Administration of the material was made by stereotactic brain surgery to place it directly in the malignant tissue. A significant decrease in tumour size and weight as well as morphologic changes in cancer cells were observed. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

  15. RNA versus DNA (NucliSENS EasyQ HIV-1 v1.2 versus Amplicor HIV-1 DNA test v1.5) for early diagnosis of HIV-1 infection in infants in Senegal.

    PubMed

    Kébé, K; Ndiaye, O; Ndiaye, H Diop; Mengue, P Mbakob; Guindo, P M M; Diallo, S; Léye, N; Gueye, S B; Diallo, A Gaye; Kane, C Touré; Mboup, S

    2011-07-01

    The objective of this study was to compare the performance of the NucliSENS EasyQ HIV-1 v1.2 platform (bioMérieux, France) to the Amplicor HIV-1 DNA test v1.5 (Roche Molecular Systems, Switzerland) in detecting HIV-1 infection in infants using venipuncture-derived whole blood in tubes and dried blood spots. A total of 149 dried blood spots and 43 EDTA-anticoagulated peripheral blood samples were collected throughout Dakar and other areas in Senegal from infants and children aged 3 weeks to 24 months who were born to HIV-1-infected mothers. Samples were tested using the NucliSENS and Amplicor technologies. The NucliSENS and Amplicor results were 100% concordant using either EDTA-anticoagulated peripheral blood or dried blood spots. Compared to Amplicor, the sensitivity and specificity of the NucliSENS test were 100%. The NucliSENS EasyQ HIV-1 RNA assay performed as well as the Amplicor HIV-1 DNA test in detecting HIV-1 infection in infants. In addition, this platform can give an indication of the viral load baseline. The NucliSENS EasyQ platform is a good alternative for early infant diagnosis of HIV-1 infection.

  16. Impact of genetic counseling and DNA testing on individuals with colorectal cancer with a positive family history: a population-based study.

    PubMed

    Loader, Starlene; Shields, Cleveland; Rowley, Peter T

    2005-01-01

    What is the impact of a genetic evaluation for colorectal cancer susceptibility? We previously reported a study of individuals diagnosed with colorectal cancer at 60 years of age or less in a five-county area of New York including Rochester. Subjects reporting at least one first- or second-degree relative with colorectal cancer were invited to receive genetic counseling and DNA testing. Of the 37 persons tested, we previously reported that 6 were found to have deleterious mutations in MSH2 or MLH1. A mutation has since been found in a seventh subject in another laboratory. To evaluate the impact of the testing experience, we followed-up on 36 of these 37 subjects at 3 and 12 months after communicating their DNA test result. We ascertained their knowledge of colon cancer, whether they told relatives their test result, their surveillance behavior, and their mental health. Three months after receiving their result, subjects knew more about colon cancer if they had more cancer of all types in their family (p = 0.02). At 12 months, they knew more if they had been found to have a mutation (p < 0.001), were younger when DNA tested (p < 0.01), or were younger when diagnosed with cancer (p < 0.03). All but 1 of those found to have an abnormality told relatives. Relatives of 3 subjects in whom a mutation had been found came to us to be tested themselves. At 12 months, surveillance for colon and endometrial cancer was more adherent if there were more total cancers in the family (p < 0.05) or if the testee were more worried about cancer (p < 0.05). Self-assessed mental health at 12 months was better for those who were married (p < 0.05). This study suggests that individuals undergoing a genetic evaluation for an inherited susceptibility to colorectal cancer pursue recommended surveillance and inform relatives of their result.

  17. 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

  18. De-novo protein function prediction using DNA binding and RNA binding proteins as a test case.

    PubMed

    Peled, Sapir; Leiderman, Olga; Charar, Rotem; Efroni, Gilat; Shav-Tal, Yaron; Ofran, Yanay

    2016-11-21

    Of the currently identified protein sequences, 99.6% have never been observed in the laboratory as proteins and their molecular function has not been established experimentally. Predicting the function of such proteins relies mostly on annotated homologs. However, this has resulted in some erroneous annotations, and many proteins have no annotated homologs. Here we propose a de-novo function prediction approach based on identifying biophysical features that underlie function. Using our approach, we discover DNA and RNA binding proteins that cannot be identified based on homology and validate these predictions experimentally. For example, FGF14, which belongs to a family of secreted growth factors was predicted to bind DNA. We verify this experimentally and also show that FGF14 is localized to the nucleus. Mutating the predicted binding site on FGF14 abrogated DNA binding. These results demonstrate the feasibility of automated de-novo function prediction based on identifying function-related biophysical features.

  19. De-novo protein function prediction using DNA binding and RNA binding proteins as a test case

    PubMed Central

    Peled, Sapir; Leiderman, Olga; Charar, Rotem; Efroni, Gilat; Shav-Tal, Yaron; Ofran, Yanay

    2016-01-01

    Of the currently identified protein sequences, 99.6% have never been observed in the laboratory as proteins and their molecular function has not been established experimentally. Predicting the function of such proteins relies mostly on annotated homologs. However, this has resulted in some erroneous annotations, and many proteins have no annotated homologs. Here we propose a de-novo function prediction approach based on identifying biophysical features that underlie function. Using our approach, we discover DNA and RNA binding proteins that cannot be identified based on homology and validate these predictions experimentally. For example, FGF14, which belongs to a family of secreted growth factors was predicted to bind DNA. We verify this experimentally and also show that FGF14 is localized to the nucleus. Mutating the predicted binding site on FGF14 abrogated DNA binding. These results demonstrate the feasibility of automated de-novo function prediction based on identifying function-related biophysical features. PMID:27869118

  20. New Tests for Syphilis: Rational Design of a PCR Method for Detection of Treponema pallidum in Clinical Specimens Using Unique Regions of the DNA Polymerase I Gene

    PubMed Central

    Liu, Hsi; Rodes, Berta; Chen, C.-Y.; Steiner, Bret

    2001-01-01

    A sensitive and specific PCR method to detect Treponema pallidum in clinical specimens was developed. PCR primers were designed based on two unique features of the DNA polymerase I gene (polA). The first distinctive characteristic is that the region codes for a high cysteine content and has low homology with similar regions of DNA polymerase I gene from known microorganisms. The second unique feature is the presence of four insertions in the gene. PCR tests using primers designed on the basis these regions reacted with various pathogenic T. pallidum subspecies but did not react with nonpathogenic treponemal species or other spirochetes. An additional 59 species of bacteria and viruses, including those that cause genital ulcers, tested negative. This PCR method is extremely robust and sensitive. The detection limit is about 10 to 25 organisms when analyzed on gel. However, the analytic sensitivity can be increased by at least 1 log, to a detection limit of a single organism, when the ABI 310 Prism Genetic Analyzer is used to detect fluorescence-labeled amplicons. We further used this test in a clinical setting and compared the results with results from a previously reported multiplex-PCR test (for T. pallidum, Haemophilus ducreyi, and herpes simplex virus). We tested 112 genital ulcer specimens by the polA PCR, obtaining a sensitivity of 95.8% and a specificity of 95.7%. These results suggest that the polA PCR is applicable as a routine clinical diagnostic test for syphilis. PMID:11326018

  1. Evaluation of the new test VERSANT CT/GC DNA 1.0 assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens.

    PubMed

    Marangoni, Antonella; Foschi, Claudio; Nardini, Paola; D'Antuono, Antonietta; Banzola, Nicoletta; Di Francesco, Antonietta; Cevenini, Roberto

    2012-02-01

    Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) are the two most common sexually transmitted bacterial infections in developed countries. The purpose of the present study was evaluating a new system for CT/GC detection in urine specimens. A total of 700 urine specimens were obtained from patients attending the STD Outpatients Clinic of St. Orsola University Hospital, Bologna, Italy. Samples were tested by VERSANT® CT/GC DNA 1.0 Assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY), a multiplex Real-Time PCR assay, for simultaneous CT/GC detection. Results obtained by VERSANT assay were compared with those obtained by culturing genital secretions of the same patients. Moreover, urine specimens testing positive in VERSANT assay were retested by in-house PCR assays, used as confirmatory tests. VERSANT® CT/GC DNA 1.0 Assay performed with 99.4% and 99.2% of specificity for GC and CT detection, respectively, whereas sensitivity was 100% both for CT and GC. Culture methods were 100% specific, but far less sensitive than VERSANT assay. VERSANT® CT/GC DNA 1.0 Assay demonstrated to be a highly sensitive and specific technique for CT/GC detection.

  2. Selection of appropriate serological tests to measure the incidence of natural Leishmania infantum infection during DNA/MVA prime/boost canine vaccine trials.

    PubMed

    Carson, Connor; Antoniou, Maria; Christodoulou, Vasiliki; Messaritakis, Ippokratis; Quinnell, Rupert J; Blackwell, Jenefer M; Courtenay, Orin

    2009-06-10

    In response to the increasing need for field trials of experimental DNA vaccines against zoonotic visceral leishmaniasis in dogs, our aim was to validate the use of ELISA protocols which will be suitable for detection of natural infection in vaccinated dogs. We have previously demonstrated that DNA/modified vaccinia virus Ankara (MVA) vaccine expressing tryparedoxin peroxidase (TRYP) induced high titres of TRYP antigen-specific IgG in immunized dogs. Here we report our findings that seroconversion to an unrelated diagnostic antigen rK39 did not occur in vaccinated dogs, and that responses to crude Leishmania infantum promastigote antigen (CLA) were weak and short-lived. This is in contrast to strong responses to both antigens shown in naturally infected dogs. To select an appropriate serological test for measurement of infection incidence, we also tested longitudinal samples from an immunologically well-characterized cohort of naturally infected dogs. The sensitivity of CLA ELISA was superior to that of rK39 in early stage infection (from 2 months before, to 2 months after the first detection of infection by PCR or parasitological culture), and more sensitive than rK39 in cross-sectional sampling (81.0% vs 61.9%). We conclude that CLA ELISA will provide sensitive estimates of L. infantum infection incidence in DNA/MVA vaccinated dogs, though optimal testing would include rK39, or a similar recombinant antigen, to improve overall specificity.

  3. DNA methylation based testing of 418 patients suspected of having Prader-Willi syndrome (PWS) and tentative localization of the 15q11-13 imprinting center

    SciTech Connect

    Horsthemke, B.; Dittrich, B.; Buiting, K.

    1994-09-01

    Using a test based on parent-of-origin specific DNA methylation at the D15S63 (PW71) locus, we studied 358 patients (aged 1-33 years) for diagnostic confirmation of PWS and 60 infants (aged 0-12 months) with severe hypotonia of unknown origin. 57/358 patients were examined personally. 28/57 patients showed typical clinical signs of PWS and lacked the paternal PW71 band. 29-57 patients did not fulfill the diagnostic criteria for PWS and had a normal methylation pattern. This suggests that the test detects most if not all patients with typical PWS. 301/358 samples were sent to us from outside. The test confirmed the diagnosis in 105/301 patients. Most of the other 196 patients lacked neonatal hypotonia and hypogonadism. On the other hand, 27/60 hypotonic infants tested positive for PWS. These results indicate that PWS is often not recognized in infants and wrongly diagnosed in obese children and adults. We propose to perform the PW71 methylation test in every newborn with severe hypotonia of unknown origin to avoid unnecessary diagnostic procedures. In the course of diagnostic testing we and others have identified PWS and Angelman syndrome (AS) patients with apparently normal chromosomes of biparental inheritance, but abnormal DNA methylation and gene expression. The identification of microdeletions in some of these patients suggests the existence of an imprinting center within 30 kb proximal of SNRPN, which regulates the chromatin structure, DNA methylation and gene expression. We propose that the paternal copy of 15q11-13 has a euchromatoid structure, from which the PWS genes are transcribed, and that the maternal copy has a heterochromatoid structure from which the AS gene is transcribed. Depending on the parental origin of the mutation, both chromosomes of a patient have a heterochromatoid domain, which silences the PWS genes, or a euchromatoid domain, which silences the AS gene.

  4. Evaluation of a commercial in-clinic point-of-care polymerase chain reaction test for Ehrlichia canis DNA in artificially infected dogs.

    PubMed

    Waner, Trevor; Nachum-Biala, Yaarit; Harrus, Shimon

    2014-12-01

    A novel in-clinic point-of-care (ICPOC) polymerase chain reaction (PCR) test was evaluated for its ability to detect Ehrlichia canis DNA in artificially infected dogs compared to a real-time PCR assay. Six Beagle dogs negative for E. canis antibodies and PCR negative were artificially infected with an Israeli E. canis strain (611). All dogs developed IgG antibodies 8 days post infection (PI), and clinical and hematological abnormalities on day 10 PI. Only the real-time PCR detected E. canis DNA in the blood of five dogs at days 3 and 5 PI. At day 12 PI during the acute phase of the disease, 1 day after the initiation of doxycycline treatment, the ICPOC PCR assay detected E. canis DNA in all infected dogs, which were also positive by the real-time PCR. Two days later the ICPOC PCR assay was able to detect only 3/6 infected dogs, which were all positive by the real-time PCR. At days 17 and 19 PI, the ICPOC PCR assay did not detect E. canis DNA in the dogs while the real-time PCR detected all dogs as positive on day 17 PI and two dogs on day 19 PI. In conclusion, the sensitivity of the ICPOC PCR assay was 75% for the acute phase of the disease and 30% for the whole study, suggesting that this ICPOC assay has a potential utility for the diagnosis of acute canine monocytic ehrlichiosis.

  5. An innovative test for non-invasive Kell genotyping on circulating fetal DNA by means of the allelic discrimination of K1 and K2 antigens.

    PubMed

    Cro', Fabiana; Lapucci, Cristina; Vicari, Emilio; Salsi, Ginevra; Rizzo, Nicola; Farina, Antonio

    2016-12-01

    The aim of this study was to present a new method for fetal Kell genotyping by means of the allelic discrimination of K1 and K2 in real-time polymerase chain reaction (PCR). Real-time quantitative polymerase chain reaction incorporating an allele-specific primer was developed for detecting the K allele of KEL. By means of this method, the K1/K2 genotype was able to be determined in all blood samples analyzed. Results using cell-free fetal DNA (cffDNA) from two Kell-negative pregnant women confirmed the Kell-positive genotype of fetuses. The real-time PCR analysis also allowed the determination of the fetal fraction using the quantification of Kell-positive DNA. An efficient and reliable strategy for Kell genotyping is herein presented. The method was optimized on cffDNA to create a non-invasive prenatal test which could be routinely used for the prevention of hemolytic disease of the fetus and the newborn (HDFN). © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. A significant component of ageing (DNA damage) is reflected in fading breeding colors: an experimental test using innate antioxidant mimetics in painted dragon lizards.

    PubMed

    Olsson, Mats; Tobler, Michael; Healey, Mo; Perrin, Cecile; Wilson, Mark

    2012-08-01

    A decade ahead of their time, von Schantz et al. united sexual selection and free radical biology by identifying causal links between deep-rooted physiological processes that dictate resistance to toxic waste from oxidative metabolism (reactive oxygen species, ROS), and phenotypic traits, such as ornaments. Ten years later, these ideas have still only been tested with indirect estimates of free radical levels (oxidative stress) subsequent to the action of innate and dietary antioxidants. Here, we measure net superoxide (a selection pressure for antioxidant production) and experimentally manipulate superoxide antioxidation using a synthetic mimetic of superoxide dismutase (SOD), Eukarion 134 (EUK). We then measure the toxic effect of superoxide in terms of DNA erosion and concomitant loss of male breeding coloration in the lizard, Ctenophorus pictus. Control males suffered more DNA damage than EUK males. Spectroradiometry showed that male coloration is lost in relation to superoxide and covaries with DNA erosion; in control males, these variables explained loss of color, whereas in EUK males, the fading of coloration was unaffected by superoxide and unrelated to DNA damage. Thus, EUK's powerful antioxidation removes the erosion effect of superoxide on coloration and experimentally verifies the prediction that colors reflect innate capacity for antioxidation. © 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.

  7. Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA.

    PubMed

    Yadav, Arun A; Wu, Xing; Patel, Daywin; Yalowich, Jack C; Hasinoff, Brian B

    2014-11-01

    Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds were designed, synthesized and biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topoisomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid had a mean GI(50) in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the N-mustard compounds from which they were derived. These results supported the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Using DNA to test the utility of pellet-group counts as an index of deer counts

    Treesearch

    T. J. Brinkman; D. K. Person; W. Smith; F. Stuart Chapin; K. McCoy; M. Leonawicz; K. Hundertmark

    2013-01-01

    Despite widespread use of fecal pellet-group counts as an index of ungulate density, techniques used to convert pellet-group numbers to ungulate numbers rarely are based on counts of known individuals, seldom evaluated across spatial and temporal scales, and precision is infrequently quantified. Using DNA from fecal pellets to identify individual deer, we evaluated the...

  9. Collaborative study for establishment of a European Pharmacopoei Biological Reference Preparation (BRP) for B19 virus DNA testing of plasma pools by nucleic acid amplification technique.

    PubMed

    Nübling, C M; Daas, A; Buchheit, K H

    2004-01-01

    The goal of the collaborative study was to calibrate the B19 DNA content of a candidate Biological Reference Preparation (BRP) that is intended to be used for the validation of the analytical procedure, as threshold control and/or as quantitative reference material in the Nucleic Acid Amplification Technique (NAT) test of plasma pools for detection of B19 contamination. The candidate BRP was calibrated against the 1st International Standard for B19 DNA NAT assays. According to the European Pharmacopoeia monograph Human anti-D immunoglobulin, the threshold control needs to have a titre of 10( 4) IU/ml of B19 virus DNA. The lyophilised candidate BRP was prepared from 0.5 ml aliquots of a plasma pool spiked with B19 virus. The B19 virus originated from a "B19 virus window phase" blood donation (anti-B19 negative, B19-DNA high titre positive) and was diluted in a plasma pool tested negative by both serological and NAT assays for Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus 1 to obtain a B19-DNA concentration level in the range of 10( 6) copies/ml. The residual water content of the lyophilised candidate BRP was determined as 0.98 +/- 0.65% (mean +/- relative standard deviation). Sixteen laboratories (Official Medicine Control Laboratories, manufacturers of plasma derivatives, NAT test laboratories and NAT kit manufacturers) from nine countries participated. Participants were requested to test the candidate BRP and the International Standard (99/800) in four independent test runs on different days using their in-house qualitative and/or quantitative NAT methods. Sixteen laboratories reported results. Thirteen laboratories reported results from qualitative assays and 5 laboratories reported results from quantitative assays. Two laboratories reported results from both types of assay. For the qualitative assays a weighted combined potency of 5.64 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.17 log( 10) which corresponds to 67 to 150

  10. Disentangling the Babylonian speech confusion in genetic counseling: an analysis of the reliability and validity of the nomenclature for BRCA1/2 DNA-test results other than pathogenic.

    PubMed

    Vos, Joël; van Asperen, Christi J; Wijnen, Juul T; Stiggelbout, Anne M; Tibben, Aad

    2009-10-01

    Effective communication of DNA-test results requires a sound terminology. However, the variety of terms in literature for DNA-test results other than pathogenic, may create inconsistencies between professionals, and misunderstanding in patients. Therefore, we conducted a theoretical and empirical analysis of the terms most frequently used in articles between 2002 and 2007 for BRCA 1/2-test results other than pathogenic. We analyzed the content validity of the no-pathogenic DNA-test result-terms by comparing the literal and intended meaning of the terms and by examining their clarity and the inclusion of all relevant information. We analyzed the reliability of the terms by measuring the strength of association between terms and their meanings and the consistency among different authors over time. Two hundred twenty-seven articles with 361 no-pathogenic DNA-test result-terms were found. Only two terms seemed to have acceptable validity: variant of uncertain clinical significance and no-pathogenic-DNA-test-result. Only variant of uncertain clinical significance and true negative were found to be used reliably in the literature. Current DNA nomenclature lacks validity and reliability. Transparent DNA-test result terminology should be developed covering both laboratory findings and clinical meaning.

  11. Comparative evaluation of a triplex nucleic acid test for detection of HBV DNA, HCV RNA, and HIV-1 RNA, with the Procleix Tigris System.

    PubMed

    Xiao, Xinglong; Zhai, Jianxin; Zeng, Jinfeng; Tian, Cong; Wu, Hui; Yu, Yigang

    2013-02-01

    Nucleic acid testing (NAT) is valuable for screening blood donors for occult hepatitis B virus (HBV) infection and infection during the window period in countries where HBV is endemic, such as China. An "in-house" NAT (Triplex NAT) was developed for screening for HBV DNA, hepatitis C virus (HCV) RNA, and the human immunodeficiency virus type 1 (HIV-1) RNA. Using the Triplex NAT, a head-to-head comparative clinical evaluation was carried out against the most common commercial NAT used for blood screening in China: the Procleix Tigris System. A total of 33,025 specimens which were negative for Hepatitis B surface antigen, HCV antibody and HIV-1 antibody/antigen from potential blood donors were tested for HBV DNA, HCV RNA, and HIV-1 RNA by both the in-house Triplex assay and the commercially available Procleix Tigris System. Eleven specimens were detected as HBV positive by both NATs. Twelve specimens were detected as HBV positive by the Procleix Ultrio assay and the discriminatory assays, and not the Triplex. Twenty-eight specimens were detected as HBV positive by the Triplex and not the Procleix Ultrio. This study, combined with other data obtained in China, suggest that at least 50% HBV surface antigen negative but DNA-positive blood donations would be undetected using the current commercial NATs because of their insufficient sensitivity and/or Mini-Pool formatting strategies.

  12. Detection of an acute asymptomatic HBsAg negative hepatitis B virus infection in a blood donor by HBV DNA testing.

    PubMed

    Weber, Bernard; Mühlbacher, Annelies; Melchior, Walter

    2005-01-01

    The issue of HBV DNA screening on blood donations is controversially discussed since the economic impact of post-transfusion hepatitis B is expected to be relatively low. We report on a case of HBsAg negative unapparent acute HBV infection, which was detected by HBV NAT testing on 96-member maxi-pools with a commercially available NAT assay, which has a detection threshold of 3 IU/mL of plasma. The presence of an HBsAg escape mutant could be excluded by sequencing the amplified DNA. Follow-up testing showed the presence of an acute HBV infection (anti-HBc-IgM positive) and finally anti-HBs seroconversion. Although the reduction of the diagnostic window with NAT screening on maxi-pools may be relatively low, it may help to improve the residual risk of blood donation, especially in asymptomatic HBV infection, where the HBsAg positive period may be very short and low levels of circulating surface antigen are present. It would also permit to detect occult HBV infection in chronic carriers who are HBsAg negative. Since the viral load in chronic isolated anti-HBc positive carriers is low, there is a potential risk for failure of HBV DNA detection with pool-PCR in blood donors. Anti-HBc screening would reduce the residual risk.

  13. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR

    PubMed Central

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage. PMID:27313572

  14. Novel antigen identification method for discovery of protective malaria antigens by rapid testing of DNA vaccines encoding exons from the parasite genome.

    PubMed

    Haddad, Diana; Bilcikova, Erika; Witney, Adam A; Carlton, Jane M; White, Charles E; Blair, Peter L; Chattopadhyay, Rana; Russell, Joshua; Abot, Esteban; Charoenvit, Yupin; Aguiar, Joao C; Carucci, Daniel J; Weiss, Walter R

    2004-03-01

    We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.

  15. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR.

    PubMed

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

  16. cDNA-library testing identifies transforming genes cooperating with c-myc in mouse pre-B cells.

    PubMed

    Wolf, Inge; Bouquet, Corinne; Melchers, Fritz

    2016-11-01

    While c-myc often contributes to the generation of B cell transformation, its transgenic overexpression alone does not lead to full transformation of B-lineage cells. Synergistically acting second genes must cooperate. Here, we constructed doxycycline-inducible cDNA-libraries from pre-B cell mRNA. These libraries were retrovirally transduced as single copies into single cells and overexpressed in fetal-liver-derived c-myc-overexpressing pre-B cell lines. We scored transformation by survival and/or expansion of differentiating B-lineage cells in vitro and in vivo. Only one double c-myc/cDNA-library-expressing cell line was found in less than 5 × 10(6) library-transduced pre-B cells surviving and expressing a cDNA-library-derived transcript in vitro. This transcript was identified as a shortened form of the Exosc1 gene, encoding the RNA exosome complex component CSL4. Transplantations of double c-myc/Exosc1 short-form- or c-myc/Exosc1 full-length-transgenic cells into Rag1(-/-) mice resulted in survival, differentiation to CD19(+) CD93(-) sIgM(+) CD5(low/-) CD11b(+) mature B1 cells and, surprisingly, also vigorous expansion in vivo. Strikingly, after transplantations of c-myc/cDNA-library pre-BI cells the frequencies of double-transgenic pre-B cells and their differentiated progeny, expanding in vivo to heterogeneous phenotypes, was at least tenfold higher than in vitro. In a first analysis Ptprcap, Cacybp, Ndufs7, Rpl18a, and Rpl35a were identified. This suggests a strong influence of the host on B-cell transformation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Predicting adaptation to presymptomatic DNA testing for late onset disorders: who will experience distress? Rotterdam Leiden Genetics Workgroup.

    PubMed Central

    DudokdeWit, A C; Tibben, A; Duivenvoorden, H J; Niermeijer, M F; Passchier, J

    1998-01-01

    The first comparative study on predicting post-test distress (conceptualised by intrusion and avoidance, measured with the Impact of Event Scale) after presymptomatic genetic testing for Huntington's disease (HD, n=25), cancer syndromes (familial adenomatous polyposis (FAP, n=23)), and hereditary breast and ovarian cancer (HBOC, n=10) is reported. The variables with the highest predictive potential of post-test distress are presented. Participants who were depressed before the test were more distressed after testing, but we found that those who were anxious before the test were less distressed, that is, had less intrusive thoughts post-test. Other factors associated with a higher level of post-test intrusion were gender (being a woman), having children, and pre-test intrusion. Religion and being at risk for HBOC were associated with less post-test intrusion. Participants who showed avoidance behaviour before the test and those who had many people available for support showed more avoidance behaviour post-test. The test result did not additionally contribute to post-test distress. The prima facie simple notion that the test result, as such, determines the distress experienced seems to be a misrepresentation of the complex reality. PMID:9733033

  18. Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex.

    PubMed

    Koumbaris, George; Kypri, Elena; Tsangaras, Kyriakos; Achilleos, Achilleas; Mina, Petros; Neofytou, Maria; Velissariou, Voula; Christopoulou, Georgia; Kallikas, Ioannis; González-Liñán, Alicia; Benusiene, Egle; Latos-Bielenska, Anna; Marek, Pietryga; Santana, Alfredo; Nagy, Nikoletta; Széll, Márta; Laudanski, Piotr; Papageorgiou, Elisavet A; Ioannides, Marios; Patsalis, Philippos C

    2016-06-01

    There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT. © 2016 American Association for Clinical Chemistry.

  19. In vivo assessment of DNA damage and protective effects of extracts from Miconia species using the comet assay and micronucleus test.

    PubMed

    Serpeloni, Juliana Mara; Bisarro dos Reis, Mariana; Rodrigues, Juliana; Campaner dos Santos, Lourdes; Vilegas, Wagner; Varanda, Eliana A; Dokkedal, Anne L; Cólus, Ilce Mara S

    2008-11-01

    The genus Miconia comprises approximately 1000 species belonging to the Melastomataceae family. Several crude plant extracts from Miconia and their isolated compounds have shown biological activities, such as analgesic and anti-neoplastic action; however, no studies concerning their effects on DNA are available. The present study aimed to evaluate, in vivo, the genotoxic and mutagenic effects of four species of plants from Miconia genus using the comet assay and micronucleus test. Their possible protective effects were also evaluated in experiments associating the plant extracts with cyclophosphamide (CPA). The methanolic extracts of Miconia albicans, Miconia cabucu, Miconia rubiginosa, Miconia stenostachya and the chloroformic extract of M. albicans were investigated. For genotoxic and mutagenic evaluations, three concentrations were tested, 200, 400 and 540 mg/kg body weight (bw), based on the solubility limit of the extract in distilled water. For the protective effects, only the highest dose was evaluated against 40 mg/kg bw of CPA. Blood was removed from mice tails pre- (T0) and post-treatment (T1-30 h) for the micronucleus test and 24 h post-treatment for the comet assay. The Student's t-test was used to compare data obtained at T0 and T1, the analysis of variance-Tukey test was used to compare between groups in the micronucleus test and the Kruskal-Wallis and Dunn's test were used to compare different groups in the comet assay. All the extracts induced alterations in DNA migration (comet assay); however, no mutagenic effect was observed in the micronucleus assay. All extracts showed a protective effect against CPA in both assays. Our study showed that the use of crude extracts could be more advantageous than the use of isolated compounds. The interaction between phytochemicals in the extracts showed efficacy in reducing mutagenicity and improving the protective effects.

  20. Stability Study of Cervical Specimens Collected by Swab and Stored Dry Followed by Human Papillomavirus DNA Detection Using the cobas 4800 Test.

    PubMed

    Lin, Chun-Qing; Zeng, Xi; Cui, Jian-Feng; Liao, Guang-Dong; Wu, Ze-Ni; Gao, Qian-Qian; Zhang, Xun; Yu, Xiu-Zhang; Chen, Wen; Xi, Ming-Rong; Qiao, You-Lin

    2017-02-01

    Safer, more convenient methods for cervical sample collection and storage are necessary to facilitate human papillomavirus (HPV) DNA testing in low-resource settings. Our study aimed to evaluate the stability of cervical specimens collected with dry swabs and stored dry, compared to liquid-based cytology (LBC) samples, as detected by HPV DNA testing. Women with abnormal cytological findings or HPV-positive results at colposcopy were recruited from the West China Second University Hospital, Sichuan University, between October 2013 and March 2014. From each woman, physicians collected cervical specimens with a swab placed into a Sarstedt tube and a CytoBrush placed into LBC medium. Samples were randomly assigned to be stored at uncontrolled ambient temperature for 2, 7, 14, or 28 days and then were tested for 14 high-risk HPV (HR-HPV) types using the cobas HPV test. The rates of agreement between dry swab and LBC samples for any HR-HPV type, HPV16, HPV18, and the 12 pooled HR-HPV types were 93.8%, 97.8%, 99.4%, and 93.2%, respectively, with kappa values of 0.87 (95% confidence interval [CI], 0.83 to 0.91), 0.94 (95% CI, 0.91 to 0.97), 0.94 (95% CI, 0.87 to 1.00), and 0.86 (95% CI, 0.82 to 0.90). The performance of swab samples for detection of cervical precancerous lesions by means of cobas HPV testing was equal to that of LBC samples, even with stratification by storage time. Dry storage of swab-collected cervical samples can last for 1 month without loss of test performance by cobas HPV testing, compared to LBC samples, which may offer a simple inexpensive approach for cervical cancer screening in low-resource settings.

  1. Comparing self- and provider-collected swabbing for HPV DNA testing in female-to-male transgender adult patients: a mixed-methods biobehavioral study protocol.

    PubMed

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

    2017-06-23

    Cervical cancer, nearly all cases of which are caused by one of several high-risk strains of the human papillomavirus (hr-HPV), leads to significant morbidity and mortality in individuals with a cervix. Trans masculine (TM) individuals were born with female reproductive organs and identify as male, man, transgender man, or another diverse gender identity different from their female assigned sex at birth. Routine preventive sexual health screening of TM patients is recommended, including screening for cervical cancer and other sexually transmitted infections (STIs); however, as many as one in three TM patients are not up-to-date per recommended U.S. Among cisgender (non-transgender) women, self-swab hr.-HPV DNA testing as a primary cervical cancer screening method and self-swab specimen collection for other STIs have high levels of acceptability. No study has yet been conducted to compare the performance and acceptability of self- and provider-collected swabs for hr.-HPV DNA testing and other STIs in TM patients. This article describes the study protocol for a mixed-methods biobehavioral investigation enrolling 150 sexually active TM to (1) assess the clinical performance and acceptability of a vaginal self-swab for hr.-HPV DNA testing compared to provider cervical swab and cervical cytology, and (2) gather acceptability data on self-collected specimens for other STIs. Study participation entails a one-time clinical visit at Fenway Health in Boston, MA comprised of informed consent, quantitative assessment, venipuncture for syphilis testing and HIV (Rapid OraQuick) testing, randomization, collection of biological specimens/biomarkers, participant and provider satisfaction survey, and qualitative exit interview. Participants are compensated $100. The primary study outcomes are concordance (kappa statistic) and performance (sensitivity and specificity) of self-collected vaginal HPV DNA specimens vs provider-collected cervical HPV swabs as a gold standard. This study

  2. Comparison of the clinical performances of the AdvanSure HPV Screening Real-Time PCR, the Abbott Real-Time High-Risk HPV Test, and the Hybrid Capture High-Risk HPV DNA Test for Cervical Cancer Screening.

    PubMed

    Chung, Hae-Sun; Hahm, Chorong; Lee, Miae

    2014-09-01

    The clinical performance of three human papillomavirus (HPV) DNA commercial assays for cervical cancer screening was evaluated; the AdvanSure HPV Screening Real-Time PCR (AdvanSure PCR; LG Life Sciences) that was developed recently for the detection of both high-risk and low-risk genotypes, the Abbott RealTime High-Risk HPV Test (Abbott PCR; Abbott Molecular) and the Hybrid Capture High-Risk HPV DNA test (HC2; Qiagen). The three different HPV DNA tests were compared using cytology samples obtained from 619 women who underwent routine cervical cancer screening. The gold-standard assay was histopathological confirmation of cervical intraepithelial neoplasia of grade 2 or worse. The clinical sensitivities of the AdvanSure PCR, the Abbott PCR and the HC2 for the detection of cervical intraepithelial neoplasia of grade 2 or worse were 95.5%, 95.5% and 100%, respectively, while the clinical specificities were 61.6%, 86.4% and 83.3%, respectively. There were no significant differences in the clinical sensitivities of the Abbott PCR and the AdvanSure PCR compared to the HC2. The clinical specificities of the Abbott PCR and the AdvanSure PCR for the detection of HPV types 16/18 were 97.8% and 98.5%, respectively. For cervical cancer screening, all three tests showed relatively good clinical sensitivities, but the AdvanSure PCR had lower clinical specificity than the Abbott PCR and the HC2. The AdvanSure PCR and the Abbott PCR assays have the advantage of being automated and the ability to distinguish between HPV types 16/18 and other HPV types. The two real-time PCR assays could be useful tools in HPV testing for cervical cancer screening.

  3. Prick-tests and serological tests in the diagnosis of allergic reactivity to enzymes used in washing products

    PubMed Central

    How, M. J.; Cambridge, G. W.

    1971-01-01

    How, M. J., and Cambridge, G. W. (1971).Brit. J. industr. Med.,28, 303-307. Prick-tests and serological tests in the diagnosis of allergic reactivity to enzymes used in washing products. Standardized prick-test antigens for use in the detection of allergic reactivity to enzymes (Alcalase and Maxatase) used in washing products have been prepared. These materials contained all the potentially allergenic components of the crude enzyme preparations. Tests showed that these materials were more appropriate, as prick-test antigens, than the crystalline enzyme. The presence of reagin-mediated, immediate-type allergy in factory workers was confirmed by prick-tests and passive transfer tests. Sera from these subjects and controls contained enzyme-specific haemagglutinating antibody and gave precipitin-like reactions with solutions of Alcalase and Maxatase. The latter reaction involved serum components electrophoretically similar to, or identical with, α-globulins. PMID:4997564

  4. DNA-based diagnostic tests for Salmonella species targeting agfA, the structural gene for thin, aggregative fimbriae.

    PubMed Central

    Doran, J L; Collinson, S K; Burian, J; Sarlós, G; Todd, E C; Munro, C K; Kay, C M; Banser, P A; Peterkin, P I; Kay, W W

    1993-01-01

    Salmonella enteritidis 27655-3b and a few diarrheagenic Escherichia coli strains produce morphologically and antigenically related, thin, aggregative fimbriae, collectively named GVVPQ fimbriae (S. K. Collinson, L. Emödy, T. J. Trust, and W. W. Kay, J. Bacteriol. 174:4490-4495, 1992). To determine whether GVVPQ fimbriae are common to Salmonella spp. and other enteropathogenic members of the family Enterobacteriaceae, 113 isolates were phenotypically screened for Congo red binding and aggregative colony morphology. Presumptive positive and representative negative strains were examined by Western blotting (immunoblotting) by using antiserum to SEF 17, the native GVVPQ fimbria of S. enteritidis. Only four S. enteritidis strains and six E. coli isolates possessed substantial amounts of GVVPQ fimbriae after 24 h of incubation on T medium. Following 5 days of incubation, 56 of 93 Salmonella isolates (60%) and 1 of 7 additional E. coli clinical isolates possessed detectable levels of GVVPQ fimbriae. Since variable expression of GVVPQ fimbriae was observed among Salmonella isolates and some E. coli strains produced scant amounts, as revealed by immunoelectron microscopy, the ability to produce these fimbriae was evaluated by genotypic screening. The structural gene for the SEF 17 fimbrin, agfA, was amplified by the polymerase chain reaction, cloned, and sequenced to provide a characterized DNA probe. An agfA DNA fragment hybridized strongly to 603 of 604 (99.8%) Salmonella isolates but very weakly to 31 of 266 other members of the family Enterobacteriaceae including 26 of 137 E. coli strains, 3 of 14 Citrobacter spp., and single isolates of Shigella sonnei and Enterobacter cloacae. The agfA DNA probe proved to be a valuable diagnostic tool for Salmonella isolates arrayed on hydrophobic grid membrane filters. Unique agfA sequences were targeted in the development of a polymerase chain reaction assay specific for Salmonella spp. Images PMID:8104955

  5. A Transcontinental Challenge — A Test of DNA Barcode Performance for 1,541 Species of Canadian Noctuoidea (Lepidoptera)

    PubMed Central

    Zahiri, Reza; Lafontaine, J. Donald; Schmidt, B. Christian; deWaard, Jeremy R.; Zakharov, Evgeny V.; Hebert, Paul D. N.

    2014-01-01

    This study provides a first, comprehensive, diagnostic use of DNA barcodes for the Canadian fauna of noctuoids or “owlet” moths (Lepidoptera: Noctuoidea) based on vouchered records for 1,541 species (99.1% species coverage), and more than 30,000 sequences. When viewed from a Canada-wide perspective, DNA barcodes unambiguously discriminate 90% of the noctuoid species recognized through prior taxonomic study, and resolution reaches 95.6% when considered at a provincial scale. Barcode sharing is concentrated in certain lineages with 54% of the cases involving 1.8% of the genera. Deep intraspecific divergence exists in 7.7% of the species, but further studies are required to clarify whether these cases reflect an overlooked species complex or phylogeographic variation in a single species. Non-native species possess higher Nearest-Neighbour (NN) distances than native taxa, whereas generalist feeders have lower NN distances than those with more specialized feeding habits. We found high concordance between taxonomic names and sequence clusters delineated by the Barcode Index Number (BIN) system with 1,082 species (70%) assigned to a unique BIN. The cases of discordance involve both BIN mergers and BIN splits with 38 species falling into both categories, most likely reflecting bidirectional introgression. One fifth of the species are involved in a BIN merger reflecting the presence of 158 species sharing their barcode sequence with at least one other taxon, and 189 species with low, but diagnostic COI divergence. A very few cases (13) involved species whose members fell into both categories. Most of the remaining 140 species show a split into two or three BINs per species, while Virbia ferruginosa was divided into 16. The overall results confirm that DNA barcodes are effective for the identification of Canadian noctuoids. This study also affirms that BINs are a strong proxy for species, providing a pathway for a rapid, accurate estimation of animal diversity. PMID

  6. A transcontinental challenge--a test of DNA barcode performance for 1,541 species of Canadian Noctuoidea (Lepidoptera).

    PubMed

    Zahiri, Reza; Lafontaine, J Donald; Schmidt, B Christian; Dewaard, Jeremy R; Zakharov, Evgeny V; Hebert, Paul D N

    2014-01-01

    This study provides a first, comprehensive, diagnostic use of DNA barcodes for the Canadian fauna of noctuoids or "owlet" moths (Lepidoptera: Noctuoidea) based on vouchered records for 1,541 species (99.1% species coverage), and more than 30,000 sequences. When viewed from a Canada-wide perspective, DNA barcodes unambiguously discriminate 90% of the noctuoid species recognized through prior taxonomic study, and resolution reaches 95.6% when considered at a provincial scale. Barcode sharing is concentrated in certain lineages with 54% of the cases involving 1.8% of the genera. Deep intraspecific divergence exists in 7.7% of the species, but further studies are required to clarify whether these cases reflect an overlooked species complex or phylogeographic variation in a single species. Non-native species possess higher Nearest-Neighbour (NN) distances than native taxa, whereas generalist feeders have lower NN distances than those with more specialized feeding habits. We found high concordance between taxonomic names and sequence clusters delineated by the Barcode Index Number (BIN) system with 1,082 species (70%) assigned to a unique BIN. The cases of discordance involve both BIN mergers and BIN splits with 38 species falling into both categories, most likely reflecting bidirectional introgression. One fifth of the species are involved in a BIN merger reflecting the presence of 158 species sharing their barcode sequence with at least one other taxon, and 189 species with low, but diagnostic COI divergence. A very few cases (13) involved species whose members fell into both categories. Most of the remaining 140 species show a split into two or three BINs per species, while Virbia ferruginosa was divided into 16. The overall results confirm that DNA barcodes are effective for the identification of Canadian noctuoids. This study also affirms that BINs are a strong proxy for species, providing a pathway for a rapid, accurate estimation of animal diversity.

  7. DNA-based diagnostic tests for Salmonella species targeting agfA, the structural gene for thin, aggregative fimbriae.

    PubMed

    Doran, J L; Collinson, S K; Burian, J; Sarlós, G; Todd, E C; Munro, C K; Kay, C M; Banser, P A; Peterkin, P I; Kay, W W

    1993-09-01

    Salmonella enteritidis 27655-3b and a few diarrheagenic Escherichia coli strains produce morphologically and antigenically related, thin, aggregative fimbriae, collectively named GVVPQ fimbriae (S. K. Collinson, L. Emödy, T. J. Trust, and W. W. Kay, J. Bacteriol. 174:4490-4495, 1992). To determine whether GVVPQ fimbriae are common to Salmonella spp. and other enteropathogenic members of the family Enterobacteriaceae, 113 isolates were phenotypically screened for Congo red binding and aggregative colony morphology. Presumptive positive and representative negative strains were examined by Western blotting (immunoblotting) by using antiserum to SEF 17, the native GVVPQ fimbria of S. enteritidis. Only four S. enteritidis strains and six E. coli isolates possessed substantial amounts of GVVPQ fimbriae after 24 h of incubation on T medium. Following 5 days of incubation, 56 of 93 Salmonella isolates (60%) and 1 of 7 additional E. coli clinical isolates possessed detectable levels of GVVPQ fimbriae. Since variable expression of GVVPQ fimbriae was observed among Salmonella isolates and some E. coli strains produced scant amounts, as revealed by immunoelectron microscopy, the ability to produce these fimbriae was evaluated by genotypic screening. The structural gene for the SEF 17 fimbrin, agfA, was amplified by the polymerase chain reaction, cloned, and sequenced to provide a characterized DNA probe. An agfA DNA fragment hybridized strongly to 603 of 604 (99.8%) Salmonella isolates but very weakly to 31 of 266 other members of the family Enterobacteriaceae including 26 of 137 E. coli strains, 3 of 14 Citrobacter spp., and single isolates of Shigella sonnei and Enterobacter cloacae. The agfA DNA probe proved to be a valuable diagnostic tool for Salmonella isolates arrayed on hydrophobic grid membrane filters. Unique agfA sequences were targeted in the development of a polymerase chain reaction assay specific for Salmonella spp.

  8. Ancient DNA

    PubMed Central

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole mitochondrial genome sequences of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the characterization of Pleistocene mammal populations and discoveries of DNA preserved in ancient sediments. Increasingly, ancient genetic information is providing a unique means to test assumptions used in evolutionary and population genetics studies to reconstruct the past. Initial results have revealed surprisingly complex population histories, and indicate that modern phylogeographic studies may give misleading impressions about even the recent evolutionary past. With the advent and uptake of appropriate methodologies, ancient DNA is now positioned to become a powerful tool in biological research and is also evolving new and unexpected uses, such as in the search for extinct or extant life in the deep biosphere and on other planets. PMID:15875564

  9. Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: Testing of 24 compounds

    SciTech Connect

    Mirsalis, J.C.; Tyson, C.K.; Steinmetz, K.L.; Loh, E.K.; Hamilton, C.M.; Bakke, J.P. ); Spalding, J.W. )

    1989-01-01

    The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful for studying genotoxic hepatocarcinogens. In addition, measurement of S-phase synthesis (SPS) provides an indirect indicator of hepatocellular proliferation, which may be an important mechanism in rodent carcinogenesis. This assay was used to examine 24 chemicals for their ability to induce unscheduled DNA synthesis (UDS) or SPS in Fischer-344 rats or B6C3F1 mice following in vivo treatment. Hepatocytes were isolated by liver perfusion and incubated with {sup 3}H-thymidine following in vivo treatment by gavage. Chemicals chosen for testing were from the National Toxicology Program (NTP) genetic toxicology testing program and most were also evaluated in long-term animal studies conducted by the NTP. Dinitrotoluene and Michler's Ketone induced positive UDS response in rat, while N-nitrosodiethanolamine and selenium sulfide induced equivocal UDS results in mouse and rat, respectively. BCMEE, bromoform, chloroform, PBB, 1,1,2-trichloroethane, and trichloroethylene were all potent inducers of SPS in mouse liver, while C.I. Solvent Yellow 14, and 1,1,2,2-tetrachloroethane yielded equivocal SPS results in rat and mouse, respectively. These results indicate that most of the test compounds do not induced UDS in the liver; however, the significant S-phase response induced by many of these compounds, especially the halogenated solvents, may be an important mechanism in their hepatocarinogenicity.

  10. Improved testing for CMT1A and HNPP using multiplex ligation-dependent probe amplification (MLPA) with rapid DNA preparations: comparison with the interphase FISH method.

    PubMed

    Slater, Howard; Bruno, Damien; Ren, Hua; La, Phung; Burgess, Trent; Hills, Louise; Nouri, Sara; Schouten, Jan; Choo, K H Andy

    2004-08-01

    Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are the two most common peripheral neuropathies, with incidences of about 1 in 2,500. Several techniques can be used to detect the typical 1.5-Mb duplication or deletion associated with these respective conditions, but none combines simplicity with high sensitivity. MLPA is a new technique for measuring sequence dosage. We have assessed its performance for the detection of the specific 1.5-Mb duplication/deletion by prospectively testing 50 patients referred with differential diagnoses of CMT or HNPP. Probes were designed to evaluate the TEKT3, PMP22, and COX10 genes within the CMT1A/HNPP region. We have compared the results with our existing fluorescence in situ hybridization (FISH) assay, which was performed in parallel. There was concordance of results for 49 patients. Of note, one patient showed an intermediate multiplex ligation-dependent probe amplification (MLPA) result with an abnormal FISH result, which is consistent with mosaicism. The assay works equally well with either purified DNA or rapid DNA preparations made by direct cell lysis. The use of the latter significantly reduces the cost of the assay. MLPA is a sensitive, specific, robust, and cost-effective technique suitable for fast, high-throughput testing and offers distinct advantages over other testing methods.

  11. HPV DNA test and Pap smear in detection of residual and recurrent disease following loop electrosurgical excision procedure of high-grade cervical intraepithelial neoplasia.

    PubMed

    Sarian, Luís Otávio; Derchain, Sophie F M; Andrade, Liliana A A; Tambascia, Júlia; Morais, Sirlei Siani; Syrjänen, Kari J

    2004-07-01

    We compared the performance of cervical cytology and HPV DNA test in detection of residual or recurrent disease following the treatment of cervical intraepithelial neoplasia (CIN) 2/3 with loop electrosurgical excision procedure (LEEP). A series of 107 women subjected to LEEP due to histologically confirmed CIN 2/3 between March 2001 and December 2002 were followed-up biannually until January 2004. Follow-up visits consisted of interview and gynecological examination including cervical cytology, hybrid capture II (HCII), and colposcopy. Patients presenting with abnormal colposcopy or high-grade squamous intraepithelial lesion (HSIL) smear were subjected to new excision procedure, and presence of histologically confirmed CIN 2/3 or higher was considered as residual or recurrent disease. Performance indicators were calculated for cytology and HCII assay in detecting residual or recurrent disease. Eleven (10.2%) women showed residual or recurrent disease during the follow-up. Considering HCII and Pap smear as stand-alone tests, both techniques showed similar sensitivity, detecting 100% of CIN 2/3 at the first follow-up visit. At the second follow-up visit, Pap smear showed better specificity and positive predictive value (PPV) than HCII, and both tests had fairly the same high negative predictive value (NPV) and sensitivity. The combined positive HCII and abnormal cytology had the same sensitivity as each of the tests alone, but specificity and PPV were significantly higher than those of single tests. When only one of the tests was positive, the sensitivity and the NPV of the combination remained the same, but its specificity and PPV were lower than that of the combined two positive tests and that of the individual test, at both follow-up visits. Both tests performed well in detecting residual or recurrent disease after LEEP and combination of the tests did not increase sensitivity of the single tests.

  12. Detection rates of precancerous and cancerous cervical lesions within one screening round of primary human papillomavirus DNA testing: prospective randomised trial in Finland

    PubMed Central

    Nieminen, Pekka; Lönnberg, Stefan; Malila, Nea; Hakama, Matti; Pokhrel, Arun; Laurila, Pekka; Tarkkanen, Jussi; Anttila, Ahti

    2012-01-01

    Objective To compare the detection rates of precancerous and cancerous cervical lesions by human papillomavirus (HPV) DNA testing and by conventional cytology screening. Design Prospective randomised trial. Two cohorts were followed over one screening round of five years, screened initially by primary HPV DNA testing or by primary Pap test. Setting Population based programme for cervical cancer screening in Finland. Participants Women aged 25-65 years invited for screening in 2003-07 (101 678 in HPV arm; 101 747 in conventional cytology arm). Intervention Women were randomly allocated (1:1) to primary HPV DNA screening followed by cytology triage if they had positive results, or to primary cytology screening. Screening method was disclosed at the screening visit. Trial personnel involved were aware of all test results. Main outcome measures Cumulative detection rates of cervical intraepithelial neoplasia (CIN), adenocarcinoma in situ (AIS), and invasive cervical cancer before the second screening (after five years) or before 31 December 2008. Lesions detected at screening and during the five year interval were included. Results 1010 and 701 precancerous or cancerous lesions were detected during an average follow-up of 3.6 years in the HPV and cytology arms, respectively. Among invited women, the hazard ratio was 1.53 (95% confidence interval l.28 to 1.84) for CIN grade 1, 1.54 (1.33 to 1.78) for CIN 2, 1.32 (1.09 to 1.59) for CIN 3 or AIS, and 0.81 (0.48 to 1.37) for cervical cancer. In 25-34 year old participants, the cumulative hazard (or cumulative detection rate) was 0.0057 (0.0045 to 0.0072) for HPV screening versus 0.0046 (0.0035 to 0.0059) for conventional screening; corresponding data for women aged 35 years and older were 0.0022 (0.0019 to 0.0026) and 0.0017 (0.0014 to 0.0021), respectively. Conclusions Primary HPV DNA screening detects more cervical lesions than primary cytology within one screening round of five years. Even if the detection rate of

  13. Testing DNA barcode performance in 1000 species of European lepidoptera: large geographic distances have small genetic impacts.

    PubMed

    Huemer, Peter; Mutanen, Marko; Sefc, Kristina M; Hebert, Paul D N

    2014-01-01

    This study examines the performance of DNA barcodes (mt cytochrome c oxidase 1 gene) in the identification of 1004 species of Lepidoptera shared by two localities (Finland, Austria) that are 1600 km apart. Maximum intraspecific distances for the pooled data were less than 2% for 880 species (87.6%), while deeper divergence was detected in 124 species. Despite such variation, the overall DNA barcode library possessed diagnostic COI sequences for 98.8% of the taxa. Because a reference library based on Finnish specimens was highly effective in identifying specimens from Austria, we conclude that barcode libraries based on regional sampling can often be effective for a much larger area. Moreover, dispersal ability (poor, good) and distribution patterns (disjunct, fragmented, continuous, migratory) had little impact on levels of intraspecific geographic divergence. Furthermore, the present study revealed that, despite the intensity of past taxonomic work on European Lepidoptera, nearly 20% of the species shared by Austria and Finland require further work to clarify their status. Particularly discordant BIN (Barcode Index Number) cases should be checked to ascertain possible explanatory factors such as incorrect taxonomy, hybridization, introgression, and Wolbachia infections.