Science.gov

Sample records for qualitative pcr testing

  1. HCG blood test - qualitative

    MedlinePlus

    ... qualitative Images Blood test References Lee P, Pincus MR, McPherson RA. Diagnosis and management of cancer using serologic tumor markers. In: McPherson RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory ...

  2. Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

    PubMed

    Kodama, Takashi; Kasahara, Masaki; Minegishi, Yasutaka; Futo, Satoshi; Sawada, Chihiro; Watai, Masatoshi; Akiyama, Hiroshi; Teshima, Reiko; Kurosawa, Yasunori; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2011-01-01

    Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots. PMID:21391499

  3. Testing for Genetically Modified Foods Using PCR

    ERIC Educational Resources Information Center

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  4. Development of qualitative and quantitative PCR analysis for meat adulteration from RNA samples.

    PubMed

    Cheng, Jai-Hong; Chou, Hsiao-Ting; Lee, Meng-Shiou; Sheu, Shyang-Chwen

    2016-02-01

    Total RNA samples were used to establish qualitative and quantitative PCR-based methods for assessing meat adulteration. The primers were designed based on the mRNA sequences of troponin I (TnI), mitochondrial ribosomal protein (MRP) and tropomodulin genes to distinguish chicken, pork, goat, beef and ostrich. There was no cross reaction between the primers, and the detection limit of the cDNA template was 0.01 and 20 ng in simplex PCR and multiplex PCR, respectively. In the low temperature storage test, the detection limits of cDNA template with 10 and 1 ng were determined at 4 °C and -80 °C. In quantitative assay, the precision of real-time PCR analysis expressed as a coefficient of variation (CV) ranged from 0.25% to 5.24% and the trueness, expressed as an error, ranged from 0.28% to 6.98% for adulteration. Thus, herein, we provided alternative tools for the assessment of meat adulteration using mRNA-based PCR methods. PMID:26304356

  5. A new method for robust quantitative and qualitative analysis of real-time PCR

    PubMed Central

    Shain, Eric B.; Clemens, John M.

    2008-01-01

    An automated data analysis method for real-time PCR needs to exhibit robustness to the factors that routinely impact the measurement and analysis of real-time PCR data. Robust analysis is paramount to providing the same interpretation for results regardless of the skill of the operator performing or reviewing the work. We present a new method for analysis of real-time PCR data, the maxRatio method, which identifies a consistent point within or very near the exponential region of the PCR signal without requiring user intervention. Compared to other analytical techniques that generate only a cycle number, maxRatio generates several measurements of amplification including cycle numbers and relative measures of amplification efficiency and curve shape. By using these values, the maxRatio method can make highly reliable reactive/nonreactive determination along with quantitative evaluation. Application of the maxRatio method to the analysis of quantitative and qualitative real-time PCR assays is shown along with examples of method robustness to, and detection of, amplification response anomalies. PMID:18603594

  6. Use of qualitative integrative cycler PCR (qicPCR) to identify optimal therapeutic dosing time-points in a Respiratory Syncytial Virus Human Viral Challenge Model (hVCM).

    PubMed

    Kelly, Geraldine; Laxton, Carl; Garelnabi, Mariam; Alton, Brian; Addan, Fatima; Catchpole, Andrew; Thomas, Elaine; Borley, Daryl; Dee, Kieran; Boyers, Alison; Bringas, Erica; Noulin, Nicolas; Lambkin-Williams, Rob; Murray, Edward J

    2015-11-01

    Retroscreen (hVIVO) have developed an RSV human viral challenge model (hVCM) for testing the efficacy of novel antiviral therapies by monitoring changes in viral load and symptoms. The integrated cycler technology and Simplexa™ kits (Focus Diagnostics) currently provide fast, qualitative and sensitive diagnostic testing in hospitals and other healthcare facilities for patients with well-established respiratory illness. We have developed a novel use of qualitative integrated cycler PCR (qicPCR) technology to identify onset of RSV infection enabling an informed dosing clinical protocol in the RSV hVCM. We have validated qicPCR detection of RSV in spiked nasal wash aspirates and demonstrate that the qicPCR assay is 94% concordant with RSV plaque assay data in nasal wash samples from 53 RSV inoculated human volunteers in the hVCM. The use of qicPCR for informed dosing was successfully implemented in a recent clinical trial demonstrating efficacy of the RSV entry inhibitor GS-5806 in the hVCM (NCT01756482). Comparison of qicPCR positivity in relation to nasal wash viral load measured by both RT-qPCR and plaque assay shows that the therapeutic exposure was correctly initiated prior to onset and peak of RSV viral shedding and symptoms in the majority of volunteers. PMID:26335961

  7. Inhibition Controls for Qualitative Real-Time PCR Assays: Are They Necessary for All Specimen Matrices?

    PubMed Central

    Buckwalter, S. P.; Sloan, L. M.; Cunningham, S. A.; Espy, M. J.; Uhl, J. R.; Jones, M. F.; Vetter, E. A.; Mandrekar, J.; Cockerill, F. R.; Pritt, B. S.; Patel, R.

    2014-01-01

    A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue. PMID:24740078

  8. Validation of qualitative microbiological test methods.

    PubMed

    IJzerman-Boon, Pieta C; van den Heuvel, Edwin R

    2015-01-01

    This paper considers a statistical model for the detection mechanism of qualitative microbiological test methods with a parameter for the detection proportion (the probability to detect a single organism) and a parameter for the false positive rate. It is demonstrated that the detection proportion and the bacterial density cannot be estimated separately, not even in a multiple dilution experiment. Only the product can be estimated, changing the interpretation of the most probable number estimator. The asymptotic power of the likelihood ratio statistic for comparing an alternative method with the compendial method, is optimal for a single dilution experiment. The bacterial density should either be close to two CFUs per test unit or equal to zero, depending on differences in the model parameters between the two test methods. The proposed strategy for method validation is to use these two dilutions and test for differences in the two model parameters, addressing the validation parameters specificity and accuracy. Robustness of these two parameters might still be required, but all other validation parameters can be omitted. A confidence interval-based approach for the ratio of the detection proportions for the two methods is recommended, since it is most informative and close to the power of the likelihood ratio test. PMID:25412584

  9. Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

    PubMed Central

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2016-01-01

    Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

  10. Reproducibility problems with the AMPLICOR PCR Chlamydia trachomatis test.

    PubMed Central

    Peterson, E M; Darrow, V; Blanding, J; Aarnaes, S; de la Maza, L M

    1997-01-01

    In an attempt to use an expanded "gold standard" in an evaluation of an antigen detection test for Chlamydia trachomatis, the AMPLICOR (Roche Diagnostics Systems, Inc., Branchburg, N.J.) PCR Chlamydia trachomatis test and culture were used with 591 sets of cervical specimens. Of the 591 specimens assayed, 35 were retested due to either an equivocal result by the PCR (19 samples) or a discrepancy between the results of culture, PCR, and the antigen detection method. During the repeat testing of the samples with equivocal and discrepant results, all but one interpretation change was due to the PCR result. In addition, upon repeat testing the PCR assay value measured in optical density units varied widely for 13 of these specimens. These 13 specimens were then tested in triplicate by the manufacturer with primers to the chlamydia plasmid and in duplicate with primers to the major outer membrane protein. Only 3 of the 13 specimens gave the same interpretation with these five replicates. In summary, reproducibility problems with the AMPLICOR test should be considered before it is incorporated as part of routine testing or used as an expanded gold standard for chlamydia testing. PMID:9157161

  11. High-Stakes Testing and Curricular Control: A Qualitative Metasynthesis

    ERIC Educational Resources Information Center

    Au, Wayne

    2007-01-01

    Using the method of qualitative metasynthesis, this study analyzes 49 qualitative studies to interrogate how high-stakes testing affects curriculum, defined here as embodying content, knowledge form, and pedagogy. The findings from this study complicate the understanding of the relationship between high-stakes testing and classroom practice by…

  12. Detection of genetically modified maize in processed foods sold commercially in iran by qualitative PCR.

    PubMed

    Rabiei, Maryam; Mehdizadeh, Mehrangiz; Rastegar, Hossein; Vahidi, Hossein; Alebouyeh, Mahmoud

    2013-01-01

    Detection of genetically modified organisms (GMOs) in food is an important issue for all the subjects involved in food control and customer's right. Due to the increasing number of GMOs imported to Iran during the past few years, it has become necessary to screen the products in order to determine the identity of the consumed daily foodstuffs. In this study, following the extraction of genomic DNA from processed foods sold commercially in Iran, qualitative PCR was performed to detect genetically modified maize. The recombinant DNA target sequences were detected with primers highly specific for each investigated transgene such as CaMV35s gene, Bt-11, MON810 and Bt-176 separately. Based on the gel electrophoresis results, Bt- 11 and MON810 events were detected in some maize samples, while, in none of them Bt- 176 modified gene was detected. For the first time, the results demonstrate the presence of genetically modified maize in Iranian food products, reinforcing the need for the development of labeling system and valid quantitative methods in routine analyses. PMID:24250568

  13. Detection of Genetically Modified Maize in Processed Foods Sold Commercially in Iran by Qualitative PCR

    PubMed Central

    Rabiei, Maryam; Mehdizadeh, Mehrangiz; Rastegar, Hossein; Vahidi, Hossein; Alebouyeh, Mahmoud

    2013-01-01

    Detection of genetically modified organisms (GMOs) in food is an important issue for all the subjects involved in food control and customer’s right. Due to the increasing number of GMOs imported to Iran during the past few years, it has become necessary to screen the products in order to determine the identity of the consumed daily foodstuffs. In this study, following the extraction of genomic DNA from processed foods sold commercially in Iran, qualitative PCR was performed to detect genetically modified maize. The recombinant DNA target sequences were detected with primers highly specific for each investigated transgene such as CaMV35s gene, Bt-11, MON810 and Bt-176 separately. Based on the gel electrophoresis results, Bt- 11 and MON810 events were detected in some maize samples, while, in none of them Bt- 176 modified gene was detected. For the first time, the results demonstrate the presence of genetically modified maize in Iranian food products, reinforcing the need for the development of labeling system and valid quantitative methods in routine analyses. PMID:24250568

  14. A PCR test for avian malaria in Hawaiian birds.

    PubMed

    Feldman, R A; Freed, L A; Cann, R L

    1995-12-01

    The decline of native Hawaiian forest birds since European contact is attributed to factors ranging from habitat destruction to interactions with introduced species. Remaining populations of Hawaiian honeycreepers (Fringillidae: Drepanidinae) are most abundant and diverse in high elevation refuges above the normal range of disease-carrying mosquitoes. Challenge experiments suggest that honeycreepers are highly susceptible to avian malaria (Plasmodium sp.) but resistance exists in some species. In order to detect low levels of malarial infection and quantify prevalence of Plasmodium in high elevation natural populations of Hawaiian birds, a polymerase chain reaction (PCR) based diagnostic test was developed that identifies rRNA genes of Plasmodium in avian blood samples. Quantitative competitive PCR (QC-PCR) experiments indicate that the detection limit of our test is an order of magnitude greater than that reported for human malaria DNA blot tests. Compared with standard histological methods, the PCR test detected a higher prevalence of diseased birds at mid-elevations. Malaria was detected in three species of native birds living in a high elevation wildlife refuge on the island of Hawaii and in four species from Maui. Our results show that avian malaria is more widespread in Hawaiian forests than previously thought, a finding that has important conservation implications for these threatened species. PMID:8564006

  15. Development and validation of qualitative SYBR®Green real-time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes.

    PubMed

    Barbau-Piednoir, Elodie; Botteldoorn, Nadine; Yde, Marc; Mahillon, Jacques; Roosens, Nancy H

    2013-05-01

    A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the "Definition of minimum performance requirements for analytical methods of GMO testing". The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively). PMID:23086339

  16. Qualitative and event-specific real-time PCR detection methods for Bt brinjal event EE-1.

    PubMed

    Randhawa, Gurinder Jit; Sharma, Ruchi; Singh, Monika

    2012-01-01

    Bt brinjal event EE-1 with cry1Ac gene, expressing insecticidal protein against fruit and shoot borer, is the first genetically modified food crop in the pipeline for commercialization in India. Qualitative polymerase chain reaction (PCR) along with event-specific conventional as well as real-time PCR methods to characterize the event EE-1 is reported. A multiplex (pentaplex) PCR system simultaneously amplifying cry1Ac transgene, Cauliflower Mosaic Virus (CaMV) 35S promoter, nopaline synthase (nos) terminator, aminoglycoside adenyltransferase (aadA) marker gene, and a taxon-specific beta-fructosidase gene in event EE-1 has been developed. Furthermore, construct-specific PCR, targeting the approximate 1.8 kb region of inserted gene construct comprising the region of CaMV 35S promoter and cry1Ac gene has also been developed. The LOD of developed EE-1 specific conventional PCR assay is 0.01%. The method performance of the reported real-time PCR assay was consistent with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with the LOD and LOQ values of 0.05%. The developed detection methods would not only facilitate effective regulatory compliance for identification of genetic traits, risk assessment, management, and postrelease monitoring, but also address consumer concerns and resolution of legal disputes. PMID:23451391

  17. Qualitative Aspects of Group-Only Testing

    ERIC Educational Resources Information Center

    Hanshaw, Larry G.

    2012-01-01

    This study sought to determine how students would describe their group-only cooperative testing experiences in terms of key elements of cooperative learning often cited in the literature. Written comments of 159 graduate students were analyzed and 26 related categories of comments were derived from 495 statements of students enrolled in two…

  18. Testing for spatial association of qualitative data using symbolic dynamics

    NASA Astrophysics Data System (ADS)

    Ruiz, Manuel; López, Fernando; Páez, Antonio

    2010-09-01

    Qualitative spatial variables are important in many fields of research. However, unlike the decades-worth of research devoted to the spatial association of quantitative variables, the exploratory analysis of spatial qualitative variables is relatively less developed. The objective of the present paper is to propose a new test ( Q) for spatial independence. This is a simple, consistent, and powerful statistic for qualitative spatial independence that we develop using concepts from symbolic dynamics and symbolic entropy. The Q test can be used to detect, given a spatial distribution of events, patterns of spatial association of qualitative variables in a wide variety of settings. In order to enable hypothesis testing, we give a standard asymptotic distribution of an affine transformation of the symbolic entropy under the null hypothesis of independence in the spatial qualitative process. We include numerical experiments to demonstrate the finite sample behaviour of the test, and show its application by means of an empirical example that explores the spatial association of fast food establishments in the Greater Toronto Area in Canada.

  19. Use of Treponema pallidum PCR in Testing of Ulcers for Diagnosis of Primary Syphilis1

    PubMed Central

    Sednaoui, Patrice; Lautenschlager, Stephan; Ferry, Tristan; Toutous-Trellu, Laurence; Cavassini, Matthias; Yassir, Fatima; Martinez de Tejada, Begoña; Emonet, Stéphane; Combescure, Christophe; Schrenzel, Jacques; Perneger, Thomas

    2015-01-01

    Treponema pallidum PCR (Tp-PCR) has been noted as a valid method for diagnosing syphilis. We compared Tp-PCR to a combination of darkfield microscopy (DFM), the reference method, and serologic testing in a cohort of 273 patients from France and Switzerland and found the diagnostic accuracy of Tp-PCR was higher than that for DFM. PMID:25531672

  20. Use of Treponema pallidum PCR in testing of ulcers for diagnosis of primary syphilis.

    PubMed

    Gayet-Ageron, Angèle; Sednaoui, Patrice; Lautenschlager, Stephan; Ferry, Tristan; Toutous-Trellu, Laurence; Cavassini, Matthias; Yassir, Fatima; Martinez de Tejada, Begoña; Emonet, Stéphane; Combescure, Christophe; Schrenzel, Jacques; Perneger, Thomas

    2015-01-01

    Treponema pallidum PCR (Tp-PCR) has been noted as a valid method for diagnosing syphilis. We compared Tp-PCR to a combination of darkfield microscopy (DFM), the reference method, and serologic testing in a cohort of 273 patients from France and Switzerland and found the diagnostic accuracy of Tp-PCR was higher than that for DFM. PMID:25531672

  1. HHV-6 encephalitis may complicate the early phase after allogeneic hematopoietic stem cell transplantation: Detection by qualitative multiplex PCR and subsequent quantitative real-time PCR.

    PubMed

    Inazawa, Natsuko; Hori, Tsukasa; Yamamoto, Masaki; Hatakeyama, Naoki; Yoto, Yuko; Nojima, Masanori; Yasui, Hiroshi; Suzuki, Nobuhiro; Shimizu, Norio; Tsutsumi, Hiroyuki

    2016-02-01

    Viral reactivation following hematopoietic stem cell transplantation (HSCT) can cause various complications especially viral encephalitis. In this prospective study, we investigated the correlation of post-HSCT viral reactivation in blood with CNS dysfunction. We employed a multiplex PCR that detects 13 kinds of viruses as a first-line screening test and real-time PCR for subsequent quantitative evaluation. Five hundred ninety-one whole blood samples were collected from 105 patients from before until 42 days after HSCT. Seven patients developed CNS dysfunction such as altered consciousness. In six of the seven, the multiplex PCR test detected HHV-6 DNA in at least one sample. In contrast, DNA from other viruses, such as CMV, EBV, HHV-7, adenovirus, and HBV was never detected in any of the seven patients throughout the study period. Quantitative measurement of whole blood HHV-6 DNA levels demonstrated four of the six HHV-6 DNA loads were elevated at successive time points during the CNS dysfunction. In addition, the virus DNA peaks were temporally associated with the development of CNS dysfunction. CSF was tested in two of the four patients and high HHV-6 DNA levels comparable to those in whole blood were confirmed in both. These four patients were, thus, suspected to have developed HHV-6 encephalitis, a rate of 3.8% in the study population. Our results suggest that early diagnosis of probable HHV-6 encephalitis can be improved by confirming high HHV-6 DNA load in blood. PMID:26241219

  2. Gender, Sexual Orientation, and Adolescent HIV Testing: A Qualitative Analysis

    PubMed Central

    Siegel, Karolynn; Lekas, Helen-Maria; Olson, Kari; VanDevanter, Nancy

    2010-01-01

    Using qualitative data, this article explored the circumstances leading to HIV testing among 59 HIV-infected adolescents recruited from New York City HIV clinics. Results showed differences between the heterosexual women and the gay and bisexual men. Most of the young women were tested during routine health care or self-initiated tests, and most were asymptomatic when they tested positive. Their testing decisions were sometimes based on assessments of their boyfriends’ risk behaviors, rather than their own. Many males were experiencing symptoms of illness when they tested positive, and about half of these recognized their symptoms as related to HIV and sought tests. Some young men expressed fear of learning about positive test results, which delayed their testing, and some providers did not initially recommend HIV testing for males who presented with symptoms. The article concludes that consideration of these gender and sexual orientation-related concerns can facilitate HIV testing among adolescents. PMID:20303793

  3. Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

    EPA Science Inventory

    Quantitative PCR (qPCR) techniques have been widely used to measure Dehalococcoides (Dhc) DNA in the groundwater at field sites for several years. Interpretation of these data may be complicated when different laboratories using alternate methods conduct the analysis. An...

  4. Optimized 5-hour multiplex PCR test for the detection of tinea unguium: performance in a routine PCR laboratory.

    PubMed

    Brillowska-Dabrowska, Anna; Nielsen, Sanne Søgaard; Nielsen, Henrik Vedel; Arendrup, Maiken Cavling

    2010-09-01

    We recently reported the development of a 5-hour multiplex PCR test for the detection of tinea unguium and the optimization of this test by the inclusion of an inhibition control. Here we report the performance of this procedure as used in a routine clinical laboratory as compared to conventional microscopy and culture-based techniques performed in a mycology reference laboratory. We found in processing 109 samples that 22 (20.2%) yielded fungi in culture while the suspected etiologic agents were noted microscopically in 15 (13.8%) that were negative in culture. Fungi were detected by PCR in 37 (33.9%) samples, of which only three were positive in culture. Since the majority of PCR positive but culture negative samples were positive in microscopic examinations, the increased sensitivity was not due to contamination. PCR inhibitors were present in 5% of the samples, but this was overcome by re-running the samples with a 50% reduction of sample DNA. In conclusion, the PCR test performance in the routine setting was excellent and provided a markedly reduced time to diagnosis with a higher sensitivity. PMID:20105101

  5. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

    PubMed Central

    Espy, M. J.; Uhl, J. R.; Sloan, L. M.; Buckwalter, S. P.; Jones, M. F.; Vetter, E. A.; Yao, J. D. C.; Wengenack, N. L.; Rosenblatt, J. E.; Cockerill, F. R.; Smith, T. F.

    2006-01-01

    Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory. PMID:16418529

  6. Predicting Gene Structures from Multiple RT-PCR Tests

    NASA Astrophysics Data System (ADS)

    Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

    It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

  7. [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. PMID:21735715

  8. PCR testing for diagnosis of Ichthyophonus hoferi: reply to LaPatra & Kocan (2013).

    PubMed

    Hamazaki, Toshihide; Kahler, Eryn; Borba, Bonnie M; Burton, Tamara

    2013-11-01

    LaPatra & Kocan (2013) critiqued our paper Hamazaki et al. (2013; Dis Aquat Org 105:21-25) for data not supporting the conclusions of 'PCR testing is as accurate as culture…', but they neither pointed out what part of our data did not support our conclusion, nor did they provide any contrary scientific evidence supporting their argument that PCR testing is less accurate than culture. In the absence of any contradictory data, we stand by our data and our conclusion: PCR test is as suitable as culture as a diagnostic and field surveillance tool. PMID:24192005

  9. Development of a qualitative real-time PCR method to detect 19 targets for identification of genetically modified organisms.

    PubMed

    Peng, Cheng; Wang, Pengfei; Xu, Xiaoli; Wang, Xiaofu; Wei, Wei; Chen, Xiaoyun; Xu, Junfeng

    2016-01-01

    As the amount of commercially available genetically modified organisms (GMOs) grows recent years, the diversity of target sequences for molecular detection techniques are eagerly needed. Considered as the gold standard for GMO analysis, the real-time PCR technology was optimized to produce a high-throughput GMO screening method. With this method we can detect 19 transgenic targets. The specificity of the assays was demonstrated to be 100 % by the specific amplification of DNA derived from reference material from 20 genetically modified crops and 4 non modified crops. Furthermore, most assays showed a very sensitive detection, reaching the limit of ten copies. The 19 assays are the most frequently used genetic elements present in GM crops and theoretically enable the screening of the known GMO described in Chinese markets. Easy to use, fast and cost efficient, this method approach fits the purpose of GMO testing laboratories. PMID:27386337

  10. Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs).

    PubMed

    Dörries, Hans-Henno; Remus, Ivonne; Grönewald, Astrid; Grönewald, Cordt; Berghof-Jäger, Kornelia

    2010-03-01

    The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction. PMID:19798485

  11. Diagnostic PCR tests for Microsporum audouinii, M. canis and Trichophyton infections.

    PubMed

    Brillowska-Dabrowska, Anna; Swierkowska, Aleksandra; Lindhardt Saunte, Ditte Marie; Arendrup, Maiken Cavling

    2010-05-01

    Since traditional diagnosis of dermatophyte infections is slow, we present a rapid new PCR test for detection of Trichophyton spp., Microsporum canis and M. audouinii infections. The performance of the test was evaluated with: 58 dermatophyte isolates; 10 yeast, mould and human DNA control samples; 25 routine specimens from patients suspected of having dermatophytosis; 10 hair specimens from guinea pigs experimentally infected with M. canis; and two samples from un-infected control animals. DNA was prepared by a 10-min procedure from pure cultures as previously described. The 302 bp PCR product was obtained for 35/35 Trichophyton isolates (10 species included) and the 279 bp for 3/3 M. canis and 4/4 M. audouinii samples. None of the 2 E. floccosum, 11 M. gypseum, 3 M M. persicolor or 12 control samples (yeast, mould, human DNA) were positive with either of the two PCR tests. Among the patient specimens, seven were T. rubrum positive, two for T. mentagrophytes, one was positive for T. tonsurans and 15 were dermatophyte negative by routine investigation (culture and/or pan-dermatophyte + T. rubrum multiplex PCR). The PCR results with our procedures were in 100% agreement with these results. Finally, the Microsporum PCR was positive for 10/10 guinea pig specimens from infected animals but for 0/2 of the control animal samples. The evaluation of the two PCR tests indicated excellent sensitivity and specificity. PMID:19886764

  12. Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence.

    PubMed

    Li, P; Jia, J W; Jiang, L X; Zhu, H; Bai, L; Wang, J B; Tang, X M; Pan, A H

    2012-01-01

    To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (<25%) of the GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite. PMID:22614281

  13. Identification of Streptococcus pyogenes – Phenotypic Tests vs Molecular Assay (spy1258PCR): A Comparative Study

    PubMed Central

    Abraham, Tintu

    2016-01-01

    Introduction Traditionally Group A Streptococcus pyogenes (GAS) is differentiated from other beta haemolytic streptococci (BHS) by certain presumptive tests such as bacitracin sensitivity and production of Pyrollidonyl Aryl Sulfatase (PYR). The phenotypic and genotypic confirmatory tests are Lancefield grouping for cell wall carbohydrate antigen and PCR for spy1258 gene respectively. Reliance on presumptive tests alone may lead to misidentification of isolates. Aim To compare the predictive values of routine phenotypic tests with spy1258 PCR for the identification of Streptococcus pyogenes. Materials and Methods This comparative analytical study was carried out in the Department of Microbiology, JIPMER, Puducherry, over a period of 18 months (1st November 2013 to 30th April 2015). Two hundred and six consecutive BHS isolates from various clinical samples were subjected to phenotypic tests such as bacitracin sensitivity, PYR test and Lancefield grouping. The results were compared with spy1258 PCR which was considered 95 the confirmatory test for identification. Results The sensitivity and specificity of phenotypic tests were as follows; Susceptibility to bacitracin – 95.42%, 70.96%, PYR test – 95.42%, 77.41%, Lancefield grouping- 97.71%, 80.64%. Conclusion Clinical laboratories should not depend on bacitracin sensitivity as a single presumptive test for the routine identification of GAS but should use supplemental tests such as PYR test or latex agglutination test and for best results use spy1258 PCR.

  14. Rapid identification of HPV 16 and 18 by multiplex nested PCR-immunochromatographic test.

    PubMed

    Kuo, Yung-Bin; Li, Yi-Shuan; Chan, Err-Cheng

    2015-02-01

    Human papillomavirus (HPV) types 16 and 18 are known to be high-risk viruses that cause cervical cancer. An HPV rapid testing kit that could help physicians to make early and more informed decisions regarding patient care is needed urgently but not yet available. This study aimed to develop a multiplex nested polymerase chain reaction-immunochromatographic test (PCR-ICT) for the rapid identification of HPV 16 and 18. A multiplex nested PCR was constructed to amplify the HPV 16 and 18 genotype-specific L1 gene fragments and followed by ICT which coated with antibodies to identify rapidly the different PCR products. The type-specific gene regions of high-risk HPV 16 and 18 could be amplified successfully by multiplex nested PCR at molecular sizes of approximately 99 and 101bp, respectively. The capture antibodies raised specifically against the moleculars labeled on the PCR products could be detected simultaneously both HPV 16 and 18 in one strip. Under optimal conditions, this PCR-ICT assay had the capability to detect HPV in a sample with as low as 100 copies of HPV viral DNA. The PCR-ICT system has the advantage of direct and simultaneous detection of two high-risk HPV 16 and 18 DNA targets in one sample, which suggested a significant potential of this assay for clinical application. PMID:25446515

  15. A longitudinal evaluation of Treponema pallidum PCR testing in early syphilis

    PubMed Central

    2012-01-01

    Background Syphilis is a growing public health problem among men who have sex with men (MSM) globally. Rapid and accurate detection of syphilis is vital to ensure patients and their contacts receive timely treatment and reduce ongoing transmission. Methods We evaluated a PCR assay for the diagnosis of Treponema pallidum using swabs of suspected early syphilis lesions in longitudinally assessed MSM. Results We tested 260 MSM for T pallidum by PCR on 288 occasions: 77 (26.7%) had early syphilis that was serologically confirmed at baseline or within six weeks, and 211 (73.3%) remained seronegative for syphilis. Of 55 men with primary syphilis, 49 were PCR positive, giving a sensitivity of 89.1% (95% CI: 77.8%-95.9%) and a specificity of 99.1% (95% CI: 96.5%-99.9%). Of 22 men with secondary syphilis, 11 were PCR positive, giving a sensitivity of 50% (95% CI: 28.2%-71.8%) and a specificity of 100% (95% CI: 66.4%-71.8%). Of the 77 syphilis cases, 43 (56%) were HIV positive and the sensitivity and specificity of the PCR test did not vary by HIV status. The PCR test was able to detect up to five (10%) primary infections that were initially seronegative, including one HIV positive man with delayed seroconversion to syphilis (72 to 140 days) and one HIV positive man who did not seroconvert to syphilis over 14 months follow-up. Both men had been treated for syphilis within a week of the PCR test. Conclusions T pallidum PCR is a potentially powerful tool for the early diagnosis of primary syphilis, particularly where a serological response has yet to develop. PMID:23241398

  16. Comparative Evaluation of RUT, PCR and ELISA Tests for Detection of Infection with Cytotoxigenic H. pylori

    PubMed Central

    Jalalypour, Farzaneh; Farajnia, Safar; Somi, Mohammad Hossein; Hojabri, Zoya; Yousefzadeh, Rana; Saeedi, Nazli

    2016-01-01

    Purpose: Helicobacter pylori is one of the most prevalent infectious agents in the world which causes a variety of gastrointestinal diseases including gastritis, peptic ulcer and gastric carcinoma. The objective of this study was to comparatively evaluate invasive (rapid urease test and polymerase chain reaction) and non-invasive (enzyme-linked immunosorbent assay) tests in diagnosis of infection with cytotoxigenic H. pylori. Methods: Biopsy specimens and sera were collected from 105 patients with gastric disorders. The presence of H. pylori infection in gastric biopsies was evaluated by RUT and PCR methods using chemotaxis signal transduction protein gene (CSTP), Urea C and HP-16srRNA primers. Serum samples were used for the ELISA test. Detection of infection with cag A-positive strains was performed by PCR and cag A-IgG ELISA kit. Results: Patients with at least two out of three positive results were regarded as infected. The sensitivity, specificity, predictive value and accuracy of the three different methods were evaluated. Of the 105 gastric biopsies, H. pylori were positive in 51 patients (48.57%). The best sensitivity (92.16%) belonged to RUT. The sensitivities of other tests including PCR and ELISA test were 88.24% and 90.20%, respectively. PCR showed the best specificity (94.44%), and the specificities of the other tests including RUT and ELISA test, were 90.74 % and 61.11%, respectively. Furthermore, results of PCR and cag A-IgG ELISA showed high prevalence of cag A-positive strain in the study population. Conclusion: Based on our findings, serum ELISA is a rapid noninvasive test for screening of H. pylori infection in the absence of endoscopy indication. In addition, considering the high prevalence of cytotoxigenic H. pylori strains, cag A is suggested as a promising target for PCR and non- invasive ELISA tests for detection of infection with toxigenic strains. PMID:27478790

  17. Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus.

    PubMed

    Weyer, Camilla T; Joone, Christopher; Lourens, Carina W; Monyai, Mpho S; Koekemoer, Otto; Grewar, John D; van Schalkwyk, Antoinette; Majiwa, Phelix O A; MacLachlan, N James; Guthrie, Alan J

    2015-10-01

    Blood samples collected as part of routine diagnostic investigations from South African horses with clinical signs suggestive of African horse sickness (AHS) were subjected to analysis with an AHS virus (AHSV) group specific reverse transcription quantitative polymerase chain reaction (AHSV RT-qPCR) assay and virus isolation (VI) with subsequent serotyping by plaque inhibition (PI) assays using AHSV serotype-specific antisera. Blood samples that tested positive by AHSV RT-qPCR were then selected for analysis using AHSV type specific RT-qPCR (AHSV TS RT-qPCR) assays. The TS RT-qPCR assays were evaluated using both historic stocks of the South African reference strains of each of the 9 AHSV serotypes, as well as recently derived stocks of these same viruses. Of the 503 horse blood samples tested, 156 were positive by both AHSV RT-qPCR and VI assays, whereas 135 samples that were VI negative were positive by AHSV RT-qPCR assay. The virus isolates made from the various blood samples included all 9 AHSV serotypes, and there was 100% agreement between the results of conventional serotyping of individual virus isolates by PI assay and AHSV TS RT-qPCR typing results. Results of the current study confirm that the AHSV TS RT-qPCR assays for the identification of individual AHSV serotypes are applicable and practicable and therefore are potentially highly useful and appropriate for virus typing in AHS outbreak situations in endemic or sporadic incursion areas, which can be crucial in determining appropriate and timely vaccination and control strategies. PMID:26232526

  18. [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. PMID:20208409

  19. A point-of-care PCR test for HIV-1 detection in resource-limited settings.

    PubMed

    Jangam, Sujit R; Agarwal, Abhishek K; Sur, Kunal; Kelso, David M

    2013-04-15

    A low-cost, fully integrated sample-to-answer, quantitative PCR (qPCR) system that can be used for detection of HIV-1 proviral DNA in infants at the point-of-care in resource-limited settings has been developed and tested. The system is based on a novel DNA extraction method, which uses a glass fiber membrane, a disposable assay card that includes on-board reagent storage, provisions for thermal cycling and fluorescence detection, and a battery-operated portable analyzer. The system is capable of automated PCR mix assembly using a novel reagent delivery system and performing qPCR. HIV-1 and internal control targets are detected using two spectrally separated fluorophores, FAM and Quasar 670. In this report, a proof-of-concept of the platform is demonstrated. Initial results with whole blood demonstrate that the test is capable of detecting HIV-1 in blood samples containing greater than 5000 copies of HIV-1. In resource-limited settings, a point-of-care HIV-1 qPCR test would greatly increase the number of test results that reach the infants caregivers, allowing them to pursue anti-retroviral therapy. PMID:23202333

  20. Evaluation and Field Validation of PCR Tests for Detection of Actinobacillus pleuropneumoniae in Subclinically Infected Pigs

    PubMed Central

    Fittipaldi, Nahuel; Broes, André; Harel, Josée; Kobisch, Marylène; Gottschalk, Marcelo

    2003-01-01

    Eight PCR tests were evaluated for their abilities to detect Actinobacillus pleuropneumoniae in swine tonsils. At first they were compared regarding their specificities by using A. pleuropneumoniae and related bacterial species and their analytical sensitivities by using tonsils experimentally infected in vitro. PCRs were carried out both directly with tonsil homogenates (direct PCR) and after culture of the sample (after-culture PCR). Most tests demonstrated good specificities; however, some tests gave false-positive results with some non-A. pleuropneumoniae species. High degrees of variation in the analytical sensitivities among the tests were observed for the direct PCRs (109 to 102 CFU/g of tonsil), whereas those of most of the after-culture PCRs were similar (102 CFU/g of tonsil). In a second phase, the effects of sample storage time and storage conditions were evaluated by using tonsils from experimentally infected animals. Storage at −20°C allowed the detection of the organism for at least 4 months. Finally, the omlA PCR test described by Savoye et al. (C. Savoye et al., Vet. Microbiol. 73:337-347, 2000) and the commercially available Adiavet App PCR test were further validated with field samples. Their effectiveness was compared to those of standard and immunomagnetic separation-based methods of bacterial isolation. In addition, a comparison of tonsil biopsy specimens (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted. A. pleuropneumoniae was neither isolated nor detected by PCR from a herd serologically negative for A. pleuropneumoniae. PCR was more sensitive than the standard isolation method with whole tonsils from three infected herds. After-culture PCR offered the highest degree of sensitivity (93 and 83% for the omlA and Adiavet App PCRs, respectively). The PCR detection rate was higher with whole tonsils than with tonsil biopsy specimens. Good agreement (κ = 0.65) was found between the presence of A

  1. False-Positive Results after Environmental Pinworm PCR Testing due to Rhabditid Nematodes in Corncob Bedding

    PubMed Central

    Leblanc, Mathias; Berry, Kristina; Graciano, Sandy; Becker, Brandon; Reuter, Jon D

    2014-01-01

    Modern rodent colonies are housed in individually ventilated cages to protect the animals from contamination with adventitious pathogens. Standard health monitoring through soiled-bedding sentinels does not always detect infections, especially in the context of low pathogen prevalence. Recently proposed alternatives include analyzing environmental samples from the cages or rack exhaust by PCR to improve the detection of rodent pathogens but optimal sampling strategies have not yet been established for different microorganisms. Although generally very sensitive and specific, these molecular assays are not foolproof and subject to false-positive and –negative results and should always be interpreted cautiously with an overall understanding of the intrinsic controls and all the variables that may affect the results. Here, we report a limited Aspiculuris tetraptera outbreak in a mouse barrier facility that was detected by fecal PCR in sentinels and confirmed by fecal flotation and direct cecal examination of both sentinels and colony animals. The outbreak led to a widespread survey of all facilities for pinworms by using environmental PCR from ventilated rack exhaust plenums. Environmental PCR suggested an unexpected widespread contamination of all ventilated racks holding nonautoclaved cages, but results could not be confirmed in sentinel or colony animals by fecal flotation, cecal and colonic examination, or cage PCR testing. After additional investigation, the unexpected environmental PCR results were confirmed as false-positive findings due to the nonspecificity of the assay, leading to the amplification of rhabditid nematodes, which are not infectious in rodents but which contaminated the corncob bedding. PMID:25650980

  2. False-positive results after environmental pinworm PCR testing due to Rhabditid nematodes in Corncob bedding.

    PubMed

    Leblanc, Mathias; Berry, Kristina; Graciano, Sandy; Becker, Brandon; Reuter, Jon D

    2014-11-01

    Modern rodent colonies are housed in individually ventilated cages to protect the animals from contamination with adventitious pathogens. Standard health monitoring through soiled-bedding sentinels does not always detect infections, especially in the context of low pathogen prevalence. Recently proposed alternatives include analyzing environmental samples from the cages or rack exhaust by PCR to improve the detection of rodent pathogens but optimal sampling strategies have not yet been established for different microorganisms. Although generally very sensitive and specific, these molecular assays are not foolproof and subject to false-positive and -negative results and should always be interpreted cautiously with an overall understanding of the intrinsic controls and all the variables that may affect the results. Here, we report a limited Aspiculuris tetraptera outbreak in a mouse barrier facility that was detected by fecal PCR in sentinels and confirmed by fecal flotation and direct cecal examination of both sentinels and colony animals. The outbreak led to a widespread survey of all facilities for pinworms by using environmental PCR from ventilated rack exhaust plenums. Environmental PCR suggested an unexpected widespread contamination of all ventilated racks holding nonautoclaved cages, but results could not be confirmed in sentinel or colony animals by fecal flotation, cecal and colonic examination, or cage PCR testing. After additional investigation, the unexpected environmental PCR results were confirmed as false-positive findings due to the nonspecificity of the assay, leading to the amplification of rhabditid nematodes, which are not infectious in rodents but which contaminated the corncob bedding. PMID:25650980

  3. Development, standardization and assessment of PCR systems for purity testing of avian viral vaccines.

    PubMed

    Ottiger, Hans-Peter

    2010-05-01

    The European Pharmacopoeia (Ph. Eur.) requires avian viral vaccines to be free of adventitious agents. Purity testing is an essential quality requirement of immunological veterinary medicinal products (IVMPs) and testing for extraneous agents includes monitoring for many different viruses. Conventional virus detection methods include serology or virus culture, however, molecular tests have become a valid alternative testing method. Nucleic acid testing (NAT) is fast, highly sensitive and has a higher degree of discrimination than conventional approaches. These advantages have led to the development and standardization of polymerase chain reaction (PCR) assays for the detection of avian leucosis virus, avian orthoreovirus, infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus, infectious laryngotracheitis virus, influenza A virus, Marek's disease virus, turkey rhinotracheitis virus, egg drop syndrome virus, chicken anaemia virus, avian adenovirus and avian encephalomyelitis virus. This paper reviews the development, standardization and assessment of PCR for extraneous agent testing in IVMPs with examples from an Official Medicines Control Laboratory (OMCL). PMID:20338785

  4. Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.

    PubMed

    Buh Gasparic, Meti; Tengs, Torstein; La Paz, Jose Luis; Holst-Jensen, Arne; Pla, Maria; Esteve, Teresa; Zel, Jana; Gruden, Kristina

    2010-03-01

    Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use. PMID:20087729

  5. An Improved Flame Test for Qualitative Analysis Using a Multichannel UV-Visible Spectrophotometer

    ERIC Educational Resources Information Center

    Blitz, Jonathan P.; Sheeran, Daniel J.; Becker, Thomas L.

    2006-01-01

    Qualitative analysis schemes are used in undergraduate laboratory settings as a way to introduce equilibrium concepts and logical thinking. The main component of all qualitative analysis schemes is a flame test, as the color of light emitted from certain elements is distinctive and a flame photometer or spectrophotometer in each laboratory is…

  6. Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses.

    PubMed

    Dai, Xiaohu; Chen, Sisi; Li, Ning; Yan, Han

    2016-05-15

    The aim of this study was to comprehensively validate the sonication-based DNA extraction method, in hope of the replacement of the so-called 'standard DNA extraction method' - the commercial kit method. Microbial cells in the digested sludge sample, containing relatively high amount of PCR-inhibitory substances, such as humic acid and protein, were applied as the experimental alternatives. The procedure involving solid/liquid separation of sludge sample and dilution of both DNA templates and inhibitors, the minimum templates for PCR-based analyses, and the in-depth understanding from the bias analysis by pyrosequencing technology were obtained and confirmed the availability of the sonication-based DNA extraction method. PMID:26774955

  7. Generic RT-PCR tests for detection and identification of tospoviruses.

    PubMed

    Hassani-Mehraban, A; Westenberg, M; Verhoeven, J T J; van de Vossenberg, B T L H; Kormelink, R; Roenhorst, J W

    2016-07-01

    A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories. PMID:27036502

  8. Evaluation of a PCR Test for Detection of Treponema pallidum in Swabs and Blood

    PubMed Central

    Grange, P. A.; Gressier, L.; Dion, P. L.; Farhi, D.; Benhaddou, N.; Gerhardt, P.; Morini, J. P.; Deleuze, J.; Pantoja, C.; Bianchi, A.; Lassau, F.; Avril, M. F.; Janier, M.

    2012-01-01

    Syphilis diagnosis is based on clinical observation, serological analysis, and dark-field microscopy (DFM) detection of Treponema pallidum subsp. pallidum, the etiological agent of syphilis, in skin ulcers. We performed a nested PCR (nPCR) assay specifically amplifying the tpp47 gene of T. pallidum from swab and blood specimens. We studied a cohort of 294 patients with suspected syphilis and 35 healthy volunteers. Eighty-seven of the 294 patients had primary syphilis, 103 had secondary syphilis, 40 had latent syphilis, and 64 were found not to have syphilis. The T. pallidum nPCR results for swab specimens were highly concordant with syphilis diagnosis, with a sensitivity of 82% and a specificity of 95%. Reasonable agreement was observed between the results obtained with the nPCR and DFM methods (kappa = 0.53). No agreement was found between the nPCR detection of T. pallidum in blood and the diagnosis of syphilis, with sensitivities of 29, 18, 14.7, and 24% and specificities of 96, 92, 93, and 97% for peripheral blood mononuclear cell (PBMC), plasma, serum, and whole-blood fractions, respectively. HIV status did not affect the frequency of T. pallidum detection in any of the specimens tested. Swab specimens from mucosal or skin lesions seemed to be more useful than blood for the efficient detection of the T. pallidum genome and, thus, for the diagnosis of syphilis. PMID:22219306

  9. Detection limits of the strip test and PCR for genetically modified corn in Brazil.

    PubMed

    Nascimento, V E; Von Pinho, É V R; Von Pinho, R G; do Nascimento, A D

    2012-01-01

    Brazilian legislation establishes a labeling limit for products that contain more than 1% material from genetically modified organisms (GMOs). We assessed the sensitivity of the lateral flow strip test in detection of the GMO corn varieties Bt11 and MON810 and the specificity and sensitivity of PCR techniques for their detection. For the strip test, the GMO seeds were mixed with conventional seeds at levels of 0.2, 0.4 and 0.8% for Bt11, and 0.4, 0.8 and 1.6% for MON810. Three different methodologies were assessed and whole seeds, their endosperm and embryonic axis were used. For the PCR technique, the GMO seeds of each of the two varieties were mixed with conventional seeds at levels of 20, 10, 5, 2, 1, and 0.5%. The seeds were ground and the DNA extracted. For detection of the GMO material, specific primers were used for MON810 and Bt11 and maize zein as an endogenous control. The sensitivity of the strip test varied for both maize varieties and methodologies. The test was positive for Bt11 only at 0.8%, in contrast with the detection limit of 0.4% indicated by the manufacturer. In the multiplex PCR, the primers proved to be specific for the different varieties. These varieties were detected in samples with one GMO seed in 100. Thus, this technique proved to be efficient in detecting contaminations equal to or greater than 1%. PMID:22843069

  10. Rediscovery of the Yesso scallop pathogen Perkinsus qugwadi in Canada, and development of PCR tests.

    PubMed

    Itoh, Naoki; Meyer, Gary R; Tabata, Amy; Lowe, Geoff; Abbott, Cathryn L; Johnson, Stewart C

    2013-04-29

    Perkinsus qugwadi, a pathogenic protozoan parasite of Yesso scallops Patinopecten yessoensis, is found only in cultured populations in British Columbia, Canada. This pathogen was first identified in 1988 and caused significant mortalities at some locations during the early 1990s. Prevalence of infection decreased dramatically following 1995, and the disease was last reported in 1997, leading to speculation that the Yesso scallop stocks in Canada had developed resistance to the disease, or that P. qugwadi had disappeared. However, the present study revealed that infection with P. qugwadi and associated mortality is still occurring in scallops from at least one location in British Columbia. One of the PCR tests developed for P. qugwadi detected the parasite in a 105-fold dilution of DNA extracted from a heavily infected sample and detected 52% more positive scallops than histology; however, the assay also cross-reacted with P. honshuensis and P. olseni. The other PCR test was less sensitive and detected 34% more positives, but did not react to any of the other Perkinsus species tested, suggesting that these PCR tests are powerful tools for screening for the presence of P. qugwadi. Phylogenetic analysis of 1796 bp of SSU rRNA gene sequence clearly indicated that P. qugwadi is positioned basally to other Perkinsus species. PMID:23670082

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

    PubMed

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

    2015-12-01

    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. PMID:26648102

  12. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... should consist of the following sequences: ER12JA07.005 (c) Polymerase chain reaction. (1) Treat...

  13. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... should consist of the following sequences: ER12JA07.005 (c) Polymerase chain reaction. (1) Treat...

  14. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section... Examination Procedures § 147.30 Laboratory procedure recommended for the polymerase chain reaction (PCR) test... should consist of the following sequences: ER12JA07.005 (c) Polymerase chain reaction. (1) Treat...

  15. 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.

  16. Optimization of the PCR test for the mutation causing bovine leukocyte adhesion deficiency.

    PubMed

    Mirck, M H; Von Bannisseht-Wijsmuller, T; Timmermans-Besselink, W J; Van Luijk, J H; Buntjer, J B; Lenstra, J A

    1995-07-01

    The recent emergence of the bovine leukocyte adhesion deficiency (BLAD) demonstrated the risks of narrowing the genetic basis of a population. About 6% of the Holstein-Friesian cattle now descends from one bull who was a heterozygous BLAD carrier. Crossing his descendants resulted in the birth of homozygous BLAD calves with a life expectancy of < 1 year. The BLAD syndrome is caused by a point mutation in the gene coding for CD18, a subunit of the beta 2 integrins on the surface of leukocytes. By using a PCR-RFLP test, large numbers of cattle are now being screened in several countries to eradicate the mutant allele. We describe an optimization of the PCR primer set that has led to an improvement of the test. PMID:7580848

  17. Adaptive and Qualitative Changes in Encoding Strategy with Experience: Evidence from the Test-Expectancy Paradigm

    ERIC Educational Resources Information Center

    Finley, Jason R.; Benjamin, Aaron S.

    2012-01-01

    Three experiments demonstrated learners' abilities to adaptively and qualitatively accommodate their encoding strategies to the demands of an upcoming test. Stimuli were word pairs. In Experiment 1, test expectancy was induced for either cued recall (of targets given cues) or free recall (of targets only) across 4 study-test cycles of the same…

  18. 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

  19. Pre-validation study for testing of avian viral vaccines for extraneous agents by PCR.

    PubMed

    Bruckner, L; Ottiger, H P

    2007-12-01

    The biological nature of vaccines imposes a permanent risk for contamination with extraneous agents. Therefore, testing of vaccines for freedom from extraneous agents is essential in the manufacturing process and quality control. Relevant methods for testing for extraneous agents of avian viral vaccines are specified in the monographs of the European Pharmacopoeia (Ph. Eur.). Currently, most of these methods involve the use of embryonated eggs or chickens. Polymerase chain reaction (PCR) is a widely used and suitable tool for the amplification and detection of extraneous nucleic acids. Different PCR assays have been developed for the application in routine testing of veterinary vaccines. However, before introduction of new methods in monographs of the Ph. Eur., they must undergo validation. Here we report about a pre-validation study performed in Official Medicines Control Laboratories (OMCLs). Diluted samples of avian infectious laryngotracheitis, avian infectious bronchitis and avian infectious bursal disease viruses have been analysed using standardised procedures and reagents. The study demonstrated that PCR methods can be transferred to other laboratories. The results also show that further work is warranted for full validation of the method. PMID:18413134

  20. Malaria surveillance in the Democratic Republic of the Congo: comparison of microscopy, PCR, and rapid diagnostic test.

    PubMed

    Doctor, Stephanie M; Liu, Yunhao; Whitesell, Amy; Thwai, Kyaw L; Taylor, Steve M; Janko, Mark; Emch, Michael; Kashamuka, Melchior; Muwonga, Jérémie; Tshefu, Antoinette; Meshnick, Steven R

    2016-05-01

    Malaria surveillance is critical for control efforts, but diagnostic methods frequently disagree. Here, we compare microscopy, PCR, and a rapid diagnostic test in 7137 samples from children in the Democratic Republic of the Congo using latent class analysis. PCR had the highest sensitivity (94.6%) and microscopy had the lowest (76.7%). PMID:26915637

  1. Fluorescent detection of Southern blots and PCR-based genetic typing tests

    SciTech Connect

    Mansfield, E.S.; Worley, J.M.; Zimmerman, P.A.

    1994-09-01

    The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.

  2. Adaptive and qualitative changes in encoding strategy with experience: evidence from the test-expectancy paradigm.

    PubMed

    Finley, Jason R; Benjamin, Aaron S

    2012-05-01

    Three experiments demonstrated learners' abilities to adaptively and qualitatively accommodate their encoding strategies to the demands of an upcoming test. Stimuli were word pairs. In Experiment 1, test expectancy was induced for either cued recall (of targets given cues) or free recall (of targets only) across 4 study-test cycles of the same test format, followed by a final critical cycle featuring either the expected or the unexpected test format. For final tests of both cued and free recall, participants who had expected that test format outperformed those who had not. This disordinal interaction, supported by recognition and self-report data, demonstrated not mere differences in effort based on anticipated test difficulty, but rather qualitative and appropriate differences in encoding strategies based on expected task demands. Participants also came to appropriately modulate metacognitive monitoring (Experiment 2) and study-time allocation (Experiment 3) across study-test cycles. Item and associative recognition performance, as well as self-report data, revealed shifts in encoding strategies across trials; these results were used to characterize and evaluate the different strategies that participants employed for cued versus free recall and to assess the optimality of participants' metacognitive control of encoding strategies. Taken together, these data illustrate a sophisticated form of metacognitive control, in which learners qualitatively shift encoding strategies to match the demands of anticipated tests. PMID:22103783

  3. PCR Testing of a Ventilated Caging System to Detect Murine Fur Mites

    PubMed Central

    Jensen, Eric S; Allen, Kenneth P; Henderson, Kenneth S; Szabo, Aniko; Thulin, Joseph D

    2013-01-01

    Rodents housed in microisolation caging are commonly monitored for infectious agents by the use of soiled bedding sentinels. This strategy relies on the successful transmission of rodent pathogens from the index rodents via soiled bedding to sentinel cages and the subsequent infection or colonization of sentinel rodents. When the prevalence of a pathogen is low or the target agent is not readily transmitted by soiled bedding, alternative testing methodologies should be used. Given the continued prevalence of institutions self-reporting murine fur mites and with the advent of a new sensitive and specific PCR assay for mites, we sought to determine whether the exhaust system of an individual ventilated caging (IVC) system could be used for monitoring the rack's rodent population for mites rather than relying on the responses of sentinels. We deployed single cages of mice (Mus musculus) that were known to be infested with either Radfordia affinis or Myobia musculi on a 70-cage rack, sampled the horizontal exhaust manifolds weekly, and used the new PCR assay to test these samples for mite DNA. We detected the presence of fur mites at a 94.1% probability of detection within 4 wk of placement. Therefore, we recommend swabbing and testing the shelf exhaust manifolds of IVC racks rather than relying on soiled-bedding sentinels as an indicator of the mite status of the rodents on that rack. PMID:23562030

  4. PCR testing of a ventilated caging system to detect murine fur mites.

    PubMed

    Jensen, Eric S; Allen, Kenneth P; Henderson, Kenneth S; Szabo, Aniko; Thulin, Joseph D

    2013-01-01

    Rodents housed in microisolation caging are commonly monitored for infectious agents by the use of soiled bedding sentinels. This strategy relies on the successful transmission of rodent pathogens from the index rodents via soiled bedding to sentinel cages and the subsequent infection or colonization of sentinel rodents. When the prevalence of a pathogen is low or the target agent is not readily transmitted by soiled bedding, alternative testing methodologies should be used. Given the continued prevalence of institutions self-reporting murine fur mites and with the advent of a new sensitive and specific PCR assay for mites, we sought to determine whether the exhaust system of an individual ventilated caging (IVC) system could be used for monitoring the rack's rodent population for mites rather than relying on the responses of sentinels. We deployed single cages of mice (Mus musculus) that were known to be infested with either Radfordia affinis or Myobia musculi on a 70-cage rack, sampled the horizontal exhaust manifolds weekly, and used the new PCR assay to test these samples for mite DNA. We detected the presence of fur mites at a 94.1% probability of detection within 4 wk of placement. Therefore, we recommend swabbing and testing the shelf exhaust manifolds of IVC racks rather than relying on soiled-bedding sentinels as an indicator of the mite status of the rodents on that rack. PMID:23562030

  5. Boston (WGBH) Field Testing of a Qualitative Television Rating System for Public Broadcasting.

    ERIC Educational Resources Information Center

    Myrick, Howard A.; Keegan, Carol

    A field test was conducted to examine the efficiency of a paper-and-pencil television diary for collecting viewer evaluations of public and commerical television programs. Six hundred individuals in Boston, Massachusetts, kept qualitative viewing diaries for one week. The results indicated high and enthusiastic cooperation by the total sample. The…

  6. HIV Testing among Men Who Have Sex with Men (MSM): Systematic Review of Qualitative Evidence

    ERIC Educational Resources Information Center

    Lorenc, Theo; Marrero-Guillamon, Isaac; Llewellyn, Alexis; Aggleton, Peter; Cooper, Chris; Lehmann, Angela; Lindsay, Catriona

    2011-01-01

    We conducted a systematic review of qualitative evidence relating to the views and attitudes of men who have sex with men (MSM) concerning testing for HIV. Studies conducted in high-income countries (Organisation for Economic Co-operation and Development members) since 1996 were included. Seventeen studies were identified, most of gay or bisexual…

  7. Employing Online S-P Diagnostic Table for Qualitative Comments on Test Results

    ERIC Educational Resources Information Center

    Wang, Chien-hwa; Chen, Cheng-ping

    2013-01-01

    The major concerns of adaptive testing studies have concentrated on effectiveness and efficiency of the system built for the research experiments. It has been criticised that such general information has fallen short of providing qualitative descriptions regarding learning performance. Takahiro Sato of Japan proposed an analytical diagram called…

  8. Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR

    PubMed Central

    2011-01-01

    Background This study describes the use of malaria rapid diagnostic tests (RDTs) as a source of DNA for Plasmodium species-specific real-time PCR. Methods First, the best method to recover DNA from RDTs was investigated and then the applicability of this DNA extraction method was assessed on 12 different RDT brands. Finally, two RDT brands (OptiMAL Rapid Malaria Test and SDFK60 malaria Ag Plasmodium falciparum/Pan test) were comprehensively evaluated on a panel of clinical samples submitted for routine malaria diagnosis at ITM. DNA amplification was done with the 18S rRNA real-time PCR targeting the four Plasmodium species. Results of PCR on RDT were compared to those obtained by PCR on whole blood samples. Results Best results were obtained by isolating DNA from the proximal part of the nitrocellulose component of the RDT strip with a simple DNA elution method. The PCR on RDT showed a detection limit of 0.02 asexual parasites/μl, which was identical to the same PCR on whole blood. For all 12 RDT brands tested, DNA was detected except for one brand when a low parasite density sample was applied. In RDTs with a plastic seal covering the nitrocellulose strip, DNA extraction was hampered. PCR analysis on clinical RDT samples demonstrated correct identification for single species infections for all RDT samples with asexual parasites of P. falciparum (n = 60), Plasmodium vivax (n = 10), Plasmodium ovale (n = 10) and Plasmodium malariae (n = 10). Samples with only gametocytes were detected in all OptiMAL and in 10 of the 11 SDFK60 tests. None of the negative samples (n = 20) gave a signal by PCR on RDT. With PCR on RDT, higher Ct-values were observed than with PCR on whole blood, with a mean difference of 2.68 for OptiMAL and 3.53 for SDFK60. Mixed infections were correctly identified with PCR on RDT in 4/5 OptiMAL tests and 2/5 SDFK60 tests. Conclusions RDTs are a reliable source of DNA for Plasmodium real-time PCR. This study demonstrates the best method of RDT

  9. Somatic Mutation Allelic Ratio Test Using ddPCR (SMART-ddPCR): An Accurate Method for Assessment of Preferential Allelic Imbalance in Tumor DNA.

    PubMed

    de Smith, Adam J; Walsh, Kyle M; Hansen, Helen M; Endicott, Alyson A; Wiencke, John K; Metayer, Catherine; Wiemels, Joseph L

    2015-01-01

    The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19-142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a

  10. Somatic Mutation Allelic Ratio Test Using ddPCR (SMART-ddPCR): An Accurate Method for Assessment of Preferential Allelic Imbalance in Tumor DNA

    PubMed Central

    de Smith, Adam J.; Walsh, Kyle M.; Hansen, Helen M.; Endicott, Alyson A.; Wiencke, John K.; Metayer, Catherine; Wiemels, Joseph L.

    2015-01-01

    The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19–142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a

  11. Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

    SciTech Connect

    Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

    2000-05-05

    Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCR amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.

  12. Production, Extraction, and Qualitative Testing of Penicillin: A Biochemistry Experiment for Health Science Chemistry Courses

    NASA Astrophysics Data System (ADS)

    Stevens, Richard E.; Billingsley, Kara C.

    1998-10-01

    This laboratory procedure guides students through the growth of a submerged Penicillium chrysogenum culture. Subsequent steps include extraction of the penicillin by adsorption onto activated charcoal, extraction with acetone, and qualitative testing of the drug on a bacterial culture. The laboratory procedure is designed for freshman-level health science chemistry courses. This procedure produces minimal waste, which can be disposed of by the appropriate use of an autoclave.

  13. ‘I’m fishing really’ — inflammatory marker testing in primary care: a qualitative study

    PubMed Central

    Watson, Jessica; de Salis, Isabel; Hamilton, Willie; Salisbury, Chris

    2016-01-01

    Background Inflammatory markers can be helpful as part of the diagnostic workup for specific diseases or for monitoring disease activity. A third use is as a screening and/or triage tool to differentiate between the presence or absence of disease. Most research into inflammatory markers looks at diagnosis of specific diseases and comes from secondary care. Qualitative studies to explore when and why clinicians use these tests in primary care are lacking. Aim To identify clinicians’ approaches to inflammatory marker testing in primary care. Design and setting Qualitative study with 26 GPs and nurse practitioners. Method Interviews were conducted using a semi-structured topic guide. Clinicians reviewed recent cases of inflammatory marker testing in their pathology inbox. Interviews were audiorecorded and transcribed. Qualitative analysis was conducted by two of the authors. Results Clinicians are uncertain about the appropriate use of inflammatory markers and differ in their approach to testing patients with undifferentiated symptoms. Normal or significantly elevated inflammatory markers are seen as helpful, but mildly raised inflammatory markers in the context of non-specific symptoms are difficult to interpret. Clinicians describe a tension between not wanting to ‘miss anything’ and, on the other hand, being wary of picking up borderline abnormalities that can lead to cascades of further tests. Diagnostic uncertainty is a common reason for inflammatory marker testing, with the aim to reassure; however, paradoxically, inconclusive results can generate a cycle of uncertainty and anxiety. Conclusion Further research is needed to define when inflammatory marker testing is useful in primary care and how to interpret results. PMID:26852797

  14. Patients with diffuse uveitis and inactive toxoplasmic retinitis lesions test PCR positive for Toxoplasma gondii in their vitreous and blood

    PubMed Central

    Novais, Eduardo A; Commodaro, Alessandra G; Santos, Fábio; Muccioli, Cristina; Maia, André; Nascimento, Heloisa; Moeller, Cecilia T A; Rizzo, Luiz V; Grigg, Michael E; Belfort, Rubens

    2016-01-01

    Background/aims To determine if patients with inactive chorioretinitis lesions who experience chronic toxoplasmic uveitis test PCR positive for Toxoplasma in their ocular fluids. Methods Two patients undergoing long-term anti-toxoplasmic treatment developed chronic uveitis and vitritis. They underwent therapeutic and diagnostic pars plana vitrectomy. Patient specimens were tested for toxoplasmosis by real-time PCR and nested PCR. Patient specimens were also tested for the presence of Toxoplasma antibodies that recognise allelic peptide motifs to determine parasite serotype. Results Patients tested positive for Toxoplasma by real-time PCR at the B1 gene in the vitreous and aqueous humours of patient 1, but only the vitreous of patient 2. Patients were not parasitemic by real-time PCR in plasma and blood. During surgery, only old hyperpigmented toxoplasmic scars were observed; there was no sign of active retinitis. Multilocus PCR–DNA sequence genotyping at B1, NTS2 and SAG1 loci established that two different non-archetypal Toxoplasma strains had infected patients 1 and 2. A peptide-based serotyping ELISA confirmed the molecular findings. Conclusions No active lesions were observed, but both patients possessed sufficient parasite DNA in their vitreous to permit genotyping. Several hypotheses to explain the persistence of the vitritis and anterior uveitis in the absence of active retinitis are discussed. PMID:24518074

  15. PMA treatment is an effective means to reduce false positive PCR testing results

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Traditional and real time PCR are widely used in detecting bacterial pathogens in various food matrix and environmental samples. Sometimes a positive detection using PCR can not be confirmed by subsequent culture isolation of the targeted pathogen, resulting in a potential “false positive.” False po...

  16. HIV Testing Patterns among Black Men Who Have Sex with Men: A Qualitative Typology

    PubMed Central

    Hussen, Sophia A.; Stephenson, Robert; del Rio, Carlos; Wilton, Leo; Wallace, Jermel; Wheeler, Darrell

    2013-01-01

    Background Black men who have sex with men (MSM) in the Southeastern United States are disproportionately affected by HIV. Black MSM are more likely to have unrecognized HIV infection, suggesting that testing may occur later and/or infrequently relative to current recommendations. The objective of this qualitative study was to explore the HIV testing behaviors of Black MSM in Atlanta, Georgia, who were participants in the HIV Prevention Trials Network Brothers Study (HPTN 061). Methods and Findings We conducted 29 in-depth interviews and four focus groups with a community-recruited sample. Modified grounded theory methodologies were used to guide our inductive analysis, which yielded a typology comprised of four distinct HIV testing patterns. Participants could be categorized as: (1) Maintenance Testers, who tested regularly as part of routine self-care; (2) Risk-Based Testers, whose testing depended on relationship status or sexual behavior; (3) Convenience Testers, who tested irregularly depending on what testing opportunities arose; or (4) Test Avoiders, who tested infrequently and/or failed to follow up on results. We further characterized these groups with respect to age, socioeconomic factors, identity, stigma and healthcare access. Conclusions Our findings highlight the heterogeneity of HIV testing patterns among Black MSM, and offer a framework for conceptualizing HIV testing in this group. Public health messaging must account for the diversity of Black MSM's experiences, and multiple testing approaches should be developed and utilized to maximize outreach to different types of testers. PMID:24069408

  17. Pp65 antigenemia, plasma real-time PCR and DBS test in symptomatic and asymptomatic cytomegalovirus congenitally infected newborns

    PubMed Central

    2010-01-01

    Background Many congenitally cytomegalovirus-infected (cCMV) neonates are at risk for severe consequences, even if they are asymptomatic at birth. The assessment of the viral load in neonatal blood could help in identifying the babies at risk of sequelae. Methods In the present study, we elaborated the results obtained on blood samples collected in the first two weeks of life from 22 symptomatic and 48 asymptomatic newborns with cCMV diagnosed through urine testing. We evaluated the performances of two quantitative methods (pp65 antigenemia test and plasma Real-time PCR) and the semi-quantitative results of dried blood sample (DBS) test in the aim of identifying a valid method for measuring viral load. Results Plasma qPCR and DBS tests were positive in 100% of cases, antigenemia in 81%. Only the latter test gave quantitatively different results in symptomatic versus asymptomatic children. qPCR values of 103 copies/ml were found in 52% of newborn. "Strong" DBS test positivity cases had higher median values of both pp65 positive PBL and DNA copies/ml than cases with a "weak" positivity. Conclusions As expected antigenemia test was less sensitive than molecular tests and DBS test performed better on samples with higher rates of pp65 positive PBL and higher numbers of DNA copies/ml. The prognostic significance of the results of these tests will be evaluated on completion of the ongoing collection of follow-up data of these children. PMID:20149232

  18. 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. PMID:12084490

  19. An extension of the Wilcoxon-Mann-Whitney test for analyzing RT-qPCR data.

    PubMed

    De Neve, Jan; Thas, Olivier; Ottoy, Jean-Pierre; Clement, Lieven

    2013-06-01

    Classical approaches for analyzing reverse transcription quantitative polymerase chain reaction (RT-qPCR) data commonly require normalization before assessing differential expression (DE). Normalization often has a substantial effect on the interpretation and validity of the subsequent analysis steps, but at the same time it causes a reduction in variance and introduces dependence among the normalized outcomes. These effects can be substantial, however, they are typically ignored. Most normalization techniques and methods for DE focus on mean expression and are sensitive to outliers. Moreover, in cancer studies, for example, oncogenes are often only expressed in a subsample of the populations during sampling. This primarily affects the skewness and the tails of the distribution and the mean is therefore not necessarily the best effect size measure within these experimental setups. In our contribution, we propose an extension of the Wilcoxon-Mann-Whitney test which incorporates a robust normalization, and the uncertainty associated with normalization is propagated into the final statistical summaries for DE. Our method relies on semiparametric regression models that focus on the probability P{Y ≤ Y'}, where Y and Y' denote independent responses for different subject groups. This effect size is robust to outliers, while remaining informative and intuitive when DE affects the shape of the distribution instead of only the mean. We also extend our approach for assessing DE for multiple features simultaneously. Simulation studies show that the test has a good performance, and that it is very competitive with standard methods for this platform. The method is illustrated on two neuroblastoma studies. PMID:23652635

  20. Systematic review and meta-analysis: rapid diagnostic tests versus placental histology, microscopy and PCR for malaria in pregnant women

    PubMed Central

    2011-01-01

    Background During pregnancy, malaria infection with Plasmodium falciparum or Plasmodium vivax is related to adverse maternal health and poor birth outcomes. Diagnosis of malaria, during pregnancy, is complicated by the absence or low parasite densities in peripheral blood. Diagnostic methods, other than microscopy, are needed for detection of placental malaria. Therefore, the diagnostic accuracy of rapid diagnostic tests (RDTs), detecting antigen, and molecular techniques (PCR), detecting DNA, for the diagnosis of Plasmodium infections in pregnancy was systematically reviewed. Methods MEDLINE, EMBASE and Web of Science were searched for studies assessing the diagnostic accuracy of RDTs, PCR, microscopy of peripheral and placental blood and placental histology for the detection of malaria infection (all species) in pregnant women. Results The results of 49 studies were analysed in metandi (Stata), of which the majority described P. falciparum infections. Although both placental and peripheral blood microscopy cannot reliably replace histology as a reference standard for placental P. falciparum infection, many studies compared RDTs and PCR to these tests. The proportion of microscopy positives in placental blood (sensitivity) detected by peripheral blood microscopy, RDTs and PCR are respectively 72% [95% CI 62-80], 81% [95% CI 55-93] and 94% [95% CI 86-98]. The proportion of placental blood microscopy negative women that were negative in peripheral blood microscopy, RDTs and PCR (specificity) are 98% [95% CI 95-99], 94% [95% CI 76-99] and 77% [95% CI 71-82]. Based on the current data, it was not possible to determine if the false positives in RDTs and PCR are caused by sequestered parasites in the placenta that are not detected by placental microscopy. Conclusion The findings suggest that RDTs and PCR may have good performance characteristics to serve as alternatives for the diagnosis of malaria in pregnancy, besides any other limitations and practical considerations

  1. Knowing is not enough: a qualitative report on HIV testing among heterosexual African-American men.

    PubMed

    Bond, Keosha T; Frye, Victoria; Taylor, Raekiela; Williams, Kim; Bonner, Sebastian; Lucy, Debbie; Cupid, Malik; Weiss, Linda; Koblin, Beryl A

    2015-01-01

    Despite having higher rates of HIV testing than all other racial groups, African-Americans continue to be disproportionately affected by the HIV epidemic in the United States. Knowing one's status is the key step to maintaining behavioral changes that could stop the spread of the virus, yet little is known about the individual- and socio-structural-level barriers associated with HIV testing and communication among heterosexual African-American men. To address this and inform the development of an HIV prevention behavioral intervention for heterosexual African-American men, we conducted computerized, structured interviews with 61 men, focus group interviews with 25 men in 5 different groups, and in-depth qualitative interviews with 30 men living in high HIV prevalence neighborhoods in New York City. Results revealed that HIV testing was frequent among the participants. Even with high rates of testing, the men in the study had low levels of HIV knowledge; perceived little risk of HIV; and misused HIV testing as a prevention method. Factors affecting HIV testing, included stigma, relationship dynamics and communication, and societal influences, suggesting that fear, low perception of risk, and HIV stigma may be the biggest barriers to HIV testing. These results also suggest that interventions directed toward African-American heterosexual men must address the use of "testing as prevention" as well as correct misunderstandings of the window period and the meaning of HIV test results, and interventions should focus on communicating about HIV. PMID:25298014

  2. Knowing is not enough: a qualitative report on HIV testing among heterosexual African-American men

    PubMed Central

    Bond, Keosha T.; Frye, Victoria; Taylor, Raekiela; Williams, Kim; Bonner, Sebastian; Lucy, Debbie; Cupid, Malik; Weiss, Linda; Koblin, Beryl A.

    2015-01-01

    Despite having higher rates of HIV testing than all other racial groups, African-Americans continue to be disproportionately affected by the HIV epidemic in the United States. Knowing one’s status is the key step to maintaining behavioral changes that could stop the spread of the virus, yet little is known about the individual- and socio-structural-level barriers associated with HIV testing and communication among heterosexual African-American men. To address this and inform the development of an HIV prevention behavioral intervention for heterosexual African-American men, we conducted computerized, structured interviews with 61 men, focus group interviews with 25 men in 5 different groups, and in-depth qualitative interviews with 30 men living in high HIV prevalence neighborhoods in New York City. Results revealed that HIV testing was frequent among the participants. Even with high rates of testing, the men in the study had low levels of HIV knowledge; perceived little risk of HIV; and misused HIV testing as a prevention method. Factors affecting HIV testing, included stigma, relationship dynamics and communication, and societal influences, suggesting that fear, low perception of risk, and HIV stigma may be the biggest barriers to HIV testing. These results also suggest that interventions directed toward African-American heterosexual men must address the use of “testing as prevention” as well as correct misunderstandings of the window period and the meaning of HIV test results, and interventions should focus on communicating about HIV. PMID:25298014

  3. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section 147.30 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE LIVESTOCK IMPROVEMENT AUXILIARY...

  4. 9 CFR 147.30 - Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma...

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Laboratory procedure recommended for the polymerase chain reaction (PCR) test for Mycoplasma gallisepticum and M. synoviae. 147.30 Section 147.30 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE LIVESTOCK IMPROVEMENT AUXILIARY...

  5. Evaluation of an updated real-time RT-PCR test for the identification of the H7 subtype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid detection of avian influenza (AI) virus and identification of the H5 and H7 subtypes is critical for wild bird monitoring programs. A real-time RT-PCR test for identification of the H7 subtype in North America was first reported in 2002. With the recent surveillance in wild birds it was disc...

  6. Evaluation of primer and probe mismatches in sensitivity of select RRT-PCR tests for avian influenza

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The recent outbreak of pH1N1 in animals highlighted an imperfection of the matrix real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) that has become the primary screening test for avian and swine influenza viruses. Four mismatches in one primer resulted in an important loss of sens...

  7. [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

  8. The contribution of PCR testing to influenza and pertussis notifications in Australia.

    PubMed

    Kaczmarek, M C; Ware, R S; Lambert, S B

    2016-01-01

    Influenza and pertussis are the two most common vaccine-preventable infections notified in Australia. We assessed the role of polymerase chain reaction (PCR) diagnosis in influenza and pertussis cases notified to the Australian National Notifiable Diseases Surveillance System (NNDSS). There were a total of 2 10 786 notified influenza cases (2001-2013) and 2 55 866 notified pertussis cases (1991-2013). After 1 January 2007, the majority of influenza and pertussis notifications were PCR-based (80·5% and 59·6%, respectively). Before 31 December 2006, PCR-based notifications were limited (29·1% and 11·7%, respectively). By 2013, PCR-based notifications had largely replaced all other diagnostic methods, with the exception of serology-based notifications in pertussis cases in adults aged ⩾ 25 years. PMID:26112983

  9. A Comparison of EGFR Mutation Testing Methods in Lung Carcinoma: Direct Sequencing, Real-time PCR and Immunohistochemistry

    PubMed Central

    Angulo, Bárbara; Conde, Esther; Suárez-Gauthier, Ana; Plaza, Carlos; Martínez, Rebeca; Redondo, Pilar; Izquierdo, Elisa; Rubio-Viqueira, Belén; Paz-Ares, Luis; Hidalgo, Manuel; López-Ríos, Fernando

    2012-01-01

    The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry), with direct sequencing and to investigate the limit of detection (LOD) of both PCR-based methods. We identified EGFR mutations in 21 (16%) of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%), which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%). Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+) yielded similar results. Immunohistochemistry (IHC) staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity. PMID:22952784

  10. Diagnostic Accuracy of PCR Alone and Compared to Urinary Antigen Testing for Detection of Legionella spp.: a Systematic Review

    PubMed Central

    Green, Hefziba; Steinmetz, Tali; Leibovici, Leonard; Paul, Mical

    2015-01-01

    The diagnosis of Legionnaires' disease (LD) is based on the isolation of Legionella spp., a 4-fold rise in antibodies, a positive urinary antigen (UA), or direct immunofluorescence tests. PCR is not accepted as a diagnostic tool for LD. This systematic review assesses the diagnostic accuracy of PCR in various clinical samples with a direct comparison versus UA. We included prospective or retrospective cohort and case-control studies. Studies were included if they used the Centers for Disease Control and Prevention consensus definition criteria of LD or a similar one, assessed only patients with clinical pneumonia, and reported data for all true-positive, false-positive, true-negative, and false-negative results. Two reviewers abstracted data independently. Risk of bias was assessed using Quadas-2. Summary sensitivity and specificity values were estimated using a bivariate model and reported with a 95% confidence interval (CI). Thirty-eight studies were included. A total of 653 patients had confirmed LD, and 3,593 patients had pneumonia due to other pathogens. The methodological quality of the studies as assessed by the Quadas-2 tool was poor to fair. The summary sensitivity and specificity values for diagnosis of LD in respiratory samples were 97.4% (95% CI, 91.1% to 99.2%) and 98.6% (95% CI, 97.4% to 99.3%), respectively. These results were mainly unchanged by any covariates tested and subgroup analysis. The diagnostic performance of PCR in respiratory samples was much better than that of UA. Compared to UA, PCR in respiratory samples (especially in sputum samples or swabs) revealed a significant advantage in sensitivity and an additional diagnosis of 18% to 30% of LD cases. The diagnostic performance of PCR in respiratory samples was excellent and preferable to that of the UA. Results were independent on the covariate tested. PCR in respiratory samples should be regarded as a valid tool for the diagnosis of LD. PMID:26659202

  11. Diagnostic Accuracy of PCR Alone and Compared to Urinary Antigen Testing for Detection of Legionella spp.: a Systematic Review.

    PubMed

    Avni, Tomer; Bieber, Amir; Green, Hefziba; Steinmetz, Tali; Leibovici, Leonard; Paul, Mical

    2016-02-01

    The diagnosis of Legionnaires' disease (LD) is based on the isolation of Legionella spp., a 4-fold rise in antibodies, a positive urinary antigen (UA), or direct immunofluorescence tests. PCR is not accepted as a diagnostic tool for LD. This systematic review assesses the diagnostic accuracy of PCR in various clinical samples with a direct comparison versus UA. We included prospective or retrospective cohort and case-control studies. Studies were included if they used the Centers for Disease Control and Prevention consensus definition criteria of LD or a similar one, assessed only patients with clinical pneumonia, and reported data for all true-positive, false-positive, true-negative, and false-negative results. Two reviewers abstracted data independently. Risk of bias was assessed using Quadas-2. Summary sensitivity and specificity values were estimated using a bivariate model and reported with a 95% confidence interval (CI). Thirty-eight studies were included. A total of 653 patients had confirmed LD, and 3,593 patients had pneumonia due to other pathogens. The methodological quality of the studies as assessed by the Quadas-2 tool was poor to fair. The summary sensitivity and specificity values for diagnosis of LD in respiratory samples were 97.4% (95% CI, 91.1% to 99.2%) and 98.6% (95% CI, 97.4% to 99.3%), respectively. These results were mainly unchanged by any covariates tested and subgroup analysis. The diagnostic performance of PCR in respiratory samples was much better than that of UA. Compared to UA, PCR in respiratory samples (especially in sputum samples or swabs) revealed a significant advantage in sensitivity and an additional diagnosis of 18% to 30% of LD cases. The diagnostic performance of PCR in respiratory samples was excellent and preferable to that of the UA. Results were independent on the covariate tested. PCR in respiratory samples should be regarded as a valid tool for the diagnosis of LD. PMID:26659202

  12. Evaluation of a commercial PCR test for the diagnosis of dermatophyte nail infections.

    PubMed

    Spiliopoulou, Anastasia; Bartzavali, Christina; Jelastopulu, Eleni; Anastassiou, Evangelos D; Christofidou, Myrto

    2015-01-01

    Tinea unguium, known as onychomycosis, is a dermatophyte infection of nails with worldwide distribution. Conventional methods for detecting fungi in nail specimens are either non-specific (microscopy) or insensitive (culture). PCR has been used to improve sensitivity in detecting the causative fungi in nail specimens from patients with suspected onychomycosis. Results of a commercial multiplex PCR for the detection of dermatophytes, especially Trichophyton rubrum (the main dermatophyte implicated), as compared to conventional methods are presented. A total of 418 nail scrapings obtained from dermatological outpatients were handled in the Laboratory of Microbiology between May 2010 and May 2013. Among them, multiplex PCR detected 126 (30.1 %) dermatophyte-positive samples, whereas culture revealed 44 (10.5 %). Direct microscopy revealed 63 (15.1 %) positive specimens. T. rubrum was identified in 116 out of 126 (92 %) positive PCR samples and 40 out of 44 (91 %) dermatophyte-positive cultures. Implementation of PCR increased species-specific detection of dermatophytes by 21.1 %, leading to a threefold increase as compared to culture alone. Multiplex PCR offers a time-saving diagnostic tool for tinea unguium and augments laboratory assistance to clinical evaluation for proper treatment. PMID:25418736

  13. Direct Comparison of Flow-FISH and qPCR as Diagnostic Tests for Telomere Length Measurement in Humans

    PubMed Central

    Gutierrez-Rodrigues, Fernanda; Santana-Lemos, Bárbara A.; Scheucher, Priscila S.; Alves-Paiva, Raquel M.; Calado, Rodrigo T.

    2014-01-01

    Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R2 = 0.60; p<0.0001) and patients (R2 = 0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R2 = 0.35; p<0.0001) and low correlation for patients (R2 = 0.20; p = 0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R2 = 0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R2 = 0.1, p = 0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8±7.1%) and qPCR (9.5±7.4%; p = 0.35), but the inter-assay CV was lower for flow-FISH (9.6±7.6% vs. 16±19.5%; p = 0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the

  14. Evaluation and Comparison of Multiple Test Methods, Including Real-time PCR, for Legionella Detection in Clinical Specimens

    PubMed Central

    Peci, Adriana; Winter, Anne-Luise; Gubbay, Jonathan B.

    2016-01-01

    Legionella is a Gram-negative bacterium that can cause Pontiac fever, a mild upper respiratory infection and Legionnaire’s disease, a more severe illness. We aimed to compare the performance of urine antigen, culture, and polymerase chain reaction (PCR) test methods and to determine if sputum is an acceptable alternative to the use of more invasive bronchoalveolar lavage (BAL). Data for this study included specimens tested for Legionella at Public Health Ontario Laboratories from 1st January, 2010 to 30th April, 2014, as part of routine clinical testing. We found sensitivity of urinary antigen test (UAT) compared to culture to be 87%, specificity 94.7%, positive predictive value (PPV) 63.8%, and negative predictive value (NPV) 98.5%. Sensitivity of UAT compared to PCR was 74.7%, specificity 98.3%, PPV 77.7%, and NPV 98.1%. Out of 146 patients who had a Legionella-positive result by PCR, only 66 (45.2%) also had a positive result by culture. Sensitivity for culture was the same using either sputum or BAL (13.6%); sensitivity for PCR was 10.3% for sputum and 12.8% for BAL. Both sputum and BAL yield similar results regardless testing methods (Fisher Exact p-values = 1.0, for each test). In summary, all test methods have inherent weaknesses in identifying Legionella; therefore, more than one testing method should be used. Obtaining a single specimen type from patients with pneumonia limits the ability to diagnose Legionella, particularly when urine is the specimen type submitted. Given ease of collection and similar sensitivity to BAL, clinicians are encouraged to submit sputum in addition to urine when BAL submission is not practical from patients being tested for Legionella.

  15. Understanding Referral Patterns for Bone Mineral Density Testing among Family Physicians: A Qualitative Descriptive Study

    PubMed Central

    Munce, Sarah E. P.; Allin, Sonya; Carlin, Leslie; Sale, Joanna; Hawker, Gillian; Kim, Sandra; Butt, Debra A.; Polidoulis, Irene; Tu, Karen; Jaglal, Susan B.

    2016-01-01

    Introduction. Evidence of inappropriate bone mineral density (BMD) testing has been identified in terms of overtesting in low risk women and undertesting among patients at high risk. In light of these phenomena, the objective of this study was to understand the referral patterns for BMD testing among Ontario's family physicians (FPs). Methods. A qualitative descriptive approach was adopted. Twenty-two FPs took part in a semi-structured interview lasting approximately 30 minutes. An inductive thematic analysis was performed on the transcribed data in order to understand the referral patterns for BMD testing. Results. We identified a lack of clarity about screening for osteoporosis with a tendency for baseline BMD testing in healthy, postmenopausal women and a lack of clarity on the appropriate age for screening for men in particular. A lack of clarity on appropriate intervals for follow-up testing was also described. Conclusions. These findings lend support to what has been documented at the population level suggesting a tendency among FPs to refer menopausal women (at low risk). Emphasis on referral of high-risk groups as well as men and further clarification and education on the appropriate intervals for follow-up testing is warranted. PMID:26904357

  16. Evaluation of a novel real-time PCR test based on the ssrA gene for the identification of group B streptococci in vaginal swabs

    PubMed Central

    2009-01-01

    Background Despite the implementation of prevention guidelines, early-onset group B streptococci (GBS) disease remains a cause of neonatal morbidity and mortality worldwide. Strategies to identify women who are at risk of transmitting GBS to their infant and the administration of intrapartum antibiotics have greatly reduced the incidence of neonatal GBS disease. However, there is a requirement for a rapid diagnostic test for GBS that can be carried out in a labour ward setting especially for women whose GBS colonisation status is unknown at the time of delivery. We report the design and evaluation of a real-time PCR test (RiboSEQ GBS test) for the identification of GBS in vaginal swabs from pregnant women. Methods The qualitative real-time PCR RiboSEQ GBS test was designed based on the bacterial ssrA gene and incorporates a competitive internal standard control. The analytical sensitivity of the test was established using crude lysate extracted from serial dilutions of overnight GBS culture using the IDI Lysis kit. Specificity studies were performed using DNA prepared from a panel of GBS strains, related streptococci and other species found in the genital tract environment. The RiboSEQ GBS test was evaluated on 159 vaginal swabs from pregnant women and compared with the GeneOhm™ StrepB Assay and culture for the identification of GBS. Results The RiboSEQ GBS test is specific and has an analytical sensitivity of 1-10 cell equivalents. The RiboSEQ GBS test was 96.4% sensitive and 95.8% specific compared to "gold standard" culture for the identification of GBS in vaginal swabs from pregnant women. In this study, the RiboSEQ GBS test performed slightly better than the commercial BD GeneOhm™ StrepB Assay which gave a sensitivity of 94.6% and a specificity of 89.6% compared to culture. Conclusion The RiboSEQ GBS test is a valuable method for the rapid, sensitive and specific detection of GBS in pregnant women. This study also validates the ssrA gene as a suitable and

  17. Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

    PubMed Central

    Cunha, Pricila da Silva; Pena, Heloisa B.; D'Angelo, Carla Sustek; Koiffmann, Celia P.; Rosenfeld, Jill A.; Shaffer, Lisa G.; Stofanko, Martin; Gonçalves-Dornelas, Higgor; Pena, Sérgio Danilo Junho

    2014-01-01

    Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs. PMID:24839341

  18. Qualitative risk assessment during polymer mortar test specimens preparation - methods comparison

    NASA Astrophysics Data System (ADS)

    Silva, F.; Sousa, S. P. B.; Arezes, P.; Swuste, P.; Ribeiro, M. C. S.; Baptista, J. S.

    2015-05-01

    Polymer binder modification with inorganic nanomaterials (NM) could be a potential and efficient solution to control matrix flammability of polymer concrete (PC) materials without sacrificing other important properties. Occupational exposures can occur all along the life cycle of a NM and “nanoproducts” from research through scale-up, product development, manufacturing, and end of life. The main objective of the present study is to analyse and compare different qualitative risk assessment methods during the production of polymer mortars (PM) with NM. The laboratory scale production process was divided in 3 main phases (pre-production, production and post-production), which allow testing the assessment methods in different situations. The risk assessment involved in the manufacturing process of PM was made by using the qualitative analyses based on: French Agency for Food, Environmental and Occupational Health & Safety method (ANSES); Control Banding Nanotool (CB Nanotool); Ecole Polytechnique Fédérale de Lausanne method (EPFL); Guidance working safely with nanomaterials and nanoproducts (GWSNN); Istituto Superiore per la Prevenzione e la Sicurezza del Lavoro, Italy method (ISPESL); Precautionary Matrix for Synthetic Nanomaterials (PMSN); and Stoffenmanager Nano. It was verified that the different methods applied also produce different final results. In phases 1 and 3 the risk assessment tends to be classified as medium-high risk, while for phase 2 the more common result is medium level. It is necessary to improve the use of qualitative methods by defining narrow criteria for the methods selection for each assessed situation, bearing in mind that the uncertainties are also a relevant factor when dealing with the risk related to nanotechnologies field.

  19. Validation of a qualitative immunochromatographic test for the noninvasive assessment of stress in dogs.

    PubMed

    Di Nardo, F; Anfossi, L; Ozella, L; Saccani, A; Giovannoli, C; Spano, G; Baggiani, C

    2016-08-15

    Salivary cortisol is regarded as a reliable parameter for the noninvasive assessment of the welfare of animals, because it is strictly related to stress levels. Several methods are available for salivary cortisol measurement in mammals, however rapid diagnostic test for detecting salivary cortisol are confined to humans. The availability of such non invasive diagnostic tools operable in situ would facilitate monitoring of animal welfare. The Cortisol stress™ test provides a simple and rapid tool to discriminate cortisol levels in canine saliva above or below 4ng/ml, which has been suggested as the cut-off value for distinguishing unstressed dogs from those experiencing stress. The test is based on a competitive immunochromatographic assay (ICT) using gold nanoparticles as probes, in which the color intensity of the Test line is inversely correlated to the salivary cortisol level. The qualitative result is obtained by the visual observation of the color formed on the Test line compared to that of the Control line We evaluated the accuracy of the test by determining salivary cortisol in 85 samples of canine saliva belonging to dogs with very variable age, sex, breed, and life history, and comparing the qualitative results to those obtained by a reference ELISA kit. Agreeing results were obtained through the two methods, and the ICT showed high diagnostic sensitivity, specificity and efficiency (100%, 98.4%, and 98.8%, respectively). Furthermore, we evaluated the precision of the test by an experimental design approach, which combines errors due to within-day and between-day variation with the biological variability, and demonstrated that the test could be reliably applied for correctly classifying canine samples, according to their salivary cortisol level. Moreover, we studied the shelf-life of the device in three experimental conditions. We confirmed the stability of the ICT at 4°C and 25°C for at least six months and observed similar results for an accelerated

  20. Development of a PCR test system for specific detection of Salmonella Paratyphi B in foods.

    PubMed

    Zhai, Ligong; Yu, Qian; Bie, Xiaomei; Lu, Zhaoxin; Lv, Fengxia; Zhang, Chong; Kong, Xiaohan; Zhao, Haizhen

    2014-06-01

    Salmonella enterica serotype Paratyphi B is a globally distributed human-specific pathogen causing paratyphoid fever. The aim of this study was to develop a rapid and reliable polymerase chain reaction (PCR) assay for its detection in food. The SPAB_01124 gene was found to be unique to S. Paratyphi B using comparative genomics. Primers for fragments of the SPAB_01124 gene and the Salmonella-specific invA gene were used in combination to establish a multiplex PCR assay that showed 100% specificity across 45 Salmonella strains (representing 34 serotypes) and 18 non-Salmonella strains. The detection limit was 2.2 CFU mL(-1) of S. Paratyphi B after 12-h enrichment in pure culture. It was shown that co-culture with S. Typhimurium or Escherichia coli up to concentrations of 3.6 × 10(5)  CFU and 3.3 × 10(4)  CFU, respectively, did not interfere with PCR detection of S. Paratyphi B. In artificially contaminated milk, the assay could detect as few as 62 CFU mL(-1) after 8 h of enrichment. In conclusion, comparative genomics was found to be an efficient approach to the mining of pathogen-specific target genes, and the PCR assay that was developed from this provided a rapid, specific, and sensitive method for detection of S. Paratyphi B. PMID:24725227

  1. Diagnostic Utility of a Clonality Test for Lymphoproliferative Diseases in Koreans Using the BIOMED-2 PCR Assay

    PubMed Central

    Kim, Young; Choi, Yoo Duk; Choi, Chan

    2013-01-01

    Background A clonality test for immunoglobulin (IG) and T cell receptor (TCR) is a useful adjunctive method for the diagnosis of lymphoproliferative diseases (LPDs). Recently, the BIOMED-2 multiplex polymerase chain reaction (PCR) assay has been established as a standard method for assessing the clonality of LPDs. We tested clonality in LPDs in Koreans using the BIOMED-2 multiplex PCR and compared the results with those obtained in European, Taiwanese, and Thai participants. We also evaluated the usefulness of the test as an ancillary method for diagnosing LPDs. Methods Two hundred and nineteen specimens embedded in paraffin, including 78 B cell lymphomas, 80 T cell lymphomas and 61 cases of reactive lymphadenitis, were used for the clonality test. Results Mature B cell malignancies showed 95.7% clonality for IG, 2.9% co-existing clonality, and 4.3% polyclonality. Mature T cell malignancies exhibited 83.8% clonality for TCR, 8.1% co-existing clonality, and 16.2% polyclonality. Reactive lymphadenitis showed 93.4% polyclonality for IG and TCR. The majority of our results were similar to those obtained in Europeans. However, the clonality for IGK of B cell malignancies and TCRG of T cell malignancies was lower in Koreans than Europeans. Conclusions The BIOMED-2 multiplex PCR assay was a useful adjunctive method for diagnosing LPDs. PMID:24255634

  2. New real-time PCR tests for species-specific detection of Chlamydophila psittaci and Chlamydophila abortus from tissue samples.

    PubMed

    Pantchev, Alexandra; Sting, Reinhard; Bauerfeind, Rolf; Tyczka, Judith; Sachse, Konrad

    2009-08-01

    Chlamydophila psittaci and Chlamydophila abortus are the causative agents of avian chlamydiosis (psittacosis) and ovine enzootic abortion, respectively. Both pathogens are known to possess zoonotic potential. Due to their close genetic relatedness, direct and rapid species identification is difficult. In the present study, new real-time PCR assays are reported for both species. The tests are based on highly specific probes targeting the ompA gene region and were conducted as duplex PCRs including an internal amplification control. The Cp. psittaci assay successfully passed a proficiency test at national level. Examination of field samples revealed Cp. psittaci as the dominating species in birds, but also Cp. abortus in a few psittacines. Real-time PCR assays for species-specific detection of Cp. psittaci and Cp. abortus are suited for routine diagnosis, which renders them important tools for the recognition of outbreaks of psittacosis and ovine enzootic abortion. PMID:18413292

  3. Virtual PCR

    SciTech Connect

    Gardner, S N; Clague, D S; Vandersall, J A; Hon, G; Williams, P L

    2006-02-23

    The polymerase chain reaction (PCR) stands among the keystone technologies for analysis of biological sequence data. PCR is used to amplify DNA, to generate many copies from as little as a single template. This is essential, for example, in processing forensic DNA samples, pathogen detection in clinical or biothreat surveillance applications, and medical genotyping for diagnosis and treatment of disease. It is used in virtually every laboratory doing molecular, cellular, genetic, ecologic, forensic, or medical research. Despite its ubiquity, we lack the precise predictive capability that would enable detailed optimization of PCR reaction dynamics. In this LDRD, we proposed to develop Virtual PCR (VPCR) software, a computational method to model the kinetic, thermodynamic, and biological processes of PCR reactions. Given a successful completion, these tools will allow us to predict both the sequences and concentrations of all species that are amplified during PCR. The ability to answer the following questions will allow us both to optimize the PCR process and interpret the PCR results: What products are amplified when sequence mixtures are present, containing multiple, closely related targets and multiplexed primers, which may hybridize with sequence mismatches? What are the effects of time, temperature, and DNA concentrations on the concentrations of products? A better understanding of these issues will improve the design and interpretation of PCR reactions. The status of the VPCR project after 1.5 years of funding is consistent with the goals of the overall project which was scoped for 3 years of funding. At half way through the projected timeline of the project we have an early beta version of the VPCR code. We have begun investigating means to improve the robustness of the code, performed preliminary experiments to test the code and begun drafting manuscripts for publication. Although an experimental protocol for testing the code was developed, the preliminary

  4. Patient perspectives on test result communication in primary care: a qualitative study

    PubMed Central

    Litchfield, Ian J; Bentham, Louise M; Lilford, Richard J; McManus, Richard J; Greenfield, Sheila M

    2015-01-01

    Background Although the number of blood tests ordered in primary care continues to increase, efficient systems for the communication of blood test results to patients are lacking. This is a concern in terms of both patient safety and patient satisfaction. Aim To gain an understanding of patient perspectives on organisational and technological aspects of current and prospective systems for communicating laboratory test results in primary care, and the influences that impact patients’ preferred methods for receiving results. Design and setting Qualitative study using patient focus groups in four primary care practices in Birmingham, UK. Method The primary care practices were purposively selected to ensure they varied in size, socioeconomic environment, and the default pathways they used to communicate test results. A total of 26 patients from the four practices who had had a recent blood test were recruited. Over a 6 month period in 2011, six, 1-hour focus groups were conducted at the four practices involved in the study. Results Patients expressed a preference for receiving results from the ordering GP or a clinically qualified member of staff. Suggestions for refining current systems included improved access to phlebotomy appointments, better management of patient telephone calls, and a clear, accessible protocol for the communication of results. Conclusion Despite the testing and result communication process being a core activity in primary care, it was found that practices could improve their service in a number of areas. Patients described frequent delays and inconsistency in both the level of information and the method of communication, as well as dissatisfaction with non-clinical staff relaying results. Patient preferences for result communication based on their experience of current systems have produced practical suggestions to improve processes. PMID:25733434

  5. A qualitative investigation into patients’ views on visual field testing for glaucoma monitoring

    PubMed Central

    Glen, Fiona C; Baker, Helen; Crabb, David P

    2014-01-01

    Objectives To investigate the views and experiences of patients regarding their glaucoma follow-up, particularly towards the type and frequency of visual field (VF) testing. Design A qualitative investigation using focus groups. The group discussion used broad open questions around the topics in a prompt guide relating to experiences of glaucoma follow-up, and in particular, VF monitoring. All the groups were taped, transcribed and coded using manual and computer-aided methods. Setting Three National Health Service (NHS) hospitals in England; two focus groups took place at each hospital. Participants 28 patients (mean (SD) age: 74 (9) years; 54% women) diagnosed with glaucoma for at least 2 years. Each focus group consisted of 3–6 patients. Primary and secondary outcomes (1) Attitudes and experiences of patients with glaucoma regarding VF testing. (2) Patients’ opinions about successful follow-up in glaucoma. Results These patients did not enjoy the VF test but they recognised the importance of regular monitoring for preserving their vision. These patients would agree to more frequent VF testing on their clinician's recommendation. A number of themes recurred throughout the focus groups representing perceived barriers to follow-up care. The testing environment, waiting times, efficiency of appointment booking and travel to the clinic were all perceived to influence the general clinical experience and the quality of assessment data. Patients were also concerned about aspects of patient–doctor communication, and often received little to no feedback about their results. Conclusions Patients trust the clinician to make the best decisions for their glaucoma follow-up. However, patients highlighted a number of issues that could compromise the effectiveness of VF testing. Addressing patient-perceived barriers could be an important step for devising optimal strategies for follow-up care. PMID:24413347

  6. HIV testing and disclosure: a qualitative analysis of TB patients in South Africa.

    PubMed

    Daftary, A; Padayatchi, N; Padilla, M

    2007-04-01

    In South Africa, more than 60% of TB patients have HIV co-infection. Voluntary counseling and testing (VCT) is critical to effective HIV prevention, and TB facilities are optimal venues for delivery of these services. This study employed qualitative research methods to explore the decision-making processes for HIV testing and serostatus disclosure by 21 patients hospitalized with multi/extensively-drug resistant TB (M/XDR-TB) in Durban, KwaZulu Natal. Data collected from in-depth interviews characterized 3 broad themes: HIV testing history, experiences and perceptions of stigma and disclosure, and the relationship between TB and HIV/AIDS. Fear of AIDS-related stigma, the singular stress of TB infection, the absence of partner's consent, asymptomatic or incurable disease, and uncertainty about subsequent eligibility for antiretroviral treatment while still receiving TB treatment were identified as potential barriers to the uptake of VCT. HIV serostatus disclosure was impeded by the felt stigma of a 'discreditable' infection, manifested by social rejection and discrimination. The public disclosure of TB illness helped relieve some co-infected patients' overall burden of stigma through a process of 'covering'. HIV prevention [corrected] measures such as VCTare likely to be more effective within TB facilities if greater sensitivity is paid to TB patients' specific social issues and perceptions. These patients are not only at greater risk for HIV co-infection but also for experiencing the double stigma of TB and HIV/AIDS. PMID:17453600

  7. Exploring Operational Test and Evaluation of Unmanned Aircraft Systems: A Qualitative Case Study

    NASA Astrophysics Data System (ADS)

    Saliceti, Jose A.

    The purpose of this qualitative case study was to explore and identify strategies that may potentially remedy operational test and evaluation procedures used to evaluate Unmanned Aircraft Systems (UAS) technology. The sample for analysis consisted of organizations testing and evaluating UASs (e.g., U.S. Air Force, U.S. Navy, U.S. Army, U.S. Marine Corps, U.S. Coast Guard, and Customs Border Protection). A purposeful sampling technique was used to select 15 subject matter experts in the field of operational test and evaluation of UASs. A questionnaire was provided to participants to construct a descriptive and robust research. Analysis of responses revealed themes related to each research question. Findings revealed operational testers utilized requirements documents to extrapolate measures for testing UAS technology and develop critical operational issues. The requirements documents were (a) developed without the contribution of stakeholders and operational testers, (b) developed with vague or unrealistic measures, and (c) developed without a systematic method to derive requirements from mission tasks. Four approaches are recommended to develop testable operational requirements and assist operational testers: (a) use a mission task analysis tool to derive requirements for mission essential tasks for the system, (b) exercise collaboration among stakeholders and testers to ensure testable operational requirements based on mission tasks, (c) ensure testable measures are used in requirements documents, and (d) create a repository list of critical operational issues by mission areas. The preparation of operational test and evaluation processes for UAS technology is not uniform across testers. The processes in place are not standardized, thus test plan preparation and reporting are different among participants. A standard method to prepare and report UAS technology should be used when preparing and reporting on UAS technology. Using a systematic process, such as mission

  8. Quantitative Assessment of Commutability for Clinical Viral Load Testing Using a Digital PCR-Based Reference Standard.

    PubMed

    Tang, L; Sun, Y; Buelow, D; Gu, Z; Caliendo, A M; Pounds, S; Hayden, R T

    2016-06-01

    Given recent advances in the development of quantitative standards, particularly WHO international standards, efforts to better understand the commutability of reference materials have been made. Existing approaches in evaluating commutability include prediction intervals and correspondence analysis; however, the results obtained from existing approaches may be ambiguous. We have developed a "deviation-from-ideal" (DFI) approach to evaluate commutability of standards and applied it to the assessment of Epstein-Bar virus (EBV) load testing in four quantitative PCR assays, treating digital PCR as a reference assay. We then discuss advantages and limitations of the DFI approach as well as experimental design to best evaluate the commutability of an assay in practice. PMID:27076654

  9. Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air.

    PubMed

    Libert, X; Chasseur, C; Bladt, S; Packeu, A; Bureau, F; Roosens, N H; De Keersmaecker, S C J

    2015-09-01

    Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical analytical monitoring methods, based on cultivation and microscopic identification, depend on the growth of the fungi. Consequently, they are biased by difficulties to grow some species on certain culture media and under certain conditions or by noncultivable or dead fungi that can consequently not be identified. However, they could have an impact on human health as they might be allergenic. Since molecular methods do not require a culture step, they seem an excellent alternative for the monitoring of indoor fungal contaminations. As a case study, we developed a SYBR® green real-time PCR-based assay for the specific detection and identification of Aspergillus versicolor, which is frequently observed in indoor environment and known to be allergenic. The developed primers amplify a short region of the internal transcribed spacer 1 from the 18S ribosomal DNA complex. Subsequently, the performance of this quantitative polymerase chain reaction (qPCR) method was assessed using specific criteria, including an evaluation of the selectivity, PCR efficiency, dynamic range, and repeatability. The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor. In order to demonstrate that this SYBR® green qPCR assay is a valuable alternative for monitoring indoor fungal contamination with A. versicolor, environmental samples collected in contaminated houses were analyzed and the results were compared to the ones obtained with the traditional methods. PMID:26184975

  10. PCR testing can be as accurate as culture for diagnosis of Ichthyophonus hoferi in Yukon River Chinook salmon Oncorhynchus tshawytscha .

    PubMed

    Hamazaki, Toshihide; Kahler, Eryn; Borba, Bonnie M; Burton, Tamara

    2013-07-01

    We evaluated the comparability of culture and PCR tests for detecting Ichthyophonus in Yukon River Chinook salmon Oncorhynchus tshawytscha from field samples collected at 3 locations (Emmonak, Chena, and Salcha, Alaska, USA) in 2004, 2005, and 2006. Assuming diagnosis by culture as the 'true' infection status, we calculated the sensitivity (correctly identifying fish positive for Ichthyophonus), specificity (correctly identifying fish negative for Ichthyophonus), and accuracy (correctly identifying both positive and negative fish) of PCR. Regardless of sampling locations and years, sensitivity, specificity, and accuracy exceeded 90%. Estimates of infection prevalence by PCR were similar to those by culture, except for Salcha 2005, where prevalence by PCR was significantly higher than that by culture (p < 0.0001). These results show that the PCR test is comparable to the culture test for diagnosing Ichthyophonus infection. PMID:23836767

  11. Point-Counterpoint: Large Multiplex PCR Panels Should Be First-Line Tests for Detection of Respiratory and Intestinal Pathogens

    PubMed Central

    2015-01-01

    The first FDA-approved multiplex PCR panel for a large number of respiratory pathogens was introduced in 2008. Since then, other PCR panels for detection of several respiratory and gastrointestinal pathogens have been approved by the FDA and are commercially available, and more such panels are likely to become available. These assays detect 12 to 20 pathogens, and some include pathogens that typically cause different manifestations of infection, although they infect the same organ system. Some of these tests are labor-intensive, while others require little labor, and all of them are expensive, both for the laboratory and for the patient or insurer. They include a bundle of tests with limited or no options for selecting which tests will be performed. Laboratories and hospitals have adopted different strategies for offering these assays. Some have implemented strategies to limit the use of the tests, such as limiting the frequency with which patients can be tested, restricting testing to specific groups of patients (e.g., immunocompromised patients), or providing education to encourage the use of less expensive tests before using large multiplex panels. Others have offered these assays without limiting their use, either relying on the ordering provider to exercise good judgment or because such assays are thought to be appropriate for first-line diagnostic testing. In this Point-Counterpoint, Paul Schreckenberger of Loyola University Medical Center explains why his laboratory offers these assays without restriction. Alex McAdam of Boston's Children Hospital explains the concerns about the use of these assays as first-line tests and why some limitations on their use might be appropriate. PMID:25762770

  12. Point-Counterpoint: Large Multiplex PCR Panels Should Be First-Line Tests for Detection of Respiratory and Intestinal Pathogens.

    PubMed

    Schreckenberger, Paul C; McAdam, Alexander J

    2015-10-01

    The first FDA-approved multiplex PCR panel for a large number of respiratory pathogens was introduced in 2008. Since then, other PCR panels for detection of several respiratory and gastrointestinal pathogens have been approved by the FDA and are commercially available, and more such panels are likely to become available. These assays detect 12 to 20 pathogens, and some include pathogens that typically cause different manifestations of infection, although they infect the same organ system. Some of these tests are labor-intensive, while others require little labor, and all of them are expensive, both for the laboratory and for the patient or insurer. They include a bundle of tests with limited or no options for selecting which tests will be performed. Laboratories and hospitals have adopted different strategies for offering these assays. Some have implemented strategies to limit the use of the tests, such as limiting the frequency with which patients can be tested, restricting testing to specific groups of patients (e.g., immunocompromised patients), or providing education to encourage the use of less expensive tests before using large multiplex panels. Others have offered these assays without limiting their use, either relying on the ordering provider to exercise good judgment or because such assays are thought to be appropriate for first-line diagnostic testing. In this Point-Counterpoint, Paul Schreckenberger of Loyola University Medical Center explains why his laboratory offers these assays without restriction. Alex McAdam of Boston's Children Hospital explains the concerns about the use of these assays as first-line tests and why some limitations on their use might be appropriate. PMID:25762770

  13. Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test.

    PubMed Central

    Fach, P; Popoff, M R

    1997-01-01

    A duplex PCR procedure was evaluated for the detection of Clostridium perfringens in food and biological samples and for the identification of enterotoxigenic strains. This method uses two sets of primers which amplify in the same reaction two different DNA fragments simultaneously: the 283-bp C. perfringens phospholipase C gene fragment and the 426-bp enterotoxin gene fragment. Internal primers within the two primer sets confirmed the specificity of the method by DNA-DNA hybridization with the PCR products. No cross-reaction was observed with other Clostridium species or with other bacteria routinely found in food. The detection level was approximately 10(5) C. perfringens cells per g of stool or food sample. When overnight enrichment culture was used, 10 C. perfringens cells per g was detected in 57 artificially contaminated food samples. The duplex PCR is a rapid, sensitive, and reliable method for the detection and identification of enterotoxigenic C. perfringens strains in food samples. A slide latex agglutination test was also evaluated as a rapid, simple technique for the detection of C. perfringens enterotoxin in stool samples. PMID:9361409

  14. Efficacy of species-specific recA PCR tests in the identification of Burkholderia cepacia complex environmental isolates.

    PubMed

    Dalmastri, Claudia; Pirone, Luisa; Tabacchioni, Silvia; Bevivino, Annamaria; Chiarini, Luigi

    2005-05-01

    In this study, we evaluated if recA species-specific PCR assays could be successfully applied to identify environmental isolates of the widespread Burkholderia cepacia complex (Bcc) species. A total of 729 Bcc rhizosphere isolates collected in different samplings were assigned to the species B. cepacia genomovar I (61), B. cenocepacia recA lineage IIIB (514), B. ambifaria (124) and B. pyrrocinia (30), by means of recA (RFLP) analysis, and PCR tests were performed to assess sensitivity and specificity of recA species-specific primers pairs. B. cepacia genomovar I specific primers produced the expected amplicon with all isolates of the corresponding species (sensitivity, 100%), and cross-reacted with all B. pyrrocinia isolates. On the contrary, B. cenocepacia IIIB primers did not give the expected amplicon in 164 B. cenocepacia IIIB isolates (sensitivity, 68.1%), and isolates of distinct populations showed different sensitivity. B. ambifaria primers failed to amplify a recA-specific fragment only in a few isolates of this species (sensitivity, 93.5%). The absence of specific amplification in a high number of B. cenocepacia rhizosphere isolates indicates that recA specific PCR assays can lead to an underestimation of environmental microorganisms belonging to this bacterial species. PMID:15869960

  15. SEROLOGIC SURVEY AND RESULTS OF URINARY PCR TESTING FOR LEPTOSPIROSIS IN CAPTIVE BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS).

    PubMed

    Olds, June E; Sun, Yaxuan; Baum, David H; Gauger, Phillip

    2015-12-01

    Leptospirosis is an important zoonotic disease occurring clinically and subclinically in humans and a wide variety of mammal species worldwide. Often, rodents and wild animals are identified as important reservoirs for the disease. Twenty-two captive black-tailed prairie dogs (Cynomys ludovicianus) housed within a zoo were examined as part of a routine census and preventive medicine program. During examinations, blood and urine were collected to screen for exposure to, or infection with, leptospirosis. All animals were apparently healthy at the time of examination. Leptospira microscopic agglutination test identified 12 of 22 (54.5%) prairie dogs with antibody titers ≥1 : 100 against Leptospira interrogans serovar bratislava on initial serologic examination. All prairie dogs within this collection were serologically negative for L. interrogans serovars canicola, hardjo, icterohaemorrhagiae, and pomona and Leptospira kirschneri serovar grippotyphosa. Leptospira polymerase chain reaction (PCR) testing of urine was negative in all animals tested. This report describes evidence that captive prairie dogs may be exposed to leptospirosis, most likely from wild rodent reservoirs; however, serum titers are low, and lack of leptospiral DNA detected by PCR indicates that these captive animals are unlikely to be important reservoirs for the disease. PMID:26667541

  16. Sensitive immunochemical approaches for quantitative (FPIA) and qualitative (lateral flow tests) determination of gentamicin in milk.

    PubMed

    Beloglazova, N V; Shmelin, P S; Eremin, S A

    2016-03-01

    Three kinds of immunoassays for the determination of gentamicin in milk samples were developed and validated. First, a fast and easily-performed fluorescence polarization immunoassay was used for characterization of the employed polyclonal antibody. The calculated Kaff were (1.9±0.4)×10(9)М(-1) and (6.0±0.2)×10(6)М(-1) for the high- and low-affinity fractions respectively. The assay was characterized with a good sensitivity, the limit of detection being 5μgkg(-1). Two different kinds of detection labels, i.e. colloidal gold (CG) and quantum dots (QDs), were evaluated for use in lateral-flow format with respect to rapid visual on-site testing. The cut-off levels for both qualitative formats were selected based on the maximum level for gentamicin in milk established by the European Commission, 100μgkg(-1), resulting in a 10μgkg(-1) cut-off considering sample dilution. The intra-laboratory validation was performed with sterilized milk samples artificially spiked with gentamicin at concentrations less than, equal to, and greater than the cut-off level. It was shown that milk products could be analyzed without any sample preparation, except for dilution with the buffer solution. The rates of false-positive and false-negative results were below 5% for both labels. The different developed immunoassays were tested towards gentamicin determination in artificially-spiked and naturally contaminated milk samples. PMID:26717834

  17. Identification by a Digital Gene Expression Displayer (DGED) and test by RT-PCR analysis of new mRNA candidate markers for colorectal cancer in peripheral blood.

    PubMed

    Lauriola, Mattia; Ugolini, Giampaolo; Rosati, Giancarlo; Zanotti, Simone; Montroni, Isacco; Manaresi, Alessio; Zattoni, Davide; Rivetti, Stefano; Mattei, Gabriella; Coppola, Domenico; Strippoli, Pierluigi; Taffurelli, Mario; Solmi, Rossella

    2010-08-01

    Evidence from the literature widely supports the efficacy of screening for colorectal cancer (CRC) in reducing mortality. A blood-based assay, potentially, represents a more accessible early detection tool for the identification of circulating tumour cells originating from a primary tumour site in the body. The present work aimed at identifying a set of specific mRNAs expressed in colon tissue but not in blood cells. These mRNAs may represent useful markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay, following RNA extraction from peripheral blood samples. Using a data-mining tool called cDNA digital gene expression displayer (DGED), based on serial analysis of gene expression (SAGE) from the Cancer Genome Anatomy Project (CGAP) database, 4-colon and 14-blood cDNA libraries were analyzed. We selected 7 genes expressed in colon tissue but not in blood and were able to test 6 of them by RT-PCR in peripheral blood of CRC patients and healthy controls. We present a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as candidate markers of malignancy in blood samples of patients with colon cancer. SAGE DGED provided a list of the best candidate mRNAs predicted to detect colon cells in the blood, namely those encoding the following proteins: hypothetical protein LOC644844 (LOC644844, whose cDNA was not amplifiable), fatty acid binding protein 1 (FABP1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mucin 13 cell surface associated (MUC13), guanylate cyclase activator 2A (GUCA2A), amiloride binding protein 1 (ABP1), galactoside-binding, solute carrier family 26, member 3 (SLC26A3). The mRNA expression of these genes was evaluated in 8 samples from subjects diagnosed with CRC and 9 from healthy controls. We observed the expression of 2 of the 6 investigated genes in the blood samples of the vast majority of patients considered, but also in a subset of the

  18. Comparative Application of PLS and PCR Methods to Simultaneous Quantitative Estimation and Simultaneous Dissolution Test of Zidovudine - Lamivudine Tablets.

    PubMed

    Üstündağ, Özgür; Dinç, Erdal; Özdemir, Nurten; Tilkan, M Günseli

    2015-01-01

    In the development strategies of new drug products and generic drug products, the simultaneous in-vitro dissolution behavior of oral dosage formulations is the most important indication for the quantitative estimation of efficiency and biopharmaceutical characteristics of drug substances. This is to force the related field's scientists to improve very powerful analytical methods to get more reliable, precise and accurate results in the quantitative analysis and dissolution testing of drug formulations. In this context, two new chemometric tools, partial least squares (PLS) and principal component regression (PCR) were improved for the simultaneous quantitative estimation and dissolution testing of zidovudine (ZID) and lamivudine (LAM) in a tablet dosage form. The results obtained in this study strongly encourage us to use them for the quality control, the routine analysis and the dissolution test of the marketing tablets containing ZID and LAM drugs. PMID:26085428

  19. Tests of the Dynamic Field Theory and The Spatial Precision Hypothesis: Capturing a Qualitative Developmental Transition in Spatial Working Memory

    ERIC Educational Resources Information Center

    Schutte, Anne R.; Spencer, John P.

    2009-01-01

    This study tested a dynamic field theory (DFT) of spatial working memory and an associated spatial precision hypothesis (SPH). Between 3 and 6 years of age, there is a qualitative shift in how children use reference axes to remember locations: 3-year-olds' spatial recall responses are biased toward reference axes after short memory delays, whereas…

  20. Detection of enterotoxic Bacillus cereus producing hemolytic and non hemolytic enterotoxins by PCR test.

    PubMed

    Ołtuszak-Walczak, Elzbieta; Walczak, Piotr; Modrak, Robert

    2006-01-01

    Nine strains belonging to Bacillus cereus group has been isolated from food and environmental samples. Their taxonomic position was confirmed by RFLP analysis of 16S rRNA gene digested with TaqI. The detection of DNA sequences encoding the hemolysin BL complex and enterotoxin NHE, was studied in Bacillus sp. isolates. Set of primers was used to amplify fragment of hblD gene by PCR. For the detection of nheB gene a new primer set was developed which allowed to amplify 273 bp fragment from wide number of strains belonging to B. cereus group. The hblD gene was present in 7 out of 9 isolates whereas nheB gene occurred in all of them. Reference strains of B. cereus LOCK 0807, and B. thuringiensis NCAIM 01262 contained both genes. Strains of B. subtilis ATCC 6633 and B. pumilus LOCK 0814 do not contain both genes. Obtained results showed that B. thuringiensis NCAIM 01262 contains both genes and therefore may be harmful for human beings. PMID:17419288

  1. Identification of Eastern United States Reticulitermes Termite Species via PCR-RFLP, Assessed Using Training and Test Data

    PubMed Central

    Garrick, Ryan C.; Collins, Benjamin D.; Yi, Rachel N.; Dyer, Rodney J.; Hyseni, Chaz

    2015-01-01

    Reticulitermes termites play key roles in dead wood decomposition and nutrient cycling in forests. They also damage man-made structures, resulting in considerable economic loss. In the eastern United States, five species (R. flavipes, R. virginicus, R. nelsonae, R. hageni and R. malletei) have overlapping ranges and are difficult to distinguish morphologically. Here we present a molecular tool for species identification. It is based on polymerase chain reaction (PCR) amplification of a section of the mitochondrial cytochrome oxidase subunit II gene, followed by a three-enzyme restriction fragment length polymorphism (RFLP) assay, with banding patterns resolved via agarose gel electrophoresis. The assay was designed using a large set of training data obtained from a public DNA sequence database, then evaluated using an independent test panel of Reticulitermes from the Southern Appalachian Mountains, for which species assignments were determined via phylogenetic comparison to reference sequences. After refining the interpretive framework, the PCR-RFLP assay was shown to provide accurate identification of four co-occurring species (the fifth species, R. hageni, was absent from the test panel, so accuracy cannot yet be extended to training data). The assay is cost- and time-efficient, and will help improve knowledge of Reticulitermes species distributions. PMID:26463202

  2. Evaluation of two singleplex reverse transcription-Insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus.

    PubMed

    Zhang, Jianqiang; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chen, Qi; Zhang, Yan; Chiang, Cheng-Jen; Shen, Yu-Han; Li, Fu-Chun; Chang, Hsiao-Fen Grace; Gauger, Phillip C; Harmon, Karen M; Wang, Hwa-Tang Thomas

    2016-08-01

    Recent outbreaks of porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) in multiple countries have caused significant economic losses and remain a serious challenge to the swine industry. Rapid diagnosis is critical for the implementation of efficient control strategies before and during PEDV and PDCoV outbreaks. Insulated isothermal PCR (iiPCR) on the portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, a singleplex PEDV RT-iiPCR, a singleplex PDCoV RT-iiPCR, and a duplex PEDV/PDCoV real-time RT-PCR (rRT-PCR) commercial reagents targeting the M gene were compared to an N gene-based PEDV rRT-PCR and an M gene-based PDCoV rRT-PCR that were previously published and used as reference PCRs. All PCR assays were highly specific and did not cross react with other porcine enteric pathogens. Analytical sensitivities of the PEDV RT-iiPCR, PDCoV RT-iiPCR and duplex PEDV/PDCoV rRT-PCR were determined using in vitro transcribed RNA as well as viral RNA extracted from ten-fold serial dilutions of PEDV and PDCoV cell culture isolates. Performance of each PCR assay was further evaluated using 170 clinical samples (86 fecal swabs, 24 feces, 19 intestines, and 41 oral fluids). Compared to the reference PEDV rRT-PCR, the sensitivity, specificity and accuracy of the PEDV RT-iiPCR were 97.73%, 98.78%, and 98.24%, respectively, and those of the duplex PEDV/PDCoV rRT-PCR were 98.86%, 96.34%, and 97.65%, respectively. Compared to the reference PDCoV rRT-PCR, the sensitivity, specificity and accuracy of the PDCoV RT-iiPCR were 100%, 100%, and 100%, respectively, and those of the PEDV/PDCoV duplex rRT-PCR were 96.34%, 100%, and 98.24%, respectively. Overall, all three new PCR assays were comparable to the reference rRT-PCRs for detection of PEDV and/or PDCoV. The PEDV and PDCoV RT-iiPCRs are potentially useful tools for on-site detection and the duplex PEDV/PDCoV rRT-PCR provides a convenient method to simultaneously detect

  3. The development and testing of a qualitative instrument designed to assess critical thinking

    NASA Astrophysics Data System (ADS)

    Clauson, Cynthia Louisa

    This study examined a qualitative approach to assess critical thinking. An instrument was developed that incorporates an assessment process based on Dewey's (1933) concepts of self-reflection and critical thinking as problem solving. The study was designed to pilot test the critical thinking assessment process with writing samples collected from a heterogeneous group of students. The pilot test included two phases. Phase 1 was designed to determine the validity and inter-rater reliability of the instrument using two experts in critical thinking, problem solving, and literacy development. Validity of the instrument was addressed by requesting both experts to respond to ten questions in an interview. The inter-rater reliability was assessed by analyzing the consistency of the two experts' scorings of the 20 writing samples to each other, as well as to my scoring of the same 20 writing samples. Statistical analyses included the Spearman Rho and the Kuder-Richardson (Formula 20). Phase 2 was designed to determine the validity and reliability of the critical thinking assessment process with seven science teachers. Validity was addressed by requesting the teachers to respond to ten questions in a survey and interview. Inter-rater reliability was addressed by comparing the seven teachers' scoring of five writing samples with my scoring of the same five writing samples. Again, the Spearman Rho and the Kuder-Richardson (Formula 20) were used to determine the inter-rater reliability. The validity results suggest that the instrument is helpful as a guide for instruction and provides a systematic method to teach and assess critical thinking while problem solving with students in the classroom. The reliability results show the critical thinking assessment instrument to possess fairly high reliability when used by the experts, but weak reliability when used by classroom teachers. A major conclusion was drawn that teachers, as well as students, would need to receive instruction

  4. Performance of Galactomannan, Beta-d-Glucan, Aspergillus Lateral-Flow Device, Conventional Culture, and PCR Tests with Bronchoalveolar Lavage Fluid for Diagnosis of Invasive Pulmonary Aspergillosis

    PubMed Central

    Prattes, J.; Spiess, B.; Wagner, J.; Prueller, F.; Raggam, R. B.; Posch, V.; Duettmann, W.; Hoenigl, K.; Wölfler, A.; Koidl, C.; Buzina, W.; Reinwald, M.; Thornton, C. R.; Krause, R.

    2014-01-01

    Galactomannan detection in bronchoalveolar lavage (BAL) fluid samples (GM test) is currently considered the gold standard test for diagnosing invasive pulmonary aspergillosis (IPA). The limitations, however, are the various turnaround times and availability of testing. We compared the performance of GM testing with that of conventional culture, an Aspergillus lateral-flow-device (LFD) test, a beta-d-glucan (BDG) test, and an Aspergillus PCR assay by using BAL fluid samples from immunocompromised patients. A total of 78 BAL fluid samples from 78 patients at risk for IPA (74 samples from Graz and 4 from Mannheim) collected between December 2012 and May 2013 at two university hospitals in Austria and Germany were included. Three patients had proven IPA, 14 probable IPA, and 17 possible IPA, and 44 patients had no IPA. The diagnostic accuracies of the different methods for probable/proven IPA were evaluated. The diagnostic odds ratios were the highest for the GM, PCR, and LFD tests. The sensitivities for the four methods (except culture) were between 70 and 88%. The combination of the GM (cutoff optical density index [ODI], >1.0) and LFD tests increased the sensitivity to 94%, while the combination of the GM test (>1.0) and PCR resulted in 100% sensitivity (specificity for probable/proven IPA, 95 to 98%). The performance of conventional culture was limited by low sensitivity, while that of the BDG test was limited by low specificity. We evaluated established and novel diagnostic methods for IPA and found that the Aspergillus PCR, LFD, and GM tests were the most useful methods for diagnosing the disease by using BAL fluid samples. In particular, the combination of the GM test and PCR or, if PCR is not available, the LFD test, allows for sensitive and specific diagnosis of IPA. PMID:24671798

  5. Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study

    PubMed Central

    El Khattabi, Laïla Allach; Rouillac-Le Sciellour, Christelle; Le Tessier, Dominique; Luscan, Armelle; Coustier, Audrey; Porcher, Raphael; Bhouri, Rakia; Nectoux, Juliette; Sérazin, Valérie; Quibel, Thibaut; Mandelbrot, Laurent; Tsatsaris, Vassilis

    2016-01-01

    Objective NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. Method After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. Results The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample’s trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. Conclusion Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women. PMID:27167625

  6. Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR.

    PubMed

    Aldous, Wade K; Pounder, June I; Cloud, Joann L; Woods, Gail L

    2005-05-01

    Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5-3 h for the QIAGEN extraction. PMID:15872286

  7. Hybrid Capture 2 is as Effective as PCR Testing for High Risk Human Papillomavirus in Head and Neck Cancers

    PubMed Central

    Hooper, Jody E.; Hebert, Jessica F.; Schilling, Amy; Gross, Neil D.; Schindler, Joshua S.; Lagowski, James P.; Kulesz-Martin, Molly; Corless, Christopher L.; Morgan, Terry K.

    2014-01-01

    High risk human papillomavirus (HPV) infection is a common cause of oropharyngeal squamous cell carcinoma, especially in young male nonsmokers. Accurately diagnosing HPV-associated oral cancers is important, because they have a better prognosis and may be treated differently than smoking-related oral carcinomas. Various methods have been validated to test for high risk HPV in cervical tissue samples and they are in routine clinical use to detect dysplasia before it progresses to invasive disease. Similarly, future screening for HPV-mediated oropharyngeal dysplasia may identify patients before it progresses. Our objective was to compare four of these methods in a retrospective series of 87 oral and oropharyngeal squamous cell carcinomas that had archived fresh-frozen and paraffin-embedded tissue for evaluation. Patient age, gender, smoking history, and tumor location were also recorded. DNA prepared from fresh-frozen tissue was tested for HPV genotypes by multiplex PCR analysis (Diatherix), and high risk HPV screening was done with Hybrid Capture 2 (Qiagen hc2) and Cervista (Hologic). Histologic sections were immunostained for p16 (mtm/Roche). HPV positive outcome was defined as agreement between at least two of the three genetic tests and used for X2 analysis and calculations of diagnostic predictive value. As expected, high risk HPV-positive oral cancers were most common in the tonsil and base of tongue (oropharynx) of younger male (55 years vs 65 years) (p=0.0002) non-smokers (p=0.01). Most positive cases were HPV16 (33/36, 92%). Hybrid Capture 2 and Cervista were as sensitive as PCR and had fewer false positives than p16 immunohistochemistry. PMID:25839700

  8. Real-time PCR testing of pooled (1:5) fecal samples comparison to HEYM culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increased sample submission for laboratory testing for the detection of Mycobacterium subsp avium paratuberculosis (MAP) in bovine fecal has necessitated utilization of techniques to enhance efficiency of MAP detection. Pooling of fecal samples offers a method to enhance diagnostic efficiency when c...

  9. COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing.

    PubMed

    Castellanos-Rizaldos, Elena; Milbury, Coren A; Guha, Minakshi; Makrigiorgos, G Mike

    2014-01-01

    Detection of low-level mutations is important for cancer biomarker and therapy targets discovery, but reliable detection remains a technical challenge. The newly developed method of CO-amplification at Lower Denaturation temperature PCR (COLD-PCR) helps to circumvent this issue. This PCR-based technology preferentially enriches minor known or unknown variants present in samples with a high background of wild type DNA which often hampers the accurate identification of these minority alleles. This is a simple process that consists of lowering the temperature at the denaturation step during the PCR-cycling protocol (critical denaturation temperature, T c) and inducing DNA heteroduplexing during an intermediate step. COLD-PCR in its simplest forms does not need additional reagents or specific instrumentation and thus, can easily replace conventional PCR and at the same time improve the mutation detection sensitivity limit of downstream technologies. COLD-PCR can be applied in two basic formats: fast-COLD-PCR that can enrich T m-reducing mutations and full-COLD-PCR that can enrich all mutations, though it requires an intermediate cross-hybridization step that lengthens the thermocycling program. An improved version of full-COLD-PCR (improved and complete enrichment, ice-COLD-PCR) has also been described. Finally, most recently, we developed yet another form of COLD-PCR, temperature-tolerant-COLD-PCR, which gradually increases the denaturation temperature during the COLD-PCR reaction, enriching diverse targets using a single cycling program. This report describes practical considerations for application of fast-, full-, ice-, and temperature-tolerant-COLD-PCR for enrichment of mutations prior to downstream screening. PMID:24259002

  10. Qualitative Organic Analysis: An Efficient, Safer, and Economical Approach to Preliminary Tests and Functional Group Analysis

    ERIC Educational Resources Information Center

    Dhingra, Sunita; Angrish, Chetna

    2011-01-01

    Qualitative organic analysis of an unknown compound is an integral part of the university chemistry laboratory curriculum. This type of training is essential as students learn to approach a problem systematically and to interpret the results logically. However, considerable quantities of waste are generated by using conventional methods of…

  11. Using an interview guide to identify effective nurse managers. Phase 1, Qualitative tool construction and testing.

    PubMed

    Everson-Bates, S; Fosbinder, D

    1994-04-01

    Hiring effective nurse managers is key to the success of today's healthcare institutions. The authors describe the development of a pre-employment interview guide to assist in the selection process of nurse managers. Characteristics and competencies of effective nurse managers are delineated from the qualitative data. PMID:8151434

  12. The Fecal Virome of Children with Hand, Foot, and Mouth Disease that Tested PCR Negative for Pathogenic Enteroviruses

    PubMed Central

    Linsuwanon, Piyada; Poovorawan, Yong; Li, Linlin; Deng, Xutao; Vongpunsawad, Sompong; Delwart, Eric

    2015-01-01

    Hand, foot, and mouth disease (HFMD) affects infant and young children. A viral metagenomic approach was used to identify the eukaryotic viruses in fecal samples from 29 Thai children with clinical diagnosis of HFMD collected during the 2012 outbreak. These children had previously tested negative by PCR for enterovirus 71 and coxsackievirus A16 and A6. Deep sequencing revealed nine virus families: Picornaviridae, Astroviridae, Parvoviridae, Caliciviridae, Paramyxoviridae, Adenoviridae, Reoviridae, Picobirnaviridae, and Polyomaviridae. The highest number of viral sequences belonged to human rhinovirus C, astrovirus-MLB2, and coxsackievirus A21. Our study provides an overview of virus community and highlights a broad diversity of viruses found in feces from children with HFMD. PMID:26288145

  13. Barriers to Point-of-Care Testing in India: Results from Qualitative Research across Different Settings, Users and Major Diseases

    PubMed Central

    Engel, Nora; Ganesh, Gayatri; Patil, Mamata; Yellappa, Vijayashree; Pant Pai, Nitika; Vadnais, Caroline; Pai, Madhukar

    2015-01-01

    Background Successful point-of-care testing, namely ensuring the completion of the test and treat cycle in the same encounter, has immense potential to reduce diagnostic and treatment delays, and impact patient outcomes. However, having rapid tests is not enough, as many barriers may prevent their successful implementation in point-of-care testing programs. Qualitative research on diagnostic practices may help identify such barriers across different points of care in health systems. Methods In this exploratory qualitative study, we conducted 78 semi-structured interviews and 13 focus group discussions in an urban and rural area of Karnataka, India, with healthcare providers (doctors, nurses, specialists, traditional healers, and informal providers), patients, community health workers, test manufacturers, laboratory technicians, program managers and policy-makers. Participants were purposively sampled to represent settings of hospitals, peripheral labs, clinics, communities and homes, in both the public and private sectors. Results In the Indian context, the onus is on the patient to ensure successful point-of-care testing across homes, clinics, labs and hospitals, amidst uncoordinated providers with divergent and often competing practices, in settings lacking material, money and human resources. We identified three overarching themes affecting point-of-care testing: the main theme is ‘relationships’ among providers and between providers and patients, influenced by the cross-cutting theme of ‘infrastructure’. Challenges with both result in ‘modified practices’ often favouring empirical (symptomatic) treatment over treatment guided by testing. Conclusions Even if tests can be conducted on the spot and infrastructure challenges have been resolved, relationships among providers and between patients and providers are crucial for successful point-of-care testing. Furthermore, these barriers do not act in isolation, but are interlinked and need to be examined

  14. Concordance between RT-PCR-based detection of respiratory viruses from nasal swabs collected for viral testing and nasopharyngeal swabs collected for bacterial testing

    PubMed Central

    Grijalva, Carlos G.; Griffin, Marie R.; Edwards, Kathryn M.; Johnson, Monika; Gil, Ana I.; Verastegui, Héctor; Lanata, Claudio F.; Williams, John V.

    2014-01-01

    Background Epidemiologic studies of respiratory infections frequently rely on separate sample collections for the detection of bacteria and viruses. The requirement for two specimens presents cost, logistical, and acceptability challenges. Objectives To determine the agreement in detection of respiratory viruses using RT-PCR between two different types of samples collected on the same day: nasal swabs preserved in viral transport medium (NS) and nasopharyngeal swabs preserved in skim milk-tryptone-glucose-glycerol [STGG] medium (NP), the current standard for pneumococcal colonization studies. Study design Paired NS and NP samples were collected between May 2009 and September 2011 as part of the RESPIRA-PERU study, a large prospective cohort of Andean children <3 years of age. NS samples used polyester swabs and viral transport medium whereas NP samples used rayon wire-handled swabs and STGG medium. Samples were tested for influenza, human metapneumovirus (MPV), respiratory syncytial virus (RSV), human rhinovirus (HRV), parainfluenza virus 3 (PIV3) and adenovirus (ADV) using real-time RT-PCR. We calculated the agreement, and compared cycle thresholds (CT) between NP and NS samples. Results Among 226 paired NP-NS samples, we observed very high agreement with a Kappa statistic ranging from 0.71 for ADV to 0.97 for MPV. CT values were similar for both strategies. Conclusions NP samples preserved in STGG provide a simple and reliable strategy for identification of both pneumococcus and respiratory viruses. This single specimen collection strategy could be used for epidemiologic studies, especially in resource-limited settings. Furthermore, archived NP-STGG specimens from previous studies could be reliably tested by RT-PCR for viruses. PMID:24875136

  15. Cross-Institute Evaluations of Inhibitor-Resistant PCR Reagents for Direct Testing of Aerosol and Blood Samples Containing Biological Warfare Agent DNA

    PubMed Central

    Minogue, Timothy D.; Rachwal, Phillip A.; Trombley Hall, Adrienne; Koehler, Jeffery W.

    2014-01-01

    Rapid pathogen detection is crucial for the timely introduction of therapeutics. Two groups (one in the United Kingdom and one in the United States) independently evaluated inhibitor-resistant PCR reagents for the direct testing of substrates. In the United Kingdom, a multiplexed Bacillus anthracis (target) and Bacillus subtilis (internal-control) PCR was used to evaluate 4 reagents against 5 PCR inhibitors and down-selected the TaqMan Fast Virus 1-Step master mix (Life Technologies Inc.). In the United States, four real-time PCR assays (targeting B. anthracis, Brucella melitensis, Venezuelan equine encephalitis virus [VEEV], and Orthopoxvirus spp.) were used to evaluate 5 reagents (plus the Fast Virus master mix) against buffer, blood, and soil samples and down-selected the KAPA Blood Direct master mix (KAPA Biosystems Inc.) with added Platinum Taq (Life Technologies). The down-selected reagents underwent further testing. In the United Kingdom experiments, both reagents were tested against seven contrived aerosol collector samples containing B. anthracis Ames DNA and B. subtilis spores from a commercial formulation (BioBall). In PCR assays with reaction mixtures containing 40% crude sample, an airfield-collected sample induced inhibition of the B. subtilis PCR with the KAPA reagent and complete failure of both PCRs with the Fast Virus reagent. However, both reagents allowed successful PCR for all other samples—which inhibited PCRs with a non-inhibitor-resistant reagent. In the United States, a cross-assay limit-of-detection (LoD) study in blood was conducted. The KAPA Blood Direct reagent allowed the detection of agent DNA (by four PCRs) at higher concentrations of blood in the reaction mixture (2.5%) than the Fast Virus reagent (0.5%), although LoDs differed between assays and reagent combinations. Across both groups, the KAPA Blood Direct reagent was determined to be the optimal reagent for inhibition relief in PCR. PMID:24334660

  16. The silicon-glass microreactor with embedded sensors—technology and results of preliminary qualitative tests, toward intelligent microreaction plant

    NASA Astrophysics Data System (ADS)

    Knapkiewicz, P.

    2013-03-01

    The technology and preliminary qualitative tests of silicon-glass microreactors with embedded pressure and temperature sensors are presented. The concept of microreactors for leading highly exothermic reactions, e.g. nitration of hydrocarbons, and design process-included computer-aided simulations are described in detail. The silicon-glass microreactor chip consisting of two micromixers (multistream micromixer), reaction channels, cooling/heating chambers has been proposed. The microreactor chip was equipped with a set of pressure and temperature sensors and packaged. Tests of mixing quality, pressure drops in channels, heat exchange efficiency and dynamic behavior of pressure and temperature sensors were documented. Finally, two applications were described.

  17. Mitochondrial D-loop {open_quotes}signatures{close_quotes} produced by low-stringency single specific primer PCR constitute a simple comparative human identity test

    SciTech Connect

    Barreto, G.; Vago, A.R.; Pena, S.D.J.

    1996-03-01

    We have developed a technique called {open_quotes}LSSP-PCR{close_quotes} (low-stringency single specific primer PCR) that detects single or multiple mutations in DNA. A purified DNA fragment is submitted to PCR by using a single primer specific for one of the extremities of the fragment, under conditions of very low stringency. The primer hybridizes specifically to its complementary extremity and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner. A complex set of reaction products is thus created that, when separated by electrophoresis, constitutes a unique {open_quotes}gene signature.{close_quotes} We here report the application of LSSP-PCR to the detection of sequence variation in the control (D-loop) region of human mtDNA, which is known to differ significantly between unrelated individuals. We prepared human DNA samples from blood and amplified a 1,024-bp portion of the mtDNA control region, using primers L15996 and H408. The amplified mtDNA fragments were then reamplified under LSSP-PCR conditions by using L15996 or H408 as drivers to produce complex signatures that always differed between unrelated individuals and yet were highly reproducible. In contrast, all mother-child pairs tested were identical, as expected from the matrilineal inheritance of mtDNA. Thus, the use of LSSP-PCR to produce D-loop signatures constitutes a powerful new technique for mtDNA-based comparative identity testing. 18 refs., 7 figs.

  18. Performance of Roche CAP/CTM HIV-1 qualitative test version 2.0 using dried blood spots for early infant diagnosis.

    PubMed

    Gueye, Sokhna Bousso; Diop-Ndiaye, Halimatou; Diallo, Mamadou Malick; Ly, Omar; Sow-Ndoye, Aissatou; Diagne-Gueye, Ndèye Diabou; Kébé-Fall, Khady; Diop, Fatou; Gaye-Diallo, Aïssatou; Belec, Laurent; Mboup, Souleymane; Touré-Kane, Coumba

    2016-03-01

    In the context of early infant diagnosis (EID) decentralization in sub-Saharan Africa, dried blood spot (DBS) is now widely used for HIV proviral DNA detection in resource-limited settings. A new version of CAP/CTM (version 2) has been introduced, recently by Roche Diagnosis as a new real-time PCR assay to replace previous technologies on qualitative detection of HIV-1 DNA using whole blood and DBS samples. The objective of this study was to evaluate CAP/CTM version 2 compared to CAP/CTM version 1 and Amplicor on DBS. A total of 261 DBS were collected from children aged 4 weeks to 17 months born from HIV-seropositive mothers and tested by the three techniques. CAP/CTM version 2 showed 100% of agreement with Amplicor including 74 positive results and 187 negative results. CAP/CTM version 2 versus CAP/CTM version 1 as well as CAP/CTM version 1 versus Amplicor showed two discordant results giving a sensitivity of 98.6%, specificity of 99.5%, positive predictive value of 98.6% and negative predictive value of 99.5%. The concordance was 99.12% (95% of confidence interval) giving a Kappa coefficient of 0.97 (p<0.001). These findings confirmed the expected good performance of CAP/CTM version 2 for HIV-1 EID. PMID:26706730

  19. Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing

    PubMed Central

    Waggoner, Jesse J.; Balassiano, Ilana; Mohamed-Hadley, Alisha; Vital-Brazil, Juliana Magalhães; Sahoo, Malaya K.; Pinsky, Benjamin A.

    2015-01-01

    Background Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens. Methods/Principal Findings 478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%). Conclusions/Significance This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize

  20. Comparison of a Real-Time PCR Method with Serology and Blood Smear Analysis for Diagnosis of Human Anaplasmosis: Importance of Infection Time Course for Optimal Test Utilization

    PubMed Central

    Meece, J. K.; Ivacic, L. C.; Bertz, P. D.; Zhang, K.; Weiler, T.; Uphoff, T. S.; Fritsche, T. R.

    2013-01-01

    Anaplasmosis and ehrlichiosis are emerging tick-borne diseases with clinically similar presentations caused by closely related pathogens. Currently, laboratories rely predominantly on blood smear analysis (for the detection of intracellular morulae) and on serologic tests, both of which have recognized limitations, for diagnostic purposes. We compared the performance of a published real-time PCR assay that incorporates melt curve analysis to differentiate Anaplasma and Ehrlichia species with blood smear and serologic methods in an upper Midwest population. Overall, 38.5% of the specimens selected for evaluation had one or more tests that were positive for anaplasmosis. The PCR positivity for all specimens was maximal (21.2%; 29/137) during the early acute phase of illness (0 to 4 days since illness onset) and significantly less frequent (11.5%; 20/174) during later phases (>4 days since illness onset). All positive specimens were Anaplasma phagocytophilum; no Ehrlichia species were identified. The real-time PCR detected 100% of infections that were detected by blood smear analysis (14/14) and broadened the detection window from a maximum of 14 days for smear positivity to 30 days for PCR. Additional infections were detected by real-time PCR in 12.9% (11/85) of smear-negative patients. There was poor agreement between the real-time PCR assay and serologic test results: 19.8% (19/96) and 13.7% (29/212) of seropositive and -negative patients, respectively, were PCR positive. Seropositivity increased with increasing days of illness, demonstrating that serologic detection methods are best utilized during presumed convalescence. Our results indicate that the optimal performance and utilization of laboratory tests for the diagnosis of anaplasmosis require knowledge regarding time of symptom onset or days of illness. PMID:23637292

  1. A proof of concept study to assess the potential of PCR testing to detect natural Mycobacterium bovis infection in South American camelids

    PubMed Central

    2014-01-01

    Background Cases of Mycobacterium bovis infection South American camelids have been increasing in Great Britain. Current antemortem immunological tests have some limitations. Cases at post mortem examination frequently show extensive pathology. The feasibility of detecting Mycobacterium bovis DNA in clinical samples was investigated. Findings A sensitive extraction methodology was developed and used on nasal swabs and faeces taken post-mortem to assess the potential for a PCR test to detect Mycobacterium bovis in clinical samples. The gross pathology of the studied South American camelids was scored and a significantly greater proportion of South American camelids with more severe pathology were positive in both the nasal swab and faecal PCR tests. A combination of the nasal swab and faecal PCR tests detected 63.9% of all the South American camelids with pathology that were tested. Conclusions The results suggest that antemortem diagnosis of Mycobacterium bovis in South American camelids may be possible using a PCR test on clinical samples, however more work is required to determine sensitivity and specificity, and the practicalities of applying the test in the field. PMID:24507471

  2. A systematic review of qualitative findings on factors enabling and deterring uptake of HIV testing in Sub-Saharan Africa

    PubMed Central

    2013-01-01

    Background Despite Sub-Saharan Africa (SSA) being the epicenter of the HIV epidemic, uptake of HIV testing is not optimal. While qualitative studies have been undertaken to investigate factors influencing uptake of HIV testing, systematic reviews to provide a more comprehensive understanding are lacking. Methods Using Noblit and Hare’s meta-ethnography method, we synthesised published qualitative research to understand factors enabling and deterring uptake of HIV testing in SSA. We identified 5,686 citations out of which 56 were selected for full text review and synthesised 42 papers from 13 countries using Malpass’ notion of first-, second-, and third-order constructs. Results The predominant factors enabling uptake of HIV testing are deterioration of physical health and/or death of sexual partner or child. The roll-out of various HIV testing initiatives such as ‘opt-out’ provider-initiated HIV testing and mobile HIV testing has improved uptake of HIV testing by being conveniently available and attenuating fear of HIV-related stigma and financial costs. Other enabling factors are availability of treatment and social network influence and support. Major barriers to uptake of HIV testing comprise perceived low risk of HIV infection, perceived health workers’ inability to maintain confidentiality and fear of HIV-related stigma. While the increasingly wider availability of life-saving treatment in SSA is an incentive to test, the perceived psychological burden of living with HIV inhibits uptake of HIV testing. Other barriers are direct and indirect financial costs of accessing HIV testing, and gender inequality which undermines women’s decision making autonomy about HIV testing. Despite differences across SSA, the findings suggest comparable factors influencing HIV testing. Conclusions Improving uptake of HIV testing requires addressing perception of low risk of HIV infection and perceived inability to live with HIV. There is also a need to continue

  3. Latent class analysis of bulk tank milk PCR and ELISA testing for herd level diagnosis of Mycoplasma bovis.

    PubMed

    Nielsen, Per Kantsø; Petersen, Mette Bisgaard; Nielsen, Liza Rosenbaum; Halasa, Tariq; Toft, Nils

    2015-10-01

    The bacterium Mycoplasma bovis causes disease in cattle of all ages. An apparent increase in the occurrence of M. bovis associated outbreaks among Danish dairy cattle herds since 2011 has prompted a need for knowledge regarding herd-level diagnostic performance. Therefore, the objective of this study was to evaluate the herd-level diagnostic performance of an indirect ELISA test by comparison to a real-time PCR test when diagnosing M. bovis in cattle herds of bulk tank milk. Bulk tank milk samples from Danish dairy herds (N=3437) were analysed with both the antibody detecting BIO K 302 M. bovis ELISA kit and the antigen detecting PathoProof Mastitis Major-3 kit. As none of these are considered a gold standard test for herd-level diagnostics we applied a series of Bayesian latent class analyses for a range of ELISA cut-off values. The negative and positive predictive values were calculated for hypothetical true national prevalences (1, 5, 10, 15 and 20%) of infected herds. We estimated that the ELISA test had a median sensitivity and specificity of 60.4 [37.5-96.2 95% Posterior Credibility Interval] and 97.3 [94.0-99.8 95% PCI] at the currently recommended cut-off (37% Optical density Coefficient). These changed to 43.5 [21.1-92.5 95% PCI] and 99.6 [98.8-100 95% PCI] if the cut-off was increased to 50 ODC%. In addition, herd-level diagnosis by ELISA would result in fewer false positives at a cut-off value of 50 ODC% compared to 37 ODC% without compromising the negative predictive value. PMID:26342789

  4. Stability Test and Quantitative and Qualitative Analyses of the Amino Acids in Pharmacopuncture Extracted from Scolopendra subspinipes mutilans

    PubMed Central

    Cho, GyeYoon; Han, KyuChul; Yoon, JinYoung

    2015-01-01

    Objectives: Scolopendra subspinipes mutilans (S. subspinipes mutilans) is known as a traditional medicine and includes various amino acids, peptides and proteins. The amino acids in the pharmacopuncture extracted from S. subspinipes mutilans by using derivatization methods were analyzed quantitatively and qualitatively by using high performance liquid chromatography (HPLC) over a 12 month period to confirm its stability. Methods: Amino acids of pharmacopuncture extracted from S. subspinipes mutilans were derived by using O-phthaldialdehyde (OPA) & 9-fluorenyl methoxy carbonyl chloride (FMOC) reagent and were analyzed using HPLC. The amino acids were detected by using a diode array detector (DAD) and a fluorescence detector (FLD) to compare a mixed amino acid standard (STD) to the pharmacopuncture from centipedes. The stability tests on the pharmacopuncture from centipedes were done using HPLC for three conditions: a room temperature test chamber, an acceleration test chamber, and a cold test chamber. Results: The pharmacopuncture from centipedes was prepared by using the method of the Korean Pharmacopuncture Institute (KPI) and through quantitative analyses was shown to contain 9 amino acids of the 16 amino acids in the mixed amino acid STD. The amounts of the amino acids in the pharmacopuncture from centipedes were 34.37 ppm of aspartate, 123.72 ppm of arginine, 170.63 ppm of alanine, 59.55 ppm of leucine and 57 ppm of lysine. The relative standard deviation (RSD %) results for the pharmacopuncture from centipedes had a maximum value of 14.95% and minimum value of 1.795% on the room temperature test chamber, the acceleration test chamber and the cold test chamber stability tests. Conclusion: Stability tests on and quantitative and qualitative analyses of the amino acids in the pharmacopuncture extracted from centipedes by using derivatization methods were performed by using HPLC. Through research, we hope to determine the relationship between time and the

  5. When Are Qualitative Testing Results Sufficient To Predict a Reduction in Illnesses in a Microbiological Food Safety Risk Assessment?

    PubMed

    Ebel, Eric D; Williams, Michael S

    2015-08-01

    Process models that include the myriad pathways that pathogen-contaminated food may traverse before consumption and the dose-response function to relate exposure to likelihood of illness may represent a "gold standard" for quantitative microbial risk assessment. Nevertheless, simplifications that rely on measuring the change in contamination occurrence of a raw food at the end of production may provide reasonable approximations of the effects measured by a process model. In this study, we parameterized three process models representing different product-pathogen pairs (i.e., chicken-Salmonella, chicken-Campylobacter, and beef-E. coli O157:H7) to compare with predictions based on qualitative testing of the raw product before consideration of mixing, partitioning, growth, attenuation, or dose-response processes. The results reveal that reductions in prevalence generated from qualitative testing of raw finished product usually underestimate the reduction in likelihood of illness for a population of consumers. Qualitative microbial testing results depend on the test's limit of detection. The negative bias is greater for limits of detection that are closer to the center of the contamination distribution and becomes less as the limit of detection is moved further into the right tail of the distribution. Nevertheless, a positive bias can result when the limit of detection refers to very high contamination levels. Changes in these high levels translate to larger consumed doses for which the slope of the dose-response function is smaller compared with the larger slope associated with smaller doses. Consequently, in these cases, a proportional reduction in prevalence of contamination results in a less than proportional reduction in probability of illness. The magnitudes of the biases are generally less for nonscalar (versus scalar) adjustments to the distribution. PMID:26219357

  6. A Qualitative Study of Barriers to the Utilization of HIV Testing Services Among Rural African American Cocaine Users

    PubMed Central

    Wright, Patricia B.; Stewart, Katharine E.; Curran, Geoffrey M.; Booth, Brenda M.

    2013-01-01

    This qualitative study is about barriers to the utilization of HIV testing as perceived by African Americans who have recently used cocaine and who live in the rural Delta region of Arkansas. Affordability, physical accessibility, and geographic availability were not perceived as barriers to HIV testing in this sample, yet acceptability was still perceived as poor. Acceptability due to social mores and norms was a major barrier. Many said testing was unacceptable because of fear of social costs. Many were confident of being HIV-negative based on risky assumptions about testing and the notification process. Small-town social and sexual networks added to concerns about reputation and risk. System approaches may fail if they focus solely on improving access to HIV services but do not take into consideration deeply internalized experiences of rural African Americans as well as involvement of the community in developing programs and services. PMID:24039279

  7. A Qualitative Study of Barriers to the Utilization of HIV Testing Services Among Rural African American Cocaine Users.

    PubMed

    Wright, Patricia B; Stewart, Katharine E; Curran, Geoffrey M; Booth, Brenda M

    2013-07-01

    This qualitative study is about barriers to the utilization of HIV testing as perceived by African Americans who have recently used cocaine and who live in the rural Delta region of Arkansas. Affordability, physical accessibility, and geographic availability were not perceived as barriers to HIV testing in this sample, yet acceptability was still perceived as poor. Acceptability due to social mores and norms was a major barrier. Many said testing was unacceptable because of fear of social costs. Many were confident of being HIV-negative based on risky assumptions about testing and the notification process. Small-town social and sexual networks added to concerns about reputation and risk. System approaches may fail if they focus solely on improving access to HIV services but do not take into consideration deeply internalized experiences of rural African Americans as well as involvement of the community in developing programs and services. PMID:24039279

  8. A lottery incentive system to facilitate dialogue and social support for workplace HIV counselling and testing: A qualitative inquiry

    PubMed Central

    Weihs, Martin; Meyer-Weitz, Anna

    2014-01-01

    Abstract Despite South African mid-sized companies' efforts to offer HIV counselling and testing (HCT) in the workplace, companies report relatively poor uptake rates. An urgent need for a range of different interventions aimed at increasing participation in workplace HCT has been identified. The aim of this study was to explore qualitatively the influence of a lottery incentive system (LIS) as an intervention to influence shop-floor workers' workplace HIV testing behaviour. A qualitative study was conducted among 17 shop-floor workers via convenience sampling in two mid-sized South African automotive manufacturing companies in which an LIS for HCT was implemented. The in-depth interviews employed a semi-structured interview schedule and thematic analysis was used to analyse the data. The interviews revealed that the LIS created excitement in the companies and renewed employees' personal interest in HCT. The excitement facilitated social interactions that resulted in a strong group cohesion pertaining to HCT that mitigated the burden of HIV stigma in the workplace. Open discussions allowed for the development of supportive social group pressure to seek HCT as a collective in anticipation of a reward. Lotteries were perceived as a supportive and innovative company approach to workplace HCT. The study identified important aspects for consideration by companies when using an LIS to enhance workplace HIV testing. The significance of inter- and intra-player dialogue in activating supportive social norms for HIV testing in collectivist African contexts was highlighted. PMID:25023208

  9. A lottery incentive system to facilitate dialogue and social support for workplace HIV counselling and testing: a qualitative inquiry.

    PubMed

    Weihs, Martin; Meyer-Weitz, Anna

    2014-01-01

    Despite South African mid-sized companies' efforts to offer HIV counselling and testing (HCT) in the workplace, companies report relatively poor uptake rates. An urgent need for a range of different interventions aimed at increasing participation in workplace HCT has been identified. The aim of this study was to explore qualitatively the influence of a lottery incentive system (LIS) as an intervention to influence shop-floor workers' workplace HIV testing behaviour. A qualitative study was conducted among 17 shop-floor workers via convenience sampling in two mid-sized South African automotive manufacturing companies in which an LIS for HCT was implemented. The in-depth interviews employed a semi-structured interview schedule and thematic analysis was used to analyse the data. The interviews revealed that the LIS created excitement in the companies and renewed employees' personal interest in HCT. The excitement facilitated social interactions that resulted in a strong group cohesion pertaining to HCT that mitigated the burden of HIV stigma in the workplace. Open discussions allowed for the development of supportive social group pressure to seek HCT as a collective in anticipation of a reward. Lotteries were perceived as a supportive and innovative company approach to workplace HCT. The study identified important aspects for consideration by companies when using an LIS to enhance workplace HIV testing. The significance of inter- and intra-player dialogue in activating supportive social norms for HIV testing in collectivist African contexts was highlighted. PMID:25023208

  10. Real-time PCR Tests in Dutch Exotic Mosquito Surveys; Implementation of Aedes aegypti and Aedes albopictus Identification Tests, and the Development of Tests for the Identification of Aedes atropalpus and Aedes japonicus japonicus (Diptera: Culicidae).

    PubMed

    van de Vossenberg, B T L H; Ibáñez-Justicia, A; Metz-Verschure, E; van Veen, E J; Bruil-Dieters, M L; Scholte, E J

    2015-05-01

    Since 2009, The Netherlands Food and Consumer Product Safety Authority carries out surveys focusing on, amongst others, the presence of invasive mosquito species (IMS). Special attention is given to exotic container-breeding Aedes species Aedes aegypti (L.), Aedes albopictus (Skuse), Aedes atropalpus (Coquillett), and Aedes japonicus japonicus (Theobald). This study describes the implementation of real-time PCR tests described by Hill et al. (2008) for the identification of Ae. aegypti and Ae. albopictus, and the development of two novel real-time PCR tests for the identification of Ae. atropalpus and Ae. j. japonicus. Initial test showed that optimization of elements of the Ae. aegypti and Ae. albopictus tests was needed. Method validation tests were performed to determine if the implemented and newly developed tests are fit for routine diagnostics. Performance criteria of analytical sensitivity, analytical specificity, selectivity, repeatability, and reproducibility were determined. In addition, experiments were performed to determine the influence of environmental conditions on the usability of DNA extracted from mosquito specimens trapped in BG-Sentinel traps. The real-time PCR tests were demonstrated to be sensitive, specific, repeatable, reproducible, and are less prone to false negative results compared to partial cytochrome c oxidase I gene sequencing owing to the DNA fragmentation caused by environmental influences. PMID:26334807

  11. Analysis of the Reasons for Non-Uptake of Predictive Testing for Huntington's Disease in Spain: A Qualitative Study.

    PubMed

    Rivera-Navarro, Jesús; Cubo, Esther; Mariscal, Natividad

    2015-12-01

    Children of persons affected by Huntington's disease (HD) have a 50% chance of inheriting the disease. Genetic testing in Spain is offered to individuals (presymptomatic test) or mothers of fetuses (prenatal) who run the risk of suffering from HD. The objective of this study is to analyze the factors that influence the decisions of adult children of persons affected with HD regarding predictive testing. A qualitative research methodology was used involving 4 focus groups (FGs) made up of adult children of persons with HD in different cities in Spain. The results of the study showed that over half of the focus group participants were inclined to decline genetic testing. The main explanatory determinants for taking or not taking the predictive test are: Maturity of the individual at risk, which was directly related to age; Ability to cope with a positive test result; Experience of living with HD sufferers; Information about testing and psychological support; Attitude of the family; Social visibility of genetic testing; Personality and temperament of each subject at risk of HD. These results imply that these factors should be analyzed in more detail in quantitative studies in order to help the Spanish Department of Health understand why some children of parents with HD decline genetic testing, so that they may and apply these data when creating specific policy regarding this issue. PMID:25921556

  12. The influence of antiretroviral treatment on willingness to test: a qualitative study in rural KwaZulu-Natal, South Africa.

    PubMed

    Phakathi, Zipho; Van Rooyen, Heidi; Fritz, Katherine; Richter, Linda

    2011-06-01

    Previous quantitative studies suggest a mutually reinforcing relationship between HIV counselling and testing (HCT) and antiretroviral treatment (ART). HCT is the entry into ART, and access to ART appears to increase HIV-testing uptake in settings with historically low uptake. Adopting a qualitative approach, this study examined the influence of ART on willingness to test for HIV, in a rural community in South Africa. Ninety-six in-depth interviews from a large community-based HIV-prevention trial were analysed. The data provide insight into the community members' views, perceptions and experiences regarding ART, and how they draw on these in making decisions about HIV testing. Several key factors that supported a positive relationship between ART and HIV testing were noted. These included the beliefs that ART brings hope and that it prolongs life; the powerful positive effect of witnessing the recovery of someone on treatment; and that ART encourages early HIV-testing behaviour. A few negative factors that could potentially weaken the effects of this positive relationship between ART and HCT uptake were the disclosure difficulties experienced by those enrolled in treatment, beliefs that ART does not cure HIV disease, and the travel distance to testing and treatment facilities from where people live and work. HIV/AIDS-service providers and programme planners should actively draw on these observations, to encourage increased HIV testing in communities and to ensure that the maximum number of people get the HIV treatment and care services that they require. PMID:25859740

  13. Men "missing" from population-based HIV testing: insights from qualitative research.

    PubMed

    Camlin, Carol S; Ssemmondo, Emmanuel; Chamie, Gabriel; El Ayadi, Alison M; Kwarisiima, Dalsone; Sang, Norton; Kabami, Jane; Charlebois, Edwin; Petersen, Maya; Clark, Tamara D; Bukusi, Elizabeth A; Cohen, Craig R; R Kamya, Moses; Havlir, Diane

    2016-01-01

    Men's uptake of HIV testing is critical to the success of "test and treat" strategies in generalized epidemics. This study sought to identify cultural factors and community processes that influence men's HIV testing uptake in the baseline year of an ongoing test-and-treat trial among 334,479 persons in eastern Africa (SEARCH, NCT#01864603). Data were collected using participant observation at mobile community health campaigns (CHCs) (n = 28); focus group discussions (n = 8 groups) with CHC participants; and in-depth interviews with care providers (n = 50), leaders (n = 32), and members (n = 112) of eight communities in Kenya and Uganda. An 8-person research team defined analytical codes and iteratively refined them during data collection using grounded theoretical approaches, and textual data were coded using Atlas.ti software. Structural and cultural barriers, including men's mobility and gender norms valorizing risk-taking and discouraging health-seeking behavior, were observed, and contributed to men's lower participation in HIV testing relative to women. Men's labor opportunities often require extended absences from households: during planting season, men guarded fields from monkeys from dawn until nightfall; lake fishermen traveled long distances and circulated between beaches. Men often tested "by proxy", believing their wives' HIV test results to be their status. Debates about HIV risks were vigorous, with many men questioning "traditional" masculine gender norms that enhanced risks. The promise of antiretroviral therapy (ART) to prolong health was a motivating factor for many men to participate in testing. Flexibility in operating hours of HIV testing including late evening and weekend times along with multiple convenient locations that moved were cited as facilitating factors enhancing male participating in HIV testing. Mobile testing reduced but did not eliminate barriers to men's participation in a large-scale "test & treat" effort

  14. The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety and quality assurance in the beef industry. The Biocontrol GDS and the DuPont Qualicon BAX®-RT rapid detection systems are two commercial tests based on...

  15. Analytical validation of a real-time RT-PCR test for Pan-American lineage H7 subtype avian influenza viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid detection of avian influenza virus and identification of the H5 and H7 hemagglutinin subtypes some of which are associated with high pathogenicity in poultry is critical for clinical diagnosis and wild bird monitoring programs. A real-time RT-PCR test for identification of the H7 subtype in N...

  16. Instructions for additional qualitative scoring of the initial-letter Word-association Test.

    PubMed

    Zivković, M

    1994-04-01

    An additional scoring method is based on grouping test-words according to whether the same sign is given by subjects to the test-words. In this way five test-word categories are formed, Eros (test-words with double plus signs), demi-Eros (single plus sign), demi-Thanatos (single minus), Thanatos (double minus), and Deviant (+/- and theta signs). The next step in scoring is to count the number of test-words in a given scoring category whose meanings do not conform. The greater the discrepancy between the test-word category and its meaning, the less well adapted is the subject. Several illustrative protocols are discussed. PMID:8022674

  17. Using Rapid Diagnostic Tests as a Source of Viral RNA for Dengue Serotyping by RT-PCR - A Novel Epidemiological Tool

    PubMed Central

    Vongsouvath, Manivanh; Phommasone, Koukeo; Sengvilaipaseuth, Onanong; Kosoltanapiwat, Nathamon; Chantratita, Narisara; Blacksell, Stuart D.; Lee, Sue J.; de Lamballerie, Xavier; Mayxay, Mayfong; Keomany, Sommay; Newton, Paul N.; Dubot-Pérès, Audrey

    2016-01-01

    Background Dengue virus infection causes major public health problems in tropical and subtropical areas. In many endemic areas, including the Lao PDR, inadequate access to laboratory facilities is a major obstacle to surveillance and study of dengue epidemiology. Filter paper is widely used for blood collection for subsequent laboratory testing for antibody and nucleic acid detection. For the first time, we demonstrate that dengue viral RNA can be extracted from dengue rapid diagnostic tests (RDT) and then submitted to real-time RT-PCR for serotyping. Methodology/Principal Findings We evaluated the Standard Diagnostics (SD) Bioline Dengue Duo RDT, a commonly used test in dengue endemic areas. First, using the QIAamp RNA kit, dengue RNA was purified from the sample pad of the NS1 RDT loaded with virus isolates of the four serotypes, then quantified by RT-PCR. We observed greater recovery of virus, with a mean of 27 times more RNA recovered from RDT, than from filter paper. Second, we evaluated dengue NS1 RDTs from patients at Mahosot Hospital, Vientiane, (99 patients) and from rural Salavan Provincial Hospital (362 patients). There was good agreement between dengue RT-PCR from NS1 RDT with RT-PCR performed on RNA extracted from patient sera, either using RDT loaded with blood (82.8% and 91.4%, in Vientiane and Salavan, respectively) or serum (91.9% and 93.9%). There was 100% concordance between RDT and serum RT-PCR of infecting dengue serotype. Conclusions/Significance Therefore, the collection of NS1 positive RDTs, which do not require cold storage, may be a novel approach for dengue serotyping by RT-PCR and offers promising prospects for the collection of epidemiological data from previously inaccessible tropical areas to aid surveillance and public health interventions. PMID:27159058

  18. Women's understanding of abnormal cervical smear test results: a qualitative interview study.

    PubMed Central

    Kavanagh, A. M.; Broom, D. H.

    1997-01-01

    OBJECTIVE: To describe how women interpret their experiences of diagnosis and treatment of a cervical abnormality and how healthcare services for such women can be improved. DESIGN: Qualitative study using detailed individual interviews. SETTING: Australian gynaecology clinics. SUBJECTS: 29 Women who had a cervical cytological abnormality and who attended a gynaecologist. MAIN OUTCOME MEASURES: Women's views on their diagnosis and their information needs. RESULTS: Most women wanted to participate in decisions about their care but found it difficult to get the information they required from doctors because they were confused by what their doctors told them and felt unable to ask questions in the consultation. Medical terms such as wart virus and precancer were difficult to understand. Not being able to see their cervix also made it hard for women to understand what their abnormality meant and what treatment entailed. Most women tried to make sense of their abnormality in the context of their everyday lives. For some women their gynaecological care was not consistent with the way they understood their abnormality. CONCLUSIONS: The inherent power structure of medical practice combined with time pressures often make it difficult for doctors to give the detailed information and reassurance patients need when a diagnosis is distressing or when investigation and treatment are strange and upsetting. PMID:9161314

  19. Bayesian estimation of test characteristics of real-time PCR, bacteriological culture and California mastitis test for diagnosis of intramammary infections with Staphylococcus aureus in dairy cattle at routine milk recordings.

    PubMed

    Mahmmod, Yasser S; Toft, Nils; Katholm, Jørgen; Grønbæk, Carsten; Klaas, Ilka C

    2013-11-01

    Danish farmers can order a real-time PCR mastitis diagnostic test on routinely taken cow-level samples from milk recordings. Validation of its performance in comparison to conventional mastitis diagnostics under field conditions is essential for efficient control of intramammary infections (IMI) with Staphylococcus aureus (S. aureus). Therefore, the objective of this study was to estimate the sensitivity (Se) and specificity (Sp) of real-time PCR, bacterial culture (BC) and California mastitis test (CMT) for the diagnosis of the naturally occurring IMI with S. aureus in routinely collected milk samples using latent class analysis (LCA) to avoid the assumption of a perfect reference test. Using systematic random sampling, a total of 609 lactating dairy cows were selected from 6 dairy herds with bulk tank milk PCR cycle threshold (Ct) value ≤39 for S. aureus. At routine milk recordings, automatically obtained cow-level (composite) milk samples were analyzed by PCR and at the same milking, 2436 quarter milk samples were collected aseptically for BC and CMT. Results showed that 140 cows (23%) were positive for S. aureus IMI by BC while 170 cows (28%) were positive by PCR. Estimates of Se and Sp for PCR were higher than test estimates of BC and CMT. SeCMT was higher than SeBC however, SpBC was higher than SpCMT. SePCR was 91%, while SeBC was 53%, and SeCMT was 61%. SpPCR was 99%, while SpBC was 89%, and SpCMT was 65%. In conclusion, PCR has a higher performance than the conventional diagnostic tests (BC and CMT) suggesting its usefulness as a routine test for accurate diagnosis of S. aureus IMI from dairy cows at routine milk recordings. The use of LCA provided estimates of the test characteristics for two currently diagnostic tests (BC, CMT) and a novel technique (real-time PCR) for diagnosing S. aureus IMI under field conditions at routine milk recordings in Denmark. PMID:23992955

  20. Evaluation of a Rapid Optical Immunoassay for Influenza Viruses (FLU OIA Test) in Comparison with Cell Culture and Reverse Transcription-PCR

    PubMed Central

    Boivin, Guy; Hardy, Isabelle; Kress, Andrew

    2001-01-01

    The FLU OIA test was evaluated with 146 throat swab specimens from subjects with a flu-like illness in six Canadian clinics during the 1999–2000 flu season. The rate of positivity of the FLU OIA test (41.5%) was significantly lower than that of cell culture (55.2%) or reverse transcription-PCR (55.9%) during a season in which only influenza A virus was detected. PMID:11158137

  1. Routine antenatal HIV testing: the responses and perceptions of pregnant women and the viability of informed consent. A qualitative study

    PubMed Central

    de Zulueta, Paquita; Boulton, Mary

    2007-01-01

    This qualitative cross‐sectional survey, undertaken in the antenatal booking clinics of a hospital in central London, explores pregnant women's responses to routine HIV testing, examines their reasons for declining or accepting the test, and assesses how far their responses fulfil standard criteria for informed consent. Of the 32 women interviewed, only 10 participants were prepared for HIV testing at their booking interview. None of the women viewed themselves as being particularly at risk for HIV infection. The minority (n = 6) of the participants who declined testing differed from those who accepted, by interpreting test acceptance as risky behaviour, privileging the negative outcomes of HIV positivity and expressing an inability to cope with these, should they occur. Troublingly, only a minority of women (n = 9) had a broad understanding of the rationale for the test, and none fulfilled the standard criteria for informed consent. This study suggests that, although routine screening combined with professional recommendation may be successful in increasing uptake, this may be at the cost of eroding informed consent. Protecting third parties (notably fetuses) from a preventable disease may outweigh the moral duty of respecting autonomy, enshrined in Western bioethical tradition. Nevertheless, such a policy should be made transparent, debated in the public domain and negotiated with women seeking antenatal care. PMID:17526682

  2. The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen.

    PubMed

    Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Johari, Jefree; Abd-Jamil, Juraina; Hooi, Poh-Sim; AbuBakar, Sazaly

    2016-01-01

    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2-94.8%) than in those from primary dengue (21.7-64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples. PMID:27278716

  3. The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen

    PubMed Central

    Teoh, Boon-Teong; Sam, Sing-Sin; Tan, Kim-Kee; Johari, Jefree; Abd-Jamil, Juraina; Hooi, Poh-Sim; AbuBakar, Sazaly

    2016-01-01

    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2–94.8%) than in those from primary dengue (21.7–64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples. PMID:27278716

  4. Accessing HIV testing and treatment among men who have sex with men in China: a qualitative study.

    PubMed

    Wei, Chongyi; Yan, Hongjing; Yang, Chuankun; Raymond, H Fisher; Li, Jianjun; Yang, Haitao; Zhao, Jinkou; Huan, Xiping; Stall, Ron

    2014-01-01

    Barriers to HIV testing and HIV care and treatment pose significant challenges to HIV prevention among men who have sex with men (MSM) in China. We carried out a qualitative study to identify barriers and facilitators to HIV testing and treatment among Chinese MSM. In 2012, seven focus group (FG) discussions were conducted with 49 MSM participants in Nanjing, China. Purposive sampling was used to recruit a diverse group of MSM participants. Semi-structured interviews were conducted to collect FG data. Major barriers to testing included gay- and HIV-related stigma and discrimination, relationship type and partner characteristics, low perception of risk or threat, HIV is incurable or equals death, concerns of confidentiality, unaware that testing is offered for free, and name-based testing. Key facilitators of testing included engaging in high-risk sex, sense of responsibility for partner, collectivism, testing as a part of standard/routine medical care, MSM-friendly medical personnel, increased acceptance of gay/bisexual men by the general public, legal recognition and protection of homosexuals, and home self-testing. Barriers to treatment included negative coping, nondisclosure to families, misconceptions of domestically produced antiretroviral drugs (ARVs) and the benefits of treatment, and costs associated with long-term treatment. Facilitators of treatment included sense of hopefulness that a cure would be found, the cultural value of longevity, peer social support and professional psychological counseling, affordable and specialized treatment and care, and reduced HIV-related stigma and discrimination. Finally, for both testing and treatment, more educational and promotional activities within MSM communities and among the general public are needed. PMID:23909807

  5. Tests of the Dynamic Field Theory and the Spatial Precision Hypothesis: Capturing a Qualitative Developmental Transition in Spatial Working Memory

    PubMed Central

    Schutte, Anne R.; Spencer, John P.

    2009-01-01

    This study tested a dynamic field theory (DFT) of spatial working memory and an associated spatial precision hypothesis (SPH). Between three and six years of age there is a qualitative shift in how children use reference axes to remember locations: 3-year-olds’ spatial recall responses are biased toward reference axes after short memory delays, whereas 6-year-olds’ responses are biased away from reference axes. According to the DFT and the SPH, quantitative improvements over development in the precision of excitatory and inhibitory working memory processes lead to this qualitative shift. Simulations of the DFT in Experiment 1 predict that improvements in precision should cause the spatial range of targets attracted toward a reference axis to narrow gradually over development with repulsion emerging and gradually increasing until responses to most targets show biases away from the axis. Results from Experiment 2 with 3- to 5-year-olds support these predictions. Simulations of the DFT in Experiment 3 quantitatively fit the empirical results and offer insights into the neural processes underlying this developmental change. PMID:19968430

  6. General practitioners’ perceptions of introducing near-patient testing for common infections into routine primary care: A qualitative study

    PubMed Central

    Butler, Christopher C.; Simpson, Sharon; Wood, Fiona

    2008-01-01

    Objective Near-patient tests are promoted for guiding management of common infections in primary care with a view to enhancing the effectiveness of prescribing decisions and containing antimicrobial resistance. Changes in clinical practice should be based on appraisals of the factors that might influence change, viewed from the perspective of those expected to implement the change. We therefore explored the views of general practitioners concerning the possible introduction of near-patient tests for managing common infections. Design Qualitative semi-structured interview study. Interviews were recorded and analysed using thematic content analysis. Setting General practices in south-east Wales, UK. Subjects A total of 26 general practitioners (GPs) from high fluroquinolone antibiotics prescribing practices and 14 GPs from practices that prescribed fluroquinolones close to the south-east Wales mean. Results There was strong enthusiasm for a hypothetical near-patient, finger-prick blood tests that could distinguish viral from bacterial infections. Many GPs emphasized that such tests would be valuable in “selling” decisions not to prescribe antibiotics to patients. Concerns included limited additional useful information to guide prescribing above clinical diagnosis alone, that patients might deteriorate even if the tests correctly identified a viral aetiology, and that GPs would need to be convinced by research evidence supporting uptake. Several indicated that tests would be useful only for a limited number of patients and they were concerned by time pressures, apparatus maintenance and quality control, cost, and possible objections from patients, especially children. Conclusions Despite GP enthusiasm for the concept of a rapid test to distinguish viral from bacterial infection, strategies to promote uptake would be enhanced if concerns were addressed regarding the importance and feasibility of such tests in daily practice. PMID:18297558

  7. Hybrid capture II, a new sensitive test for human papillomavirus detection. Comparison with hybrid capture I and PCR results in cervical lesions.

    PubMed Central

    Clavel, C; Masure, M; Putaud, I; Thomas, K; Bory, J P; Gabriel, R; Quereux, C; Birembaut, P

    1998-01-01

    AIM: To test a new assay for the detection of human papillomavirus (HPV) DNA, hybrid capture II (HC II), compared with the previous commercialized hybrid capture I (HC I) and polymerase chain reaction (PCR) results on cervical scrapes from fresh cone excision biopsy samples. METHODS: The three methods were used on cervical scrapes from 42 fresh cone excision biopsy samples. There were nine metaplastic and inflammatory lesions, five low grade lesions, and 28 high grade lesions. PCR was performed using the general primers GP5+/GP6+. The viral load of high risk HPV DNA was estimated by the ratio of relative light units to positive control values in the samples. RESULTS: The sensitivity of HC I for the detection of high grade lesions was 71.4%, while it was 92.8% for HC II and 96.4% for the PCR. Considering only the absence of detectable cervical in situ neoplasia, the specificity was 88.9% for HC I, 66.7% for HC II, and 66.7% for PCR. With HC II, for a ratio of cervical sample to normal control of > 200, the sensitivity for the detection of high grade lesion was only 34.6% with a specificity of 66.7%. CONCLUSIONS: HPV detection with the HC II assay is more sensitive than the previous HC I and represents a more convenient and easier test than PCR for routine use. Nevertheless the viral load estimated with this test cannot be a reliable predictive indicator of high grade lesions. PMID:10023335

  8. Y-chromosomal testing of brown bears (Ursus arctos): Validation of a multiplex PCR-approach for nine STRs suitable for fecal and hair samples.

    PubMed

    Aarnes, Siv Grethe; Hagen, Snorre B; Andreassen, Rune; Schregel, Julia; Knappskog, Per M; Hailer, Frank; Stenhouse, Gordon; Janke, Axel; Eiken, Hans Geir

    2015-11-01

    High-resolution Y-chromosomal markers have been applied to humans and other primates to study population genetics, migration, social structures and reproduction. Y-linked markers allow the direct assessment of the genetic structure and gene flow of uniquely male inherited lineages and may also be useful for wildlife conservation and forensics, but have so far been available only for few wild species. Thus, we have developed two multiplex PCR reactions encompassing nine Y-STR markers identified from the brown bear (Ursus arctos) and tested them on hair, fecal and tissue samples. The multiplex PCR approach was optimized and analyzed for species specificity, sensitivity and stutter-peak ratios. The nine Y-STRs also showed specific STR-fragments for male black bears and male polar bears, while none of the nine markers produced any PCR products when using DNA from female bears or males from 12 other mammals. The multiplex PCR approach in two PCR reactions could be amplified with as low as 0.2 ng template input. Precision was high in DNA templates from hairs, fecal scats and tissues, with standard deviations less than 0.14 and median stutter ratios from 0.04 to 0.63. Among the eight di- and one tetra-nucleotide repeat markers, we detected simple repeat structures in seven of the nine markers with 9-25 repeat units. Allelic variation was found for eight of the nine Y-STRs, with 2-9 alleles for each marker and a total of 36 alleles among 453 male brown bears sampled mainly from Northern Europe. We conclude that the multiplex PCR approach with these nine Y-STRs would provide male bear Y-chromosomal specificity and evidence suited for samples from conservation and wildlife forensics. PMID:26264959

  9. Creating more effective health plan quality reports for consumers: lessons from a synthesis of qualitative testing.

    PubMed Central

    Harris-Kojetin, L D; McCormack, L A; Jaël, E F; Sangl, J A; Garfinkel, S A

    2001-01-01

    OBJECTIVE: Social marketing techniques such as consumer testing have only recently been applied to develop effective consumer health insurance information. This article discusses lessons learned from consumer testing to create consumer plan choice materials. DATA SOURCES/STUDY SETTING: Data were collected from 268 publicly and privately insured consumers in three studies between 1994 and 1999. STUDY DESIGN: Iterative testing and revisions were conducted to design seven booklets to help Medicaid, Medicare, and employed consumers choose a health plan. DATA COLLECTION METHODS: Standardized protocols were used in 11 focus groups and 182 interviews to examine the content, comprehension, navigation, and utility of the booklets. PRINCIPAL FINDINGS: A method is suggested to help consumers narrow their plan choices by breaking down the process into smaller decisions using a set of guided worksheets. CONCLUSION: Implementing these lessons is challenging and not often done well. This article gives examples of evidence-based approaches to address cognitive barriers that designers of consumer health insurance information can adapt to their needs. Images Figure. 3 PMID:11482584

  10. Communicating BRCA1/2 genetic test results within the family: a qualitative analysis.

    PubMed

    Dancyger, Caroline; Wiseman, Mel; Jacobs, Chris; Smith, Jonathan A; Wallace, Melissa; Michie, Susan

    2011-08-01

    Genetic testing for BRCA1/2 mutations associated with hereditary breast and ovarian cancer reveals significant risk information about one's chances of developing cancer. It is important to study communication processes in families where members are undergoing genetic testing because the information received is crucial not just to the individual concerned but also to other members of the biological family. This study investigates family communication of BRCA1/2 test results from both the informants' and recipients' perspectives. A total of 10 female patients and 22 of their relatives were interviewed. Patients' and their relatives described feelings of responsibility for sharing genetic information within the family to enable others to reduce their risks of developing cancer. However, there were limits to an individuals' responsibility once key family members had been informed, who then had to take responsibility for continuing dissemination of information. Whilst there was an implicit responsibility to inform the family of a mutation, information was edited or withheld in the best interest of relatives, dependent upon their perceived emotional readiness, resilience and current life stage and circumstances. The pre-existing family culture and the impact previous cancer diagnoses had upon the family also influenced the process of communication. Findings are discussed in relation to extant literature and implications for clinical practice are considered. PMID:21797732

  11. A Qualitative Study of Healthcare Providers’ Perspectives on the Implications of Genome-Wide Testing in Pediatric Clinical Practice

    PubMed Central

    Reiff, Marian; Mueller, Rebecca; Mulchandani, Surabhi; Spinner, Nancy B.; Pyeritz, Reed E.; Bernhardt, Barbara A.

    2013-01-01

    The utilization of genome-wide chromosomal microarray analysis (CMA) in pediatric clinical practice provides an opportunity to consider how genetic diagnostics is evolving, and to prepare for the clinical integration of genome-wide sequencing technologies. We conducted semi-structured interviews with 15 healthcare providers (7 genetic counselors, 4 medical geneticists, and 4 non-genetics providers) to investigate the impact of CMA on clinical practice, and implications for providers, patients and families. Interviews were analyzed qualitatively using content analysis. Most providers reported that genomic testing enhanced their professional experience and was beneficial to patients, primarily due to the improved diagnostic rate compared with earlier chromosomal studies. Other effects on practice included moving towards genotype-first diagnosis and broadening indications for chromosomal testing. Opinions varied concerning informed consent and disclosure of results. The duty to disclose incidental findings (IFs) was noted; however concerns were raised about potential psychosocial harms of disclosing pre-symptomatic findings. Tensions were revealed between the need for comprehensive informed consent for all families and the challenges of communicating time-consuming and potentially anxiety-provoking information regarding uncertain and incidental findings that may be relevant only in rare cases. Genetic counselors can play an important role in liaising with families, health professionals and testing laboratories, providing education and guidance to non-genetics providers, and enabling families to receive adequate pre- and post-test information and follow-up care. PMID:24037030

  12. A Qualitative Exploration of Sexual Risk and HIV Testing Behaviors among Men Who Have Sex with Men in Beirut, Lebanon

    PubMed Central

    Wagner, Glenn J.; Aunon, Frances M.; Kaplan, Rachel L.; Rana, Yashodhara; Khouri, Danielle; Tohme, Johnny; Mokhbat, Jacques

    2012-01-01

    Men who have sex with men (MSM) may account for most new HIV infections in Lebanon, yet little is known about the factors that influence sexual risk behavior and HIV testing in this population. Qualitative interviews were conducted with 31 MSM living in Beirut, and content analysis was used to identify emergent themes. Mean age of the participants was 28.4 years, and all identified as either gay (77%) or bisexual (23%). Half reported not using condoms consistently and one quarter had not been HIV-tested. Many described not using condoms with a regular partner in the context of a meaningful relationship, mutual HIV testing, and a desire to not use condoms, suggesting that trust, commitment and intimacy play a role in condom use decisions. Condoms were more likely to be used with casual partners, partners believed to be HIV-positive, and with partners met online where men found it easier to candidly discuss HIV risk. Fear of infection motivated many to get HIV tested and use condoms, but such affect also led some to avoid HIV testing in fear of disease and social stigma if found to be infected. Respondents who were very comfortable with their sexual orientation and who had disclosed their sexuality to family and parents tended to be more likely to use condoms consistently and be tested for HIV. These findings indicate that similar factors influence the condom use and HIV testing of MSM in Beirut as those observed in studies elsewhere of MSM; hence, prevention efforts in Lebanon can likely benefit from lessons learned and interventions developed in other regions, particularly for younger, gay-identified men. Further research is needed to determine how prevention efforts may need to be tailored to address the needs of men who are less integrated into or do not identify with the gay community. PMID:23029103

  13. A qualitative exploration of sexual risk and HIV testing behaviors among men who have sex with men in Beirut, Lebanon.

    PubMed

    Wagner, Glenn J; Aunon, Frances M; Kaplan, Rachel L; Rana, Yashodhara; Khouri, Danielle; Tohme, Johnny; Mokhbat, Jacques

    2012-01-01

    Men who have sex with men (MSM) may account for most new HIV infections in Lebanon, yet little is known about the factors that influence sexual risk behavior and HIV testing in this population. Qualitative interviews were conducted with 31 MSM living in Beirut, and content analysis was used to identify emergent themes. Mean age of the participants was 28.4 years, and all identified as either gay (77%) or bisexual (23%). Half reported not using condoms consistently and one quarter had not been HIV-tested. Many described not using condoms with a regular partner in the context of a meaningful relationship, mutual HIV testing, and a desire to not use condoms, suggesting that trust, commitment and intimacy play a role in condom use decisions. Condoms were more likely to be used with casual partners, partners believed to be HIV-positive, and with partners met online where men found it easier to candidly discuss HIV risk. Fear of infection motivated many to get HIV tested and use condoms, but such affect also led some to avoid HIV testing in fear of disease and social stigma if found to be infected. Respondents who were very comfortable with their sexual orientation and who had disclosed their sexuality to family and parents tended to be more likely to use condoms consistently and be tested for HIV. These findings indicate that similar factors influence the condom use and HIV testing of MSM in Beirut as those observed in studies elsewhere of MSM; hence, prevention efforts in Lebanon can likely benefit from lessons learned and interventions developed in other regions, particularly for younger, gay-identified men. Further research is needed to determine how prevention efforts may need to be tailored to address the needs of men who are less integrated into or do not identify with the gay community. PMID:23029103

  14. Genotoxicity testing: moving beyond qualitative "screen and bin" approach towards characterization of dose-response and thresholds.

    PubMed

    Pottenger, Lynn H; Gollapudi, B Bhaskar

    2010-01-01

    For more than 40+ years, genotoxicity data have been interpreted in a qualitative, binary mode; a chemical is considered either positive or negative for a response in the test system. Although dose-response information is sometimes used in this decision, it is not routine to obtain the amount of information needed to inform risk assessment, for example to determine no-observed-genotoxic-effect-levels, primarily due to the historical view of genotoxic responses as "linear, no-threshold." Only recently have researchers begun to address this issue through robust experimental designs and application of statistical models. A growing body-of-evidence supports the existence of response thresholds for a number of mutagenic agents, in vitro and in vivo. Clearly, simple observation of a "hockey-stick" dose-response curve is not sufficient to establish a threshold. Collection of robust empirical data must be supported with an analysis of biological plausibility for the observed threshold. In this context, a chemical-specific mode-of-action (MOA) approach, which identifies key events responsible for the observed mutagenic effect, is extremely valuable. Biomarkers of key events, providing qualitative and quantitative information, can be integrated in a weight-of-evidence-based assessment of genotoxicity data from multiple test systems and used to identify data gaps to resolve/reduce uncertainties during the risk assessment process. To this end, specific recommendations on study design and data analysis are proposed. As the Environmental Mutagen Society celebrates its 40th anniversary, the field of genetic toxicology is marking a milestone on the path to a new paradigm, using a MOA, data-driven approach to answer questions about thresholds for genotoxic agents. PMID:20806283

  15. Pregnant women’s experiences of routine counselling and testing for HIV in Eastern Uganda: a qualitative study

    PubMed Central

    2013-01-01

    Background Routine HIV counselling and testing as part of antenatal care has been institutionalized in Uganda as an entry point for pregnant women into the prevention of mother-to-child transmission of HIV (PMTCT) programme. Understanding how women experience this mode of HIV testing is important to generate ideas on how to strengthen the PMTCT programme. We explored pregnant HIV positive and negative women’s experiences of routine counselling and testing in Mbale District, Eastern Uganda and formulated suggestions for improving service delivery. Methods This was a qualitative study conducted at Mbale Regional Referral Hospital in Eastern Uganda between January and May 2010. Data were collected using in-depth interviews with 30 pregnant women (15 HIV positive and 15 HIV negative) attending an antenatal clinic, six key informant interviews with health workers providing antenatal care and observations. Data were analyzed using a content thematic approach. Results Prior to attending their current ANC visit, most women knew that the hospital provided HIV counselling and testing services as part of antenatal care (ANC). HIV testing was perceived as compulsory for all women attending ANC at the hospital but beneficial, for mothers, especially those who test HIV positive and their unborn babies. Most HIV positive women were satisfied with the immediate counselling they received from health workers, but identified the need to provide follow up counselling and support after the test, as areas for improvement. However, most HIV negative women mentioned that they were given inadequate attention during post-test counselling. This left them with unanswered questions and, for some, doubts about the negative test results. Conclusions In this setting, routine HIV counselling and testing services are known and acceptable to mothers. There is need to strengthen post-test and follow up counselling for both HIV positive and negative women in order to maximize opportunities for

  16. Training Vegetable Parenting Practices Through a Mobile Game: Iterative Qualitative Alpha Test

    PubMed Central

    Beltran, Alicia; Buday, Richard; Hughes, Sheryl; O'Connor, Teresia; Baranowski, Janice; Dadabhoy, Hafza R; Diep, Cassandra S; Baranowski, Tom

    2015-01-01

    Background Vegetable consumption protects against chronic diseases, but many young children do not eat vegetables. One quest within the mobile application Mommio was developed to train mothers of preschoolers in effective vegetable parenting practices, or ways to approach getting their child to eat and enjoy vegetables. A much earlier version of the game, then called Kiddio, was alpha tested previously, but the game has since evolved in key ways. Objective The purpose of this research was to alpha test the first quest, substantiate earlier findings and obtain feedback on new game features to develop an effective, compelling parenting game. Methods Mothers of preschool children (n=20) played a single quest of Mommio 2 to 4 times, immediately after which a semi-structured interview about their experience was completed. Interviews were transcribed and double coded using thematic analysis methods. Results Mothers generally liked the game, finding it realistic and engaging. Some participants had difficulties with mechanics for moving around the 3-D environment. Tips and hints were well received, and further expansion and customization were desired. Conclusions Earlier findings were supported, though Mommio players reported more enjoyment than Kiddio players. Continued development will include more user-friendly mechanics, customization, opportunities for environment interaction, and food parenting scenarios. PMID:26208899

  17. Novel Real-Time PCR Assay for Detection of Helicobacter pylori Infection and Simultaneous Clarithromycin Susceptibility Testing of Stool and Biopsy Specimens

    PubMed Central

    Schabereiter-Gurtner, Claudia; Hirschl, Alexander M.; Dragosics, Brigitte; Hufnagl, Peter; Puz, Sonja; Kovách, Zsuzsanna; Rotter, Manfred; Makristathis, Athanasios

    2004-01-01

    A biprobe real-time PCR protocol, followed by hybridization melting point analysis, to detect point mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance was established and evaluated in a clinical study. Of 92 patients who underwent endoscopy, 45 were found to be H. pylori infected and invariably were also culture positive. Of the 45 isolates, 11 were shown to be resistant to clarithromycin by E-test. With respect to the detection of H. pylori infection, PCR showed sensitivities of 100% in biopsies and 98% in stool specimens and a specificity of 98% in both biopsy and stool samples. All clarithromycin-sensitive cases were identified as such by PCR in both biopsy and stool samples. Of the cases with a resistant strain, eight were identified as such in stool DNA and nine were identified in biopsy DNA. Failure of PCR to detect the resistant genotype in the biopsy DNA, stool DNA, or both (one case) was associated with mixed populations. In these cases, patients had not been treated for H. pylori infection before, and the sensitive population showed to be present in considerably higher numbers than the resistant population. In five of six cases in which infection with a resistant genotype only was identified by PCR, the patients had received clarithromycin-based eradication therapy in the past. Thus, the assay presented provides a highly accurate noninvasive method to detect H. pylori infection in stool and at the same time allows for culture-independent clarithromycin susceptibility testing. PMID:15472302

  18. Application of real time RT-PCR for the genetic homogeneity and stability tests of the seed candidates for live attenuated influenza vaccine production

    PubMed Central

    Shcherbik, Svetlana; Sergent, Sheila B.; Davis, William G.; Shu, Bo; Barnes, John; Kiseleva, Irina; Larionova, Natalie; Klimov, Alexander; Bousse, Tatiana

    2015-01-01

    Development and improvement of quality control tests for live attenuated vaccines are a high priority because of safety concerns. Live attenuated influenza vaccine (LAIV) viruses are 6:2 reassortants containing the hemagglutinin (HA) and neuraminidase (NA) gene segments from circulating influenza viruses to induce protective immune responses, and the six internal gene segments from a cold-adapted Master Donor Virus (MDV). LAIV candidate viruses for the 2012–2013 seasons, A/Victoria/361/2011-CDC-LV1 (LV1) and B/Texas/06/2011-CDC-LV2B (LV2B), were created by classical reassortment of A/Victoria/361/2011 and MDV-A A/Leningrad/134/17/57 (H2N2) or B/Texas/06/2011 and MDV-B B/USSR/60/69. In an attempt to provide better identity and stability testing for quality control of LV1 and LV2B, sensitive real-time RT-PCR assays (rRT-PCR) were developed to detect the presence of undesired gene segments (HA and NA from MDV and the six internal genes from the seasonal influenza viruses). The sensitivity of rRT-PCR assays designed for each gene segment ranged from 0.08 to 0.8 EID50 (50% of Egg Infectious Dose) per reaction for the detection of undesired genes in LV1 and from 0.1 to 1 EID50 per reaction for the detection of undesired genes in LV2B. No undesired genes were detected either before or after five passages of LV1 or LV2B in eggs. The complete genome sequencing of LV1 and LV2B confirmed the results of rRT-PCR, demonstrating the utility of the new rRT-PCR assays to provide the evidence for the homogeneity of the prepared vaccine candidate. PMID:24056261

  19. [Comparison of glucan and galactomannan tests with real-time PCR for diagnosis of invasive aspergillosis in a neutropenic rat model].

    PubMed

    Aydoğan, Sibel; Kuştimur, Semra; Kalkancı, Ayşe

    2010-07-01

    The incidence of aspergillosis which has high mortality rates, has increased gradually. Since invasive aspergillosis (IA) is one of the leading causes of death in immunocompromized and neutropenic patients, early and accurate diagnosis of IA is of crucial importance. The aims of this study were to compare the results of culture, real-time polymerase chain reaction (RtPCR), galactomannan (GM) antigen and glucan (GC) antigen detection tests and to evaluate their performances in view of rapid and accurate diagnosis of IA in neutropenic rat model. Female Wistar albino rats were included in the study with the consent of Animal Searching Ethical Committee and classified into three groups as healthy controls (n= 6), neutropenic controls (n= 10) and pulmonary aspergillosis (n= 35) groups. Rats were immunosuppressed with 5-flourourasil and then Aspergillus fumigatus conidia were inoculated intranasally. On the seventh day of the infection, blood, bronchoalveolar lavage (BAL) and lung tissue samples were collected from the animals, and control and aspergillosis groups were compared in terms of infection markers. All of the tests (culture, RtPCR, GM and BG tests) were found to be negative in controls. At the end of the study 22 rats in aspergillosis group survived. Lung tissue samples from those 22 animals were all positive for the presence of hypha on pathological preparations, while 20 (91%) yielded Aspergillus colonies on the cultures. Aspergillus DNA was detected in 7 of the 12 BAL samples (58.3%), 7 of 19 blood samples (36.8%) and 4 of 22 lung tissue samples (18%) using RtPCR method. GM antigen was detected in 7 of 20 serum samples (35%) with a commercial kit (Platelia® Aspergillus ELISA, BioRad, France). Quantitative detection of betalucan levels were investigated by using a commercial kit (Fungitell™, Cape Cod, USA) in serum and BAL samples and positive results were obtained in 11 of 22 serum (50%) and 9 of 17 BAL (52.9%) samples. In this study it was demonstrated

  20. Test me and treat me”—attitudes to vitamin D deficiency and supplementation: a qualitative study

    PubMed Central

    Kotta, Siddharth; Gadhvi, Dev; Jakeways, Niki; Saeed, Maryum; Sohanpal, Ratna; Hull, Sally; Famakin, Olufunke; Martineau, Adrian; Griffiths, Chris

    2015-01-01

    Objective Lay interest in vitamin D and the potential benefits of supplementation is considerable, but little information exists concerning lay knowledge, beliefs and attitudes towards vitamin D to inform public health initiatives and professional guidance. Design Qualitative focus group study. Participants 58 adults capturing diversity in disease status, gender, age and ethnicity. Setting A large general practice in east London. Results Many respondents lacked knowledge about vitamin D, including dietary sources and government recommendations. Most were positive about sun exposure, but confused by ambiguous health messages about risks and benefits of sunshine. Medicalised views of vitamin D were prominent, notably from those in favour of supplementation, who talked of “doses”, “side effects” and “regular testing.” Fortification of food with vitamin D was controversial, with opposing utilitarian (better overall for the majority) and libertarian (freedom to choose) views. Conclusions Knowledge about vitamin D was limited. Clearer messages are needed about risks and benefits of sun exposure. Testing and supplementation by health professionals, while potentially useful in some high-risk groups, have contributed to a medicalised view of vitamin D. Health policy should address the public's need for clear information on sources and effects of vitamin D, including risks and benefits of sun exposure, and take account of divergent views on fortification. Professional guidance is needed on testing and supplementation to counter inappropriate medicalisation. PMID:26173717

  1. Autism spectrum disorders: a qualitative study of attitudes toward prenatal genetic testing and termination decisions of affected pregnancies.

    PubMed

    Chen, L S; Xu, L; Dhar, S U; Li, M; Talwar, D; Jung, E

    2015-08-01

    In the United States, prenatal genetic testing (PGT) for Autism Spectrum Disorders (ASD) is currently available via clinical genetic services. Such testing may inform parents about their unborn child's risk for ASD, prepare parents for the birth of an affected infant, and allow them to arrange for early interventions. Although PGT for autism has potential benefits, the associated ethical, legal, and social implications (ELSI) should be considered. This first qualitative study employed a hypothetical scenario to explore the attitudes toward PGT and termination decisions of 42 parents of children with ASD. Over half of the participants expressed willingness to undergo PGT for autism. Reasons included better preparation for birth, early and better treatment, termination of affected pregnancy, contribution to research, and curiosity. Of the 31 parents who were either willing or unsure about undergoing the PGT, approximately three-fourths would continue their hypothetical affected pregnancies. Explanations included preparation for birth of the child, bonding or acceptance of existing ASD-affected children, apprehensions about test limitations, and religious concerns. Parents who reported they would terminate the affected pregnancy in this hypothetical situation were primarily Asians. This study contributes to the growing understanding of the ELSI aspects of PGT in clinical practice. PMID:25251361

  2. Molecular characterization of Mycobacterium avium complex isolates giving discordant results in AccuProbe tests by PCR-restriction enzyme analysis, 16S rRNA gene sequencing, and DT1-DT6 PCR.

    PubMed Central

    Devallois, A; Picardeau, M; Paramasivan, C N; Vincent, V; Rastogi, N

    1997-01-01

    Based on cultural and biochemical tests, a total of 84 strains (72 clinical and 12 environmental isolates from the Caribbean Isles, Europe, and the Indian subcontinent) were identified as members of the Mycobacterium avium complex (MAC). They were further characterized with MAC, M. avium, and M. intracellulare probes of the AccuProbe system, and this was followed by selective amplification of DT6 and DT1 sequences. Seventy isolates gave concordant results; 63 were identified as M. avium, 5 were identified as M. intracellulare, and 24 remained untypeable by both methods. Fourteen isolates gave discrepant results, as they were DT1 positive but gave negative results by the M. intracellulare AccuProbe test. Consequently, a detailed molecular analysis of all DT1-positive isolates (14 discrepant strains plus 5 M. intracellulare strains) was performed by PCR-restriction analysis (PRA) of the hsp65 gene and 16S rRNA gene sequencing. The results confirmed the reported heterogeneity of M. intracellulare, as only 6 of 19 isolates (32%) gave PRA results compatible with published M. intracellulare profiles while the rest of the isolates were grouped in four previously unpublished profiles. 16S rRNA gene sequencing showed that only 8 of 19 isolates (42%) were related to M. intracellulare IWGMT 90247 (EMBL accession no. X88917), the rest being related to MCRO19 (EMBL accession no. X93030) and MIWGTMR10 (EMBL accession no. X88915). In conclusion, we have characterized a significant number of MAC isolates which were not identified by the AccuProbe test, PRA, or 16S rRNA sequencing. However, all of them were identifiable by DT1-DT6 PCR (they were DT6 negative and DT1 positive) and could be tentatively identified as M. intracellulare based on previously published observations. It is noteworthy that the majority of such isolates (14 of 19) were from the Indian subcontinent, with 12 of 14 being environmental isolates. Our study confirms the marked heterogeneity of M. intracellulare

  3. Crafting Appealing Text Messages to Encourage Colorectal Cancer Screening Test Completion: A Qualitative Study

    PubMed Central

    Ellis, Shellie D; Denizard-Thompson, Nancy; Kronner, Donna; Miller, David P

    2015-01-01

    texting shorthand phrases and complicated replies); they did not want messages that contain bad news or test results. They wanted the ability to choose alternative options such as email or phone calls. Conclusions Older adults are receptive to receiving cancer screening text messages from health care providers. Sharing sample messages with patients may increase acceptance of this tool in the clinic setting. Supportive tailored text messaging reminders could enhance uptake of colorectal cancer screening by enhancing patient self-efficacy and providing cues to action to complete colonoscopy or fecal occult blood testing. PMID:26537553

  4. Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test.

    PubMed

    Worbs, Sylvia; Fiebig, Uwe; Zeleny, Reinhard; Schimmel, Heinz; Rummel, Andreas; Luginbühl, Werner; Dorner, Brigitte G

    2015-12-01

    In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as "gold standard" for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay. PMID:26703724

  5. Culture Qualitatively but Not Quantitatively Influences Performance in the Boston Naming Test in a Chinese-Speaking Population

    PubMed Central

    Chen, Ting-Bin; Lin, Chi-Ying; Lin, Ker-Neng; Yeh, Yen-Chi; Chen, Wei-Ta; Wang, Kuo-Shu; Wang, Pei-Ning

    2014-01-01

    Background/Aims The Boston Naming Test (BNT) is the most frequently administered confrontational naming test, but the cultural background of the patients may influence their performance in the BNT. The aim of this study was to identify differences in performance in the BNT between a Chinese population in Taiwan, Chinese populations in other areas and a Caucasian population. Methods A total of 264 native, Chinese-speaking, cognitively normal elders aged >60 years were enrolled in our study and conducted the 30-item Chinese version of the BNT. Another 10 BNT studies were categorized, analyzed and compared with the present study. Results Higher education was associated with higher scores, whereas age and gender had no effect on performance in the BNT. The score of the Chinese-speaking population was equivalent to the English-speaking population. A disparity in difficulties with items was not only apparent between the Taiwanese and Caucasian populations, but also between the Chinese-speaking populations in the different geographic areas. Conclusion For the most part, the impact of culture on performance in the BNT may not be quantitative but qualitative. Attention should be paid to a potential effect of culture on difficulties with items when administering the BNT to non-English-speaking populations. Understanding differences in performance in the BNT in distinct cultural settings improves the clinical application of the BNT. PMID:24847347

  6. The education and practice program for medical students with quantitative and qualitative fit test for respiratory protective equipment.

    PubMed

    Myong, Jun-Pyo; Byun, JunSu; Cho, YounMo; Seo, Hye-Kyung; Baek, Jung-Eun; Koo, Jung-Wan; Kim, Hyunwook

    2016-01-01

    Tuberculosis infection is prevalent in Korea and health care workers are vulnerable to tuberculosis infection in the hospital. The aims of this study were to develop and validate an education program that teaches senior medical students how to wear and choose the proper size and type of respiratory protective equipment (RPE), which may help reduce the risk of contracting Mycobacterium tuberculosis (MTB) from patients. Overall, 50 senior medical students participated in this education program. Methods of choosing the proper type of RPE, performing a fit check of the RPE, and choosing a suitable mask size were taught by certified instructors using the real-time quantitative fit test (QNFT). The validity of education program was evaluated with qualitative fit test (QLFT) before and after the education as pass or fail. The education program was effective, as shown by the significantly pass rate (increased 30 to 74%) in the QLFT after the education program (p<0.05). Among study participants, changing mask size from medium to small significantly increased the pass rate (p<0.001). Incorporation of this program into the medical school curriculum may help reduce risk of MTB infection in medical students working in the hospital. PMID:26538001

  7. Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

    PubMed Central

    Worbs, Sylvia; Fiebig, Uwe; Zeleny, Reinhard; Schimmel, Heinz; Rummel, Andreas; Luginbühl, Werner; Dorner, Brigitte G.

    2015-01-01

    In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay. PMID:26703724

  8. The education and practice program for medical students with quantitative and qualitative fit test for respiratory protective equipment

    PubMed Central

    MYONG, Jun-Pyo; BYUN, JunSu; CHO, YounMo; SEO, Hye-Kyung; BAEK, Jung-Eun; KOO, Jung-Wan; KIM, Hyunwook

    2015-01-01

    Tuberculosis infection is prevalent in Korea and health care workers are vulnerable to tuberculosis infection in the hospital. The aims of this study were to develop and validate an education program that teaches senior medical students how to wear and choose the proper size and type of respiratory protective equipment (RPE), which may help reduce the risk of contracting Mycobacterium tuberculosis (MTB) from patients. Overall, 50 senior medical students participated in this education program. Methods of choosing the proper type of RPE, performing a fit check of the RPE, and choosing a suitable mask size were taught by certified instructors using the real-time quantitative fit test (QNFT). The validity of education program was evaluated with qualitative fit test (QLFT) before and after the education as pass or fail. The education program was effective, as shown by the significantly pass rate (increased 30 to 74%) in the QLFT after the education program (p<0.05). Among study participants, changing mask size from medium to small significantly increased the pass rate (p<0.001). Incorporation of this program into the medical school curriculum may help reduce risk of MTB infection in medical students working in the hospital. PMID:26538001

  9. easyPAC: A Tool for Fast Prediction, Testing and Reference Mapping of Degenerate PCR Primers from Alignments or Consensus Sequences

    PubMed Central

    Rosenkranz, David

    2012-01-01

    The PCR-amplification of unknown homologous or paralogous genes generally relies on PCR primers predicted from multi sequence alignments. But increasing sequence divergence can induce the need to use degenerate primers which entails the problem of testing the characteristics, unwanted interactions and potential mispriming of degenerate primers. Here I introduce easyPAC, a new software for the prediction of degenerate primers from multi sequence alignments or single consensus sequences. As a major innovation, easyPAC allows to apply all customary primer test procedures to degenerate primer sequences including fast mapping to reference files. Thus, easyPAC simplifies and expedites the designing of specific degenerate primers enormously. Degenerate primers suggested by easyPAC were used in PCR amplification with subsequent de novo sequencing of TDRD1 exon 11 homologs from several representatives of the haplorrhine primate phylogeny. The results demonstrate the efficient performance of the suggested primers and therefore show that easyPAC can advance upcoming comparative genetic studies.

  10. How Well Does Physician Selection of Microbiologic Tests Identify Clostridium difficile and other Pathogens in Paediatric Diarrhoea? Insights Using Multiplex PCR-Based Detection

    PubMed Central

    Stockmann, Chris; Rogatcheva, Margarita; Harrel, Brian; Vaughn, Mike; Crisp, Rob; Poritz, Mark; Thatcher, Stephanie; Korgenski, Ernest K; Barney, Trenda; Daly, Judy; Pavia, Andrew T

    2014-01-01

    The objective of this study was to compare the aetiologic yield of standard of care microbiologic testing ordered by physicians with that of a multiplex PCR platform. Stool specimens obtained from children and young adults with gastrointestinal illness were evaluated by standard laboratory methods and a developmental version of the FilmArray Gastrointestinal Diagnostic System (FilmArray GI Panel), a rapid multiplex PCR platform that detects 23 bacterial, viral, and protozoal agents. Results were classified according to the microbiologic tests requested by the treating physician. A median of 3 (range 1-10) microbiologic tests were performed by the clinical laboratory during 378 unique diarrhoeal episodes. A potential aetiologic agent was identified in 46% of stool specimens by standard laboratory methods and in 65% of specimens tested using the FilmArray GI Panel (P<0.001). For those patients who only had Clostridium difficile testing requested, an alternative pathogen was identified in 29% of cases with the FilmArray GI Panel. Notably, 11 (12%) cases of norovirus were identified among children who only had testing for C. difficile ordered. Among those who had C. difficile testing ordered in combination with other tests, an additional pathogen was identified in 57% of stool specimens with the FilmArray GI Panel. For patients who had no C. difficile testing performed, the FilmArray GI Panel identified a pathogen in 63% of cases, including C. difficile in 8%. Physician-specified laboratory testing may miss important diarrhoeal pathogens. Additionally, standard laboratory testing is likely to underestimate co-infections with multiple infectious diarrhoeagenic agents. PMID:25599941

  11. Assessment of antiretroviral therapy by plasma viral load testing: standard and ICD HIV-1 p24 antigen and viral RNA (QC-PCR) assays compared.

    PubMed

    Kappes, J C; Saag, M S; Shaw, G M; Hahn, B H; Chopra, P; Chen, S; Emini, E A; McFarland, R; Yang, L C; Piatak, M

    1995-10-01

    To assess the utility of quantitative competitive-polymerase chain reaction (QC-PCR) measurements of plasma human immunodeficiency virus type 1 (HIV-1) RNA and other viral load markers for assessment of antiretroviral therapy, we used archived cryopreserved specimens from a randomized controlled clinical trial of 135 patients (CD4+ T cell count < or = 500/mm3), comparing zidovudine (500 mg/day) versus the nonnucleoside reverse transcriptase inhibitor L-697, 661 (50, 300, or 1,000 mg daily). We evaluated treatment-associated changes in plasma viral load by standard and immune complex-dissociated (ICD) HIV-1 p24 antigen assays, and, in a representative subset of patients (n = 46), by QC-PCR determination of virion-associated HIV-1 RNA. At baseline, HIV-1 RNA was quantifiable by QC-PCR in all patients tested (100%), whereas standard and ICD HIV-1 p24 antigen tests were positive (> or = 30 pg/ml) in 42% and 56%, respectively. All viral load parameters showed significant decreases from baseline within 1 week of initiation of zidovudine, as measured by standard p24 antigen assay, ICD p24 assay, and QC-PCR. At 1 week, patients treated with either 300 or 1,000 mg/day of L-697,661 showed significant decreases from baseline in plasma standard and ICD p24 antigen and QC-PCR-determined HIV-1 RNA levels. Whereas viral load decreases seen with zidovudine were sustained for the duration of treatment, plasma viral markers often returned to pretreatment levels despite ongoing L-697,661 treatment, with evidence of the emergence of drug-resistant virus. Whereas standard p24, ICD p24, and viral RNA levels changed similarly in response to treatment, the superior sensitivity and available dynamic range of plasma viral RNA assays like QC-PCR analysis provide an advantage for clinical monitoring of plasma viral load, allowing tracking of treatment-related changes even in patients with earlier stage disease and lower levels of viral load. PMID:7552477

  12. The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test

    SciTech Connect

    Gardner, R.J.; Bobrow, M.; Roberts, R.G.

    1995-08-01

    The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frameshifting deletions in the dystrophin gene that are detectable by multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be mutation in the ORF. We believe that reverse-transcription-PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion. 29 refs., 2 figs., 3 tabs.

  13. A qualitative study into the difficulties experienced by healthcare decision makers when reading a Cochrane diagnostic test accuracy review

    PubMed Central

    2013-01-01

    Background Cochrane reviews are one of the best known and most trusted sources of evidence-based information in health care. While steps have been taken to make Cochrane intervention reviews accessible to a diverse readership, little is known about the accessibility of the newcomer to the Cochrane library: diagnostic test accuracy reviews (DTARs). The current qualitative study explored how healthcare decision makers, who varied in their knowledge and experience with test accuracy research and systematic reviews, read and made sense of DTARs. Methods A purposive sample of clinicians, researchers and policy makers (n = 21) took part in a series of think-aloud interviews, using as interview material the first three DTARs published in the Cochrane library. Thematic qualitative analysis of the transcripts was carried out to identify patterns in participants’ ‘reading’ and interpretation of the reviews and the difficulties they encountered. Results Participants unfamiliar with the design and methodology of DTARs found the reviews largely inaccessible and experienced a range of difficulties stemming mainly from the mismatch between background knowledge and level of explanation provided in the text. Experience with systematic reviews of interventions did not guarantee better understanding and, in some cases, led to confusion and misinterpretation. These difficulties were further exacerbated by poor layout and presentation, which affected even those with relatively good knowledge of DTARs and had a negative impact not only on their understanding of the reviews but also on their motivation to engage with the text. Comparison between the readings of the three reviews showed that more accessible presentation, such as presenting the results as natural frequencies, significantly increased participants’ understanding. Conclusions The study demonstrates that authors and editors should pay more attention to the presentation as well as the content of Cochrane DTARs

  14. The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests.

    PubMed

    Wang, Rong; Schmidt, John W; Arthur, Terrance M; Bosilevac, Joseph M

    2013-04-01

    Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety assurance. The Biocontrol GDS(®) and DuPont Qualicon BAX(®)-RT rapid detection systems are two commercial tests based on real-time PCR amplification with potential applications for quantification of specific E. coli O157:H7 gene targets in enriched meat samples. However, there are arguments surrounding the use of these tests to predict pre-enrichment concentrations of E. coli O157:H7, as well as arguments pertaining to the influence of non-viable cells causing false positive results. The present study attempts to illustrate the effects of different bacterial physiologic states and the presence of non-viable cells on the ability of these systems to accurately measure contamination levels of E. coli O157:H7 in ground beef. While the PCR threshold cycle (C(T)) values of these assays showed a direct correlation with the number of bacteria present in pure cultures, this was not the case for ground beef samples spiked with various levels of injured or healthy cells. Furthermore, comparison of post-enrichment cell densities of bacteria did not correlate with injured or healthy cell numbers inoculated before enrichment process. Ground beef samples spiked with injured or healthy cells at different doses could not be distinguished by C(T) values from either assay. In addition, the contribution of nonviable cells in generating positive real-time PCR signals was investigated using both assays on pre-enriched and post-enriched beef samples, but only if inoculated at levels of 10(6) cells/sample or higher, which are levels not typically seen in ground beef. PMID:23200653

  15. The role of HIV testing, counselling, and treatment in coping with HIV/AIDS in Uganda: a qualitative analysis.

    PubMed

    Nyanzi-Wakholi, Barbara; Lara, Antonieta Medina; Watera, Christine; Munderi, Paula; Gilks, Charles; Grosskurth, Heiner

    2009-07-01

    HIV/AIDS has had a devastating impact at individual, household and community levels. This qualitative research investigates the role of HIV voluntary counselling and testing (VCT) and treatment in enabling HIV-positive Ugandans to cope with this disease. Twelve predetermined focus group discussions (FGDs) were conducted; six with men and six with women. Half of the men and women's groups were receiving antiretroviral therapy (ART) and half were not. An FGD was held with the health care providers administering ART. Testing for HIV was perceived as soliciting a death warrant. Participants affirmed that the incentive for testing was the possibility of accessing free ART. They described experiencing gender-variant stigma and depression on confirming their HIV status and commended the role of counselling in supporting them to adopt positive living. For those receiving ART, counselling reinforced treatment adherence. The findings also revealed gender differences in treatment adherence strategies. ART was described to reduce disease symptoms and restore physical health allowing them to resume their daily activities. Additionally, ART was preferred over traditional herbal treatment because it had clear dosages, expiry dates and was scientifically manufactured. Those that were not receiving ART bore myths and misconceptions about the effectiveness and side effects of ART, delaying the decision to seek treatment. Stigma and the attached concern of HIV/AIDS-related swift death, is a major barrier for VCT. Based on this study's findings, ensuring the provision of quality assured and gender conscious VCT and ART delivery services will enhance positive living and enforce compliance to ART programmes. PMID:20024747

  16. Rapid visual identification of PCR amplified nucleic acids by centrifugal gel separation: Potential use for molecular point-of-care tests.

    PubMed

    Hwang, Sang-Hyun; Kim, Dong-Eun; Im, Ji-Hyun; Kang, Su-Jin; Lee, Do-Hoon; Son, Sang Jun

    2016-05-15

    Recently, nucleic acid amplification and detection techniques have progressed based on advances in in microfluidics, microelectronics, and optical systems. Nucleic acids amplification based point-of-care test (POCT) in resource-limited settings requires simple visual detection methods. Several biosensing methods including lateral flow immunoassays (LFIA) were previously used to visually detect nucleic acids. However, prolonged assay time, several washing steps, and a need for specific antibodies limited their use. Here we developed a novel, rapid method to visualize amplified nucleic acids with naked eyes in clinical samples. First, we optimized conditions based on separation using very low centrifugal force and a density medium to detect human papillomavirus (HPV)-16 DNA in cervical specimens. After DNA extraction, HPV16 PCR was performed with biotin-labeled forward primer and Cy3-labeled reverse primer. PCR amplicon was mixed with streptavidin-magnetic beads, introduced into the density medium. After two-minute centrifugation, the result was visually identified. This system showed identical results with commercial HPV real-time PCR for 30 clinical samples and could detect up to 10(2)copies/mL of HPV DNA without any optical instruments. This robust and sensitive visual detection system is suitable for non-specialist personnel and point-of-care diagnosis in low-resource settings. PMID:26774997

  17. Development of a Multiplex PCR Test with Automated Genotyping Targeting E7 for Detection of Six High-Risk Human Papillomaviruses

    PubMed Central

    de Assis, Angela Maria

    2015-01-01

    Cervical cancer is caused by high-risk human papillomaviruses (HPV) and viral detection tests aid in the diagnosis of precursor lesions. In the present study, a molecular test for detection of high-risk HPV DNA, called E7-HPV, was standardized and assessed in samples from women with pre-cancerous lesions. The development of the E7-HPV test for detection and genotyping of six high-risk HPV (types 16, 18, 31, 33, 45 and 52), consisted of evaluating primer quality and adjusting the multiplex PCR conditions. Primer design was based on the E7 region of each HPV, and the fluorochrome 6-FAM was added to PCR primers. Viral detection was performed by capillary electrophoresis in automated sequencer in samples obtained from 60 women (55 with ASC-H/HSIL cytology) from August to September 2013. A non-inferiority analysis was conducted with the cobas HPV test as a reference and following international guidelines for the development of new tests. The two tests had a high concordance rate in HPV16 detection (kappa=0.972), with only one discordant case (cervical intraepithelial neoplasia grade 3, negaive with cobas and positive for HPV16 by E7-HPV) and complete agreement in HPV18 detection. When comparing detection of all high-risk HPV, three cases were positive with cobas but negative with E7-HPV, and another three cases were negative with cobas but positive with E7-HPV (HPV16, 31 and 52). When we evaluate the cases initially suspected by cytology, the two tests had the same sensitivity in detection CIN2 or worse. In conclusion, the E7-HPV test has satisfactory initial results, and its development can be continued. PMID:26087285

  18. 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. PMID:27313572

  19. 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

  20. Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy.

    PubMed

    Bruijnesteijn van Coppenraet, L E S; Wallinga, J A; Ruijs, G J H M; Bruins, M J; Verweij, J J

    2009-09-01

    Molecular detection of gastrointestinal protozoa is more sensitive and more specific than microscopy but, to date, has not routinely replaced time-consuming microscopic analysis. Two internally controlled real-time PCR assays for the combined detection of Entamoeba histolytica, Giardia lamblia, Cryptosporidium spp. and Dientamoeba fragilis in single faecal samples were compared with Triple Faeces Test (TFT) microscopy results from 397 patient samples. Additionally, an algorithm for complete parasitological diagnosis was created. Real-time PCR revealed 152 (38.3%) positive cases, 18 of which were double infections: one (0.3%) sample was positive for E. histolytica, 44 (11.1%) samples were positive for G. lamblia, 122 (30.7%) samples were positive for D. fragilis, and three (0.8%) samples were positive for Cryptosporidium. TFT microscopy yielded 96 (24.2%) positive cases, including five double infections: one sample was positive for E. histolytica/Entamoeba dispar, 29 (7.3%) samples were positive for G. lamblia, 69 (17.4%) samples were positive for D. fragilis, and two (0.5%) samples were positive for Cryptosporidium hominis/Cryptosporidium parvum. Retrospective analysis of the clinical patient information of 2887 TFT sets showed that eosinophilia, elevated IgE levels, adoption and travelling to (sub)tropical areas are predisposing factors for infection with non-protozoal gastrointestinal parasites. The proposed diagnostic algorithm includes application of real-time PCR to all samples, with the addition of microscopy on an unpreserved faecal sample in cases of a predisposing factor, or a repeat request for parasitological examination. Application of real-time PCR improved the diagnostic yield by 18%. A single stool sample is sufficient for complete parasitological diagnosis when an algorithm based on clinical information is applied. PMID:19624500

  1. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2003-01-01

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  2. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2005-05-17

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  3. Comparison of the Directigen Flu A+B Test, the QuickVue Influenza Test, and Clinical Case Definition to Viral Culture and Reverse Transcription-PCR for Rapid Diagnosis of Influenza Virus Infection

    PubMed Central

    Ruest, Annie; Michaud, Sophie; Deslandes, Sylvie; Frost, Eric H.

    2003-01-01

    The diagnostic performances of the clinical case definition of influenza virus infection based on the combination of fever and cough and of two rapid influenza diagnostic tests, the Directigen Flu A+B test (Directigen; BD Diagnostic Systems, Sparks, Md.) and the QuickVue influenza test (QuickVue; Quidel, San Diego, Calif.), were compared to those of viral culture and an in-house reverse transcription (RT)-PCR during the 2000-2001 flu season. Two hundred consecutive nasopharyngeal aspirates were analyzed from 192 patients, including 122 adults and 70 children. Viral culture identified influenza virus A in 16 samples and influenza virus B in 55 samples, whereas RT-PCR identified influenza virus A in 21 samples and influenza virus B in 64 samples. When RT-PCR was used as the reference standard, the likelihood ratios for a positive test were 40.0 for Directigen, 8.6 for QuickVue, and 1.4 for the combination of fever and cough, whereas the likelihood ratios for a negative test were 0.22, 0.16, and 0.48, respectively. Our study suggests that (i) the poor specificity (35 to 58%) and the poor positive predictive value (41 to 60%) of the clinical case definition of influenza preclude its use for prediction of influenza virus infections during epidemics, especially when infection control decision making in the hospital setting is considered; (ii) Directigen has a higher diagnostic yield than QuickVue but is associated with a larger number of invalid results; (iii) the sensitivities of the rapid diagnostic tests are significantly lower with samples from adults than with samples from children, with the rates of false-negative results reaching up to 29%; and (iv) RT-PCR detects more cases of influenza than viral culture, and this greater accuracy makes it a more useful reference standard. PMID:12904343

  4. A Qualitative Comparison of the C-Ring Test and the Jones Test as Standard Practice Test Methods for Studying Stress Corrosion Cracking in Ferritic Steels

    SciTech Connect

    Thomson, Jeffery K; Pawel, Steven J

    2015-01-01

    Creep-strength-enhanced-ferritic (CSEF) steels have been widely implemented as water wall alloy materials in the coal-fired power industry for many years. The stress corrosion cracking (SCC) behavior of this class of materials is currently of significant interest to the industry due to recent failures. To better understand the test methods used to characterize SCC behavior in the laboratory, three representative CSEF alloys (T23, T24, and T92) were subjected to two SCC test protocols: the Jones Test set forth in DIN 50915, and the C-ring SCC test set forth in ASTM G38-01. Samples were tested in either the as-received (normalized + tempered) condition or in the normalized condition (quenched from 1065 C). Samples were exposed to aerated water in one test case and de-aerated water in a second test case for a period of 7 days at 200 C. It was found that for both test protocols, the normalized condition with aerated water led to severe cracking for all three alloys, whereas no evidence of cracking was found for the other conditions.

  5. Comparison of the MChip to Viral Culture, Reverse Transcription-PCR, and the QuickVue Influenza A+B Test for Rapid Diagnosis of Influenza▿

    PubMed Central

    Mehlmann, Martin; Bonner, Aleta B.; Williams, John V.; Dankbar, Daniela M.; Moore, Chad L.; Kuchta, Robert D.; Podsiad, Amy B.; Tamerius, John D.; Dawson, Erica D.; Rowlen, Kathy L.

    2007-01-01

    The performance of a diagnostic microarray (the MChip assay) for influenza was compared in a blind study to that of viral culture, reverse transcription (RT)-PCR, and the QuickVue Influenza A+B test. The patient sample data set was composed of 102 respiratory secretion specimens collected between 29 December 2005 and 2 February 2006 at Scott & White Hospital and Clinic in Temple, Texas. Samples were collected from a wide range of age groups by using direct collection, nasal/nasopharyngeal swabs, or nasopharyngeal aspiration. Viral culture and the QuickVue assay were performed at the Texas site at the time of collection. Aliquots for each sample, identified only by study numbers, were provided to the University of Colorado and Vanderbilt University teams for blinded analysis. When referenced to viral culture, the MChip exhibited a clinical sensitivity of 98% and a clinical specificity of 98%. When referenced to RT-PCR, the MChip assay exhibited a clinical sensitivity of 92% and a clinical specificity of 98%. While the MChip assay currently requires 7 to 8 h to complete the analysis, a significant advantage of the test for influenza virus-positive samples is simultaneous detection and full subtype identification for the two subtypes currently circulating in humans (A/H3N2 and A/H1N1) and avian (A/H5N1) viruses. PMID:17301287

  6. Serologic and PCR testing of persons with chronic fatigue syndrome in the United States shows no association with xenotropic or polytropic murine leukemia virus-related viruses

    PubMed Central

    2011-01-01

    In 2009, a newly discovered human retrovirus, xenotropic murine leukemia virus (MuLV)-related virus (XMRV), was reported by Lombardi et al. in 67% of persons from the US with chronic fatigue syndrome (CFS) by PCR detection of gag sequences. Although six subsequent studies have been negative for XMRV, CFS was defined more broadly using only the CDC or Oxford criteria and samples from the US were limited in geographic diversity, both potentially reducing the chances of identifying XMRV positive CFS cases. A seventh study recently found polytropic MuLV sequences, but not XMRV, in a high proportion of persons with CFS. Here we tested blood specimens from 45 CFS cases and 42 persons without CFS from over 20 states in the United States for both XMRV and MuLV. The CFS patients all had a minimum of 6 months of post-exertional malaise and a high degree of disability, the same key symptoms described in the Lombardi et al. study. Using highly sensitive and generic DNA and RNA PCR tests, and a new Western blot assay employing purified whole XMRV as antigen, we found no evidence of XMRV or MuLV in all 45 CFS cases and in the 42 persons without CFS. Our findings, together with previous negative reports, do not suggest an association of XMRV or MuLV in the majority of CFS cases. PMID:21342521

  7. How home HIV testing and counselling with follow-up support achieves high testing coverage and linkage to treatment and prevention: a qualitative analysis from Uganda

    PubMed Central

    Ware, Norma C; Wyatt, Monique A; Asiimwe, Stephen; Turyamureeba, Bosco; Tumwesigye, Elioda; van Rooyen, Heidi; Barnabas, Ruanne V; Celum, Connie L

    2016-01-01

    Introduction The successes of HIV treatment scale-up and the availability of new prevention tools have raised hopes that the epidemic can finally be controlled and ended. Reduction in HIV incidence and control of the epidemic requires high testing rates at population levels, followed by linkage to treatment or prevention. As effective linkage strategies are identified, it becomes important to understand how these strategies work. We use qualitative data from The Linkages Study, a recent community intervention trial of community-based testing with linkage interventions in sub-Saharan Africa, to show how lay counsellor home HIV testing and counselling (home HTC) with follow-up support leads to linkage to clinic-based HIV treatment and medical male circumcision services. Methods We conducted 99 semi-structured individual interviews with study participants and three focus groups with 16 lay counsellors in Kabwohe, Sheema District, Uganda. The participant sample included both HIV+ men and women (N=47) and HIV-uncircumcised men (N=52). Interview and focus group audio-recordings were translated and transcribed. Each transcript was summarized. The summaries were analyzed inductively to identify emergent themes. Thematic concepts were grouped to develop general constructs and framing propositional statements. Results Trial participants expressed interest in linking to clinic-based services at testing, but faced obstacles that eroded their initial enthusiasm. Follow-up support by lay counsellors intervened to restore interest and inspire action. Together, home HTC and follow-up support improved morale, created a desire to reciprocate, and provided reassurance that services were trustworthy. In different ways, these functions built links to the health service system. They worked to strengthen individuals’ general sense of capability, while making the idea of accessing services more manageable and familiar, thus reducing linkage barriers. Conclusions Home HTC with follow

  8. Empowering patients to link to care and treatment: qualitative findings about the role of a home-based HIV counselling, testing and linkage intervention in South Africa.

    PubMed

    Knight, Lucia C; Van Rooyen, Heidi; Humphries, Hilton; Barnabas, Ruanne V; Celum, Connie

    2015-01-01

    To explore the barriers and facilitators of linkage to and retention in care amongst persons who tested positive for HIV, qualitative research was conducted in a home-based HIV counselling and testing (HBCT) project with interventions to facilitate linkages to HIV care in rural KwaZulu-Natal, South Africa. The intervention tested 1272 adults for HIV in Vulindlela of whom 32% were HIV positive, received point-of-care (POC) CD4 testing and referral to local HIV clinics. Those testing positive also received follow-up visits from a counsellor to evaluate linkages to care. The study employed a qualitative methodology collecting data through in-depth semi-structured interviews. Respondents included 25 HIV-positive persons who had tested as part of HBCT project, 4 intervention research counsellors who delivered the HBCT intervention and 9 government clinic staff who received referrals for care. The results show that HBCT helped to facilitate linkage to care through providing education and support to help overcome fears of stigma and discrimination. The results show the perceived value of receiving a POC CD4 result during post-test counselling, both for those newly diagnosed and those previously diagnosed as HIV positive. The results also demonstrate that in-depth counselling creates an "educated consumer" facilitating engagement with clinical services. The study provides qualitative insights into the acceptability of confidential HBCT with same day POC CD4 testing and counselling as factors that influenced HIV-positive persons' decisions to link to care. This model warrants further evaluation in non-research settings to determine impact and cost-effectiveness relative to other HIV testing and referral strategies. PMID:25923366

  9. The Use of Qualitative Evaluation Methods to Test Internal Validity: An Example in a Work Site Health Promotion Program.

    ERIC Educational Resources Information Center

    Steckler, Allan

    1989-01-01

    A qualitative case study and monitoring data complemented a study of the evaluation of a health promotion program (Project LIFE) conducted in 23 rubber-producing plants in the United States. The study illustrates assessment of threats to internal validity, including the degree to which the planned intervention was actually implemented. (SLD)

  10. Digital droplet PCR on disk.

    PubMed

    Schuler, Friedrich; Trotter, Martin; Geltman, Marcel; Schwemmer, Frank; Wadle, Simon; Domínguez-Garrido, Elena; López, María; Cervera-Acedo, Cristina; Santibáñez, Paula; von Stetten, Felix; Zengerle, Roland; Paust, Nils

    2016-01-01

    Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis. PMID:26610263

  11. Hybrid Capture 2 is as effective as PCR testing for high-risk human papillomavirus in head and neck cancers.

    PubMed

    Hooper, Jody E; Hebert, Jessica F; Schilling, Amy; Gross, Neil D; Schindler, Joshua S; Lagowski, James P; Kulesz-Martin, Molly; Corless, Christopher L; Morgan, Terry K

    2015-04-01

    High-risk human papillomavirus (HPV) infection is a common cause of oropharyngeal squamous cell carcinoma, especially in young male nonsmokers. Accurately diagnosing HPV-associated oral cancers is important, because they have a better prognosis and may be treated differently than smoking-related oral carcinomas. Various methods have been validated to test for high-risk HPV in cervical tissue samples, and they are in routine clinical use to detect dysplasia before it progresses to invasive disease. Similarly, future screening for HPV-mediated oropharyngeal dysplasia may identify patients before it progresses. Our objective was to compare 4 of these methods in a retrospective series of 87 oral and oropharyngeal squamous cell carcinomas that had archived fresh-frozen and paraffin-embedded tissue for evaluation. Patient age, sex, smoking history, and tumor location were also recorded. DNA prepared from fresh-frozen tissue was tested for HPV genotypes by multiplex polymerase chain reaction analysis, and high-risk HPV screening was carried out using Hybrid Capture 2 and Cervista. Histologic sections were immunostained for p16. HPV-positive outcome was defined as agreement between at least 2 of the 3 genetic tests and used for χ analysis and calculations of diagnostic predictive value. As expected, high-risk HPV-positive oral cancers were most common in the tonsil and base of the tongue (oropharynx) of younger male (55 vs. 65 y) (P=0.0002) nonsmokers (P=0.01). Most positive cases were HPV16 (33/36, 92%). Hybrid Capture 2 and Cervista were as sensitive as polymerase chain reaction and had fewer false positives than p16 immunohistochemical staining. PMID:25839700

  12. Validation of a Commercial Insulated Isothermal PCR-based POCKIT Test for Rapid and Easy Detection of White Spot Syndrome Virus Infection in Litopenaeus vannamei

    PubMed Central

    Tsai, Yun-Long; Wang, Han-Ching; Lo, Chu-Fang; Tang-Nelson, Kathy; Lightner, Donald; Ou, Bor-Rung; Hour, Ai-Ling; Tsai, Chuan-Fu; Yen, Cheng-Chi; Chang, Hsiao-Fen Grace; Teng, Ping-Hua; Lee, Pei-Yu

    2014-01-01

    Timely pond-side detection of white spot syndrome virus (WSSV) plays a critical role in the implementation of bio-security measures to help minimize economic losses caused by white spot syndrome disease, an important threat to shrimp aquaculture industry worldwide. A portable device, namely POCKIT™, became available recently to complete fluorescent probe-based insulated isothermal PCR (iiPCR), and automatic data detection and interpretation within one hour. Taking advantage of this platform, the IQ Plus™ WSSV Kit with POCKIT system was established to allow simple and easy WSSV detection for on-site users. The assay was first evaluated for its analytical sensitivity and specificity performance. The 95% limit of detection (LOD) of the assay was 17 copies of WSSV genomic DNA per reaction (95% confidence interval [CI], 13 to 24 copies per reaction). The established assay has detection sensitivity similar to that of OIE-registered IQ2000™ WSSV Detection and Protection System with serial dilutions of WSSV-positive Litopenaeus vannamei DNA. No cross-reaction signals were generated from infectious hypodermal and haematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), and hepatopancreatic parvovirus (HPV) positive samples. Accuracy analysis using700 L. vannamei of known WSSV infection status shows that the established assayhassensitivity93.5% (95% CI: 90.61–95.56%) and specificity 97% (95% CI: 94.31–98.50%). Furthermore, no discrepancy was found between the two assays when 100 random L. vannamei samples were tested in parallel. Finally, excellent correlation was observed among test results of three batches of reagents with 64 samples analyzed in three different laboratories. Working in a portable device, IQ Plus™ WSSV Kit with POCKIT system allows reliable, sensitive and specific on-site detection of WSSV in L. vannamei. PMID:24625894

  13. Estimation of test characteristics of real-time PCR and bacterial culture for diagnosis of subclinical intramammary infections with Streptococcus agalactiae in Danish dairy cattle in 2012 using latent class analysis.

    PubMed

    Mahmmod, Yasser S; Toft, Nils; Katholm, Jørgen; Grønbæk, Carsten; Klaas, Ilka C

    2013-05-01

    The misdiagnosis of intramammary infections (IMI) with Streptococcus agalactiae (S. agalactiae) could lead farmers to treat or cull animals unnecessarily. The objective of this field study was to estimate the sensitivity (Se) and specificity (Sp) of real-time PCR at different cut-offs for cycle threshold (Ct) values against bacterial culture (BC) for diagnosis of S. agalactiae IMI using latent class analysis to avoid the assumption of a perfect reference test. A total of 614 dairy cows were randomly selected from 6 herds with bulk tank PCR Ct value ≤ 39 for S. agalactiae and S. aureus. At milk recording, 2456 quarter milk samples were taken aseptically for BC and the routinely taken cow level milk samples were analyzed by PCR. Results showed that 53 cows (8.6%) were positive for S. agalactiae IMI by BC. Sensitivity of PCR at cut-offs; ≤ 39, ≤ 37, ≤ 34, and ≤ 32, was 96.2%, 91.9%, 87.2% and 73.9%, while Se of BC was 25.7%, 29.9%, 59.9% and 72.1%. Specificity of PCR at cut-offs; ≤ 39, ≤ 37, ≤ 34, and ≤ 32, was 96.8%, 96.9%, 96.7%, and 97.22%, while Sp of BC was 99.7%, 99.5%, 99.2%, and 98.9%. The estimated prevalence of S. agalactiae IMI by PCR was higher than the apparent prevalence at the tested cut-offs, indicating under estimation of S. agalactiae IMI in the examined dairy cows. In conclusion, Se of PCR is always higher than Se of BC at all tested cut-offs. The lower cut-off, the more comparable becomes Se of PCR and Se of BC. The changes in Se in both PCR and BC at different Ct-value cut-offs may indicate a change in the definition of the latent infection. The similar Se of both tests at cut-off ≤ 32 may indicate high concentrations of S. agalactiae viable cells, representing a cow truly/heavily infected with S. agalactiae and thus easier to detect with BC. At cut-off ≤ 39 the latent definition of infection may reflect a more general condition of cows being positive for S. agalactiae. Our findings indicate that PCR Ct-value cut-offs should

  14. Development and testing of real-time PCR assays for determining fecal loading and source identification (cattle, human, etc.) in surface water and groundwater

    NASA Astrophysics Data System (ADS)

    McKay, L. D.; Layton, A.; Gentry, R.

    2004-12-01

    A multi-disciplinary group of researchers at the University of Tennessee is developing and testing a series of microbial assay methods based on real-time PCR to detect fecal bacterial concentrations and host sources in water samples. Real-time PCR is an enumeration technique based on the unique and conserved nucleic acid sequences present in all organisms. The first research task was development of an assay (AllBac) to detect total amount of Bacteroides, which represents up to 30 percent of fecal mass. Subsequent assays were developed to detect Bacteroides from cattle (BoBac) and humans (HuBac) using 16sRNA genes based on DNA sequences in the national GenBank, as well as sequences from local fecal samples. The assays potentially have significant advantages over conventional bacterial source tracking methods because: 1. unlike traditional enumeration methods, they do not require bacterial cultivation; 2. there are no known non-fecal sources of Bacteroides; 3. the assays are quantitative with results for total concentration and for each species expressed in mg/l; and 4. they show little regional variation within host species, meaning that they do not require development of extensive local gene libraries. The AllBac and BoBac assays have been used in a study of fecal contamination in a small rural watershed (Stock Creek) near Knoxville, TN, and have proven useful in identification of areas where cattle represent a significant fecal input and in development of BMPs. It is expected that these types of assays (and future assays for birds, hogs, etc.) could have broad applications in monitoring fecal impacts from Animal Feeding Operations, as well as from wildlife and human sources.

  15. Multi-center evaluation of the novel fully-automated PCR-based Idylla™ BRAF Mutation Test on formalin-fixed paraffin-embedded tissue of malignant melanoma.

    PubMed

    Melchior, Linea; Grauslund, Morten; Bellosillo, Beatriz; Montagut, Clara; Torres, Erica; Moragón, Ester; Micalessi, Isabel; Frans, Johan; Noten, Veerle; Bourgain, Claire; Vriesema, Renske; van der Geize, Robert; Cokelaere, Kristof; Vercooren, Nancy; Crul, Katrien; Rüdiger, Thomas; Buchmüller, Diana; Reijans, Martin; Jans, Caroline

    2015-12-01

    The advent of BRAF-targeted therapies led to increased survival in patients with metastatic melanomas harboring a BRAF V600 mutation (implicated in 46-48% of malignant melanomas). The Idylla(™) System (Idylla(™)), i.e., the real-time-PCR-based Idylla(™) BRAF Mutation Test performed on the fully-automated Idylla(™) platform, enables detection of the most frequent BRAF V600 mutations (V600E/E2/D, V600K/R/M) in tumor material within approximately 90 min and with 1% detection limit. Idylla(™) performance was determined in a multi-center study by analyzing BRAF mutational status of 148 archival formalin-fixed paraffin-embedded (FFPE) tumor samples from malignant melanoma patients, and comparing Idylla(™) results with assessments made by commercial or in-house routine diagnostic methods. Of the 148 samples analyzed, Idylla(™) initially recorded 7 insufficient DNA input calls and 15 results discordant with routine method results. Further analysis learned that the quality of 8 samples was insufficient for Idylla(™) testing, 1 sample had an invalid routine test result, and Idylla(™) results were confirmed in 10 samples. Hence, Idylla(™) identified all mutations present, including 7 not identified by routine methods. Idylla(™) enables fully automated BRAF V600 testing directly on FFPE tumor tissue with increased sensitivity, ease-of-use, and much shorter turnaround time compared to existing diagnostic tests, making it a tool for rapid, simple and highly reliable analysis of therapeutically relevant BRAF mutations, in particular for diagnostic units without molecular expertise and infrastructure. PMID:26407762

  16. Preventive Care for Women in Prison: A Qualitative Community Health Assessment of the Papanicolaou Test and Follow-Up Treatment at a California State Women’s Prison

    PubMed Central

    Magee, Catherine G.; Hult, Jen R.; Turalba, Ruby; McMillan, Shelby

    2005-01-01

    Growing evidence indicates that women in prison are particularly vulnerable to many negative health outcomes, including cervical cancer. The Papanicolaou (Pap) test is an effective tool to screen for this disease. To determine what is and is not working with the Pap test and follow-up treatment, we performed qualitative interviews with women prisoners and key informants at a California state women’s prison. Our assessment revealed that the process of administering Pap tests at this institution was not meeting the health care needs of the women interviewed. Women reported having negative experiences during the test and with their health care providers. Additionally the prison’s culture and infrastructure create obstacles that hinder prisoners from receiving quality care and providers from delivering that care. In response, women prisoners use self-and community advocacy to meet their health care needs and cope with these challenges. PMID:16186450

  17. Comparison of Leishmania OligoC-TesT PCR with conventional and real-time PCR for Diagnosis of canine Leishmania infection.

    PubMed

    Carson, Connor; Quinnell, Rupert J; Holden, Jennifer; Garcez, Lourdes M; Deborggraeve, Stijn; Courtenay, Orin

    2010-09-01

    There is a need for standardization and simplification of the existing methods for molecular detection of Leishmania infantum in the canine reservoir host. The commercially available OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic dipstick detection of PCR products and is highly sensitive for use in humans but not yet independently validated for use in dogs. Here we compare the sensitivity of OligoC-TesT with those of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer 1 (ITS-1) PCR, and a PCR-hybridization protocol, using longitudinal naturally infected canine bone marrow samples whose parasite burdens were measured by real-time quantitative PCR (qPCR). The sensitivity of OligoC-TesT for infected dogs was 70% (95% confidence interval [CI], 63 to 78%), similar to that of kDNA PCR (72%; 95% CI, 65 to 80%; P = 0.69) but significantly greater than those of PCR-hybridization (61%; 95% CI, 53 to 69%; P = 0.007) and ITS-1 nested PCR (54%; 95% CI, 45 to 62%; P < 0.001); real-time qPCR had the highest sensitivity (91%; 95% CI, 85 to 95%; P < 0.001). OligoC-TesT sensitivity was greater for polysymptomatic and oligosymptomatic dogs than for asymptomatic dogs (93%, 74%, and 61%, respectively; P = 0.005), a trend also observed for the other qualitative PCR methods tested (P Test positivity increased with increasing parasite burdens, as measured by real-time qPCR: OligoC-TesT and kDNA PCR detected 100% and 99% of positive samples when parasite burdens exceeded 74 and 49 parasites/ml, respectively. OligoC-TesT has high sensitivity for detection of canine Leishmania infections; its ease of operation and ease of interpretation are further advantages for veterinary diagnostic laboratories and for large-scale survey work in developing countries. PMID:20631112

  18. Provider-Initiated HIV Testing for Migrants in Spain: A Qualitative Study with Health Care Workers and Foreign-Born Sexual Minorities

    PubMed Central

    Navaza, Barbara; Abarca, Bruno; Bisoffi, Federico; Pool, Robert; Roura, Maria

    2016-01-01

    Introduction Provider-initiated HIV testing (PITC) is increasingly adopted in Europe. The success of the approach at identifying new HIV cases relies on its effectiveness at testing individuals most at risk. However, its suitability to reach populations facing overlapping vulnerabilities is under researched. This qualitative study examined HIV testing experiences and perceptions amongst Latin-American migrant men who have sex with men and transgender females in Spain, as well as health professionals’ experiences offering HIV tests to migrants in Barcelona and Madrid. Methods We conducted 32 in-depth interviews and 8 discussion groups with 38 Latin-American migrants and 21 health professionals. We imported verbatim transcripts and detailed field work notes into the qualitative software package Nvivo-10 and applied to all data a coding framework to examine systematically different HIV testing dimensions and modalities. The dimensions analysed were based on the World Health Organization “5 Cs” principles: Consent, Counselling, Connection to treatment, Correctness of results and Confidentiality. Results Health professionals reported that PITC was conceptually acceptable for them, although their perceived inability to adequately communicate HIV+ results and resulting bottle necks in the flow of care were recurrent concerns. Endorsement and adherence to the principles underpinning the rights-based response to HIV varied widely across health settings. The offer of an HIV test during routine consultations was generally appreciated by users as a way of avoiding the embarrassment of asking for it. Several participants deemed compulsory testing as acceptable on public health grounds. In spite of—and sometimes because of—partial endorsement of rights-based approaches, PITC was acceptable in a population with high levels of internalised stigma. Conclusion PITC is a promising approach to reach sexual minority migrants who hold high levels of internalised stigma but

  19. Laboratory Evaluations of the Enterococcus qPCR Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements

    EPA Science Inventory

    The BEACH Act of 2000 directed the U.S. EPA to establish more expeditious methods for the detection of pathogen indicators in coastal waters, as well as new water quality criteria based on these methods. Progress has been made in developing a quantitative PCR (qPCR) method for en...

  20. Comparing family members' motivations and attitudes towards genetic testing for hereditary breast and ovarian cancer: a qualitative analysis

    PubMed Central

    Dancyger, Caroline; Smith, Jonathan A; Jacobs, Chris; Wallace, Melissa; Michie, Susan

    2010-01-01

    Genetic testing for hereditary breast and ovarian cancer reveals significant risk information regarding one's chances of developing cancer that has potential implications for patients and their families. This study reports on the motivations and attitudes of index patients and their relatives towards genetic testing for hereditary breast and ovarian cancer. In total, 10 female index patients and 20 of their relatives were interviewed regarding their experiences of communicating genetic information within their families, and their motivations and attitudes towards genetic testing. The analysis found two types of ‘family groups': groups strongly committed to genetic testing and groups uncertain about testing. Within committed family groups, index patients and their relatives felt obliged to be tested for others, leading some relatives to be tested without having fully thought through their decision or the implications of knowing their mutation status. These family groups also described considerations in relation to the value of testing for themselves. In family groups uncertain about testing, relatives had not attended for predictive testing, had postponed decision making until some point in the future or had expressed ambivalence about the value of testing for themselves. Results suggest the value of explicitly acknowledging motivations for genetic testing within the context of family obligations, relationships and communication, and the possible value of involving family members in genetic counselling and decision making from a family's first contact with genetic services. PMID:20648056

  1. Qualitative indices and enhancement rate of CT pulmonary angiography in patients with suspected pulmonary embolism: Comparison between test bolus and bolus-tracking methods

    PubMed Central

    Moradi, Maryam; Khalili, Babak

    2016-01-01

    Background: The aim of the present study was to assess the qualitative indices and enhancement rate of computed tomographic pulmonary angiography (CTPA) in patients with suspected pulmonary embolism using Test bolus and Bolus-tracking techniques. Materials and Methods: Fifty-two patients with suspected pulmonary embolism that passed informed consent were randomly divided in the two groups. In each group, demographic characteristics, qualitative indices, and enhancement rate of CTPA were recorded. Results: The diagnostic result obtained in majority of the participants in the two groups (88.5 % in Test bolus group vs. 73.1% in the Bolus tracking group). In the case of quantitative variables, no statistically significant differences were found between the groups (P > 0.05). The only statistically significant difference between the two groups is average of “X-ray dose”. Conclusion: The results of our study show that there is no statistically significant difference between the Bolus Tracking and Test Bolus techniques for producing more homogeneous enhancement. PMID:27403408

  2. Conceptualising quality of life outcomes for women participating in testing for sexually transmitted infections: A systematic review and meta-synthesis of qualitative research.

    PubMed

    Jackson, Louise J; Roberts, Tracy E

    2015-10-01

    Many public health interventions have aims which are broader than health alone; this means that there are difficulties in using outcome measures that capture health effects only, such as Quality Adjusted Life Years (QALYs). Sexually Transmitted Infections (STIs) are a major public health concern both in the UK and globally, with Chlamydia trachomatis being the most common bacterial STI worldwide. There is scope for the wider use of qualitative syntheses in health-related research; in this study we highlight their potential value in informing outcome identification, particularly for public health interventions where a broad range of outcomes may need to be considered. This article presents a systematic review and meta-ethnography of qualitative studies that investigated women's experiences of thinking about and participating in testing for chlamydia. The meta-ethnography highlights issues relating to beliefs about STIs and testing, assessing risk and interpreting symptoms, emotional responses to testing, coping with diagnosis, relationship with sex partners(s), informal support, and interaction with health care services. The study findings suggest that women can experience a range of impacts on their health and quality of life. It is important that this range of effects is taken into account within evaluations, to ensure that decision makers are fully informed about the outcomes associated with screening interventions, and ultimately, to make sure that appropriate interventions are available to support women in maintaining good sexual health. PMID:26360418

  3. Qualitative Evaluation.

    ERIC Educational Resources Information Center

    Stone, James C., Ed.; James, Raymond A., Ed.

    1981-01-01

    "Qualitative evaluation" is the theme of this issue of the California Journal of Teacher Education. Ralph Tyler states that evaluation is essentially descriptive, and using numbers does not solve basic problems. Martha Elin Vernazza examines the issue of objectivity in history and its implications for evaluation. She posits that the decisive…

  4. What Do Parents Think about Chromosomal Microarray Testing? A Qualitative Report from Parents of Children with Autism Spectrum Disorders

    PubMed Central

    Mitchell, Linda Crane; Richman, Alice R.; Clawson, Kaitlyn

    2016-01-01

    Background. Chromosomal Microarray Analysis (CMA) is increasingly utilized to detect copy number variants among children and families affected with autism spectrum disorders (ASD). However, CMA is controversial due to possible ambiguous test findings, uncertain clinical implications, and other social and legal issues related to the test. Methods. Participants were parents of children with ASD residing in the North Eastern region of North Carolina, USA. We conducted individual, face-to-face interviews with 45 parents and inquired about their perceptions of CMA. Results. Three major themes dominated parents' perceptions of CMA. None of the parents had ever heard of the test before and the majority of the parents postulated positive attitudes toward the test. Parents' motivations in undergoing the test were attributed to finding a potential cause of ASD, to being better prepared for having another affected child, and to helping with future reproductive decisions. Perceived barriers included the cost of testing, risk/pain of CMA testing, and fear of test results. Conclusion. This study contributes to the understanding of psychosocial aspects and cultural influences towards adoption of genetic testing for ASD in clinical practice. Genetic education can aid informed decision-making related to CMA genetic testing among parents of children with ASD. PMID:27413549

  5. Evaluation of performances of VERSANT HCV RNA 1.0 assay (kPCR) and Roche COBAS AmpliPrep/COBAS TaqMan HCV test v2.0 at low level viremia.

    PubMed

    Mazzuti, Laura; Lozzi, Maria Antonietta; Riva, Elisabetta; Maida, Paola; Falasca, Francesca; Antonelli, Guido; Turriziani, Ombretta

    2016-09-01

    We assess the concordance between low level HCV values obtained using the VERSANT HCV RNA 1.0 Assay (kPCR) and COBAS AmpliPrep/COBAS TaqMan HCV Quantitative Test v2.0. The correlation between the values obtained by the two RT-PCR assays for samples with quantifiable HCV RNA levels revealed that viral load measured by kPCR significantly correlated with that of the CAP/CTM (R=0.644, P<0.0001). The results show a good concordance (n=126/144, 87%); discordant results were mainly observed in the assessment of values below the lower limit of detection of the assays. These variations may have an impact on clinical decisions for patients on HCV triple therapy or interferon- free regimens. It is therefore recommended to monitor individual patients with the same test throughout treatment. PMID:27602422

  6. Comparison of the Novel Partec Rapid Malaria Test to the Conventional Giemsa Stain and the Gold Standard Real-Time PCR

    PubMed Central

    Nkrumah, Bernard; Agyekum, Alex; Acquah, Samuel E. K.; May, Jürgen; Tannich, Egbert; Brattig, Norbert; Nguah, Samuel Blay; von Thien, Heidrun; Adu-Sarkodie, Yaw; Huenger, Frank

    2010-01-01

    Malaria remains the single most frequent cause of death in Africa, killing one child every 30 s, but treatment decisions are often made only on clinical diagnosis, as laboratory techniques to confirm the clinical suspicion are labor intensive and costly. In this study, we evaluated the recently developed Partec rapid malaria test (PM) for the detection of Plasmodium spp. in human blood from patients in an area where malaria is endemic and compared the results with those of thick blood film Giemsa stain (GS) in terms of its performance and operational characteristics, using real-time (RT) PCR as the gold standard. The sensitivities of the PM and the GS were 62.2% (95% CI, 56.3 to 67.8) and 61.8% (95% CI, 55.9 to 67.4), respectively, while the specificities were 96.0% (95% CI, 92.3 to 98.3) and 98% (95% CI, 95.0 to 99.5), respectively. There was an excellent agreement between the results for the PM and those of the GS (k [level of agreement] = 0.96; P < 0.001). The results for the PM were obtained more quickly and at less cost than those for the GS. The performance characteristics of the PM were almost equal to those of the GS, but the operational characteristics were better, and the PM can therefore be considered as an alternative method for GS. PMID:20554822

  7. Sex Determination Using PCR

    ERIC Educational Resources Information Center

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  8. Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil

    PubMed Central

    2014-01-01

    Background Currently there is a significant risk of infection with hepatitis B virus (HBV) during blood transfusion in high epidemic area. This is due to the pre-seroconversion window period, immunovariant viral strains and the presence of occult HBV infection (OBI). The aim of this study was to develop an in-house real-time PCR-based method, which was both ultra-sensitive and efficient offering an alternative method for nucleic acid testing (NAT). Methods A precore fragment with 109 bp was cloned and serial diluted to standard curve construction. The calibration of the HBV - DNA values was performed against OptiQuant® HBV-DNA Quantification Panel, Acrometrix Europe B.V.). Results From our in-house plasmid we prepared serial dilutions ranging from 2 × 103 – 2 × 109 copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r2 = 0.99). The limit of detection for the assay with pHBVRO standards was 2000/ml in a total reaction volume of 30 μl. We found a strong correlation between the two methods (r2 = 0.9965 and p < 0.0001). The regression line give us the following equation: Log 10 (IU/mL) = 0.9038Log 10 (copies/mL) − 1.0643, suggesting that 1 IU/mL = 15 copies/mL. Conclusions Therefore, we can affirm that the qHBVRO PCR can detect HBV DNA in individuals with hepatitis B at any stage of the disease showing high capacity for NAT screening in hepatitis b donors. This results of sensitivity could provide an advance for automation in blood banks and increasing safety of patients who receive blood transfusions. PMID:24472141

  9. HIV and Syphilis Testing Preferences among Men Who Have Sex with Men in South China: A Qualitative Analysis to Inform Sexual Health Services

    PubMed Central

    Lee, Ramon; Lo, Elaine J.; Yang, Li Gang; Yang, Bin; Peeling, Rosanna W.; Tucker, Joseph D.

    2015-01-01

    could further evaluate HIV/syphilis testing programs responsive to MSM preferences. Short Summary A qualitative study of MSM in South China found that men preferred rapid STD testing at MSM-focused test centers, but were concerned about test quality assurance and confidentiality. PMID:25875336

  10. Testing the WHO responsiveness concept in the Iranian mental healthcare system: a qualitative study of service users

    PubMed Central

    2011-01-01

    Background Individuals' experience of interacting with the healthcare system has significant impact on their overall health and well-being. To relate patients' experiences to a common set of standards, the World Health Organization (WHO) developed the concept of health system responsiveness. This study aimed to assess if the WHO responsiveness concept reflected the non-medical expectations of mental healthcare users in Teheran. Methods In this qualitative study, four mixed focus group discussions were formed, comprising 53 mental health service users in Tehran, Iran, in 2010. Content analysis was performed for data analysis. Responses were examined in relation to the eight domains of the WHO's responsiveness model. Results There were many commonalities between the findings of this study and the eight domains of the WHO responsiveness model, although some variations were found. Effective care was a new domain generated from our findings. In addition, the domain of prompt attention was included in two new labelled domains: attention and access to care. Participants could not differentiate autonomy from choice of healthcare provider, believing that free choice is part of autonomy. Therefore these domains were unified under the name of autonomy. The domains of quality of basic amenities, access to social support, dignity and confidentiality were considered to be important for the responsiveness concept. Some differences regarding how these domains should be defined were observed, however. Conclusions The results showed that the concept of responsiveness developed by the WHO is applicable to mental health services in Iran. These findings might help policy-makers' better understanding of what is useful for the improvement of mental health services. PMID:22115499

  11. Qualitative Assessment of Barriers and Facilitators of Access to HIV Testing Among Men Who Have Sex with Men in China.

    PubMed

    Liu, Yu; Sun, Xiaoyun; Qian, Han-Zhu; Yin, Lu; Yan, Zheng; Wang, Lijuan; Jiang, Shulin; Lu, Hongyan; Ruan, Yuhua; Shao, Yiming; Vermund, Sten H; Amico, K Rivet

    2015-09-01

    Diagnosis of HIV is the entry point into the continuum of HIV care; a well-recognized necessary condition for the ultimate prevention of onward transmission. In China, HIV testing rates among men who have sex with men (MSM) are low compared to other high risk subgroups, yet experiences with HIV testing among MSM in China are not well understood. To address this gap and prepare for intervention development to promote HIV testing and rapid linkage to treatment, six focus groups (FGs) were conducted with MSM in Beijing (40 HIV-positive MSM participated in one of four FGs and 20 HIV-negative or status unknown MSM participated in one of two FGs). Major themes reported as challenges to HIV testing included stigma and discrimination related to HIV and homosexuality, limited HIV knowledge, inconvenient clinic times, not knowing where to get a free test, fear of positive diagnosis or nosocomial infection, perceived low service quality, and concerns/doubts about HIV services. Key facilitators included compensation, peer support, professionalism, comfortable testing locations, rapid testing, referral and support after diagnosis, heightened sense of risk through engagement in high-risk behaviors, sense of responsibility to protect self, family and partner support, and publicity via social media. Themes and recommendations were generally consistent across HIV-positive and negative/status unknown groups, although examples of enacted stigma were more prevalent in the HIV-positive groups. Findings from our study provide policy suggestions for how to bolster current HIV prevention intervention efforts to enhance 'test-and-treat' strategies for Chinese MSM. PMID:26186029

  12. Qualitative Assessment of Barriers and Facilitators of Access to HIV Testing Among Men Who Have Sex with Men in China

    PubMed Central

    Liu, Yu; Sun, Xiaoyun; Yin, Lu; Yan, Zheng; Wang, Lijuan; Jiang, Shulin; Lu, Hongyan; Ruan, Yuhua; Shao, Yiming; Vermund, Sten H.

    2015-01-01

    Abstract Diagnosis of HIV is the entry point into the continuum of HIV care; a well-recognized necessary condition for the ultimate prevention of onward transmission. In China, HIV testing rates among men who have sex with men (MSM) are low compared to other high risk subgroups, yet experiences with HIV testing among MSM in China are not well understood. To address this gap and prepare for intervention development to promote HIV testing and rapid linkage to treatment, six focus groups (FGs) were conducted with MSM in Beijing (40 HIV-positive MSM participated in one of four FGs and 20 HIV-negative or status unknown MSM participated in one of two FGs). Major themes reported as challenges to HIV testing included stigma and discrimination related to HIV and homosexuality, limited HIV knowledge, inconvenient clinic times, not knowing where to get a free test, fear of positive diagnosis or nosocomial infection, perceived low service quality, and concerns/doubts about HIV services. Key facilitators included compensation, peer support, professionalism, comfortable testing locations, rapid testing, referral and support after diagnosis, heightened sense of risk through engagement in high-risk behaviors, sense of responsibility to protect self, family and partner support, and publicity via social media. Themes and recommendations were generally consistent across HIV-positive and negative/status unknown groups, although examples of enacted stigma were more prevalent in the HIV-positive groups. Findings from our study provide policy suggestions for how to bolster current HIV prevention intervention efforts to enhance ‘test-and-treat’ strategies for Chinese MSM. PMID:26186029

  13. Improving design and conduct of randomised trials by embedding them in qualitative research: ProtecT (prostate testing for cancer and treatment) study

    PubMed Central

    Donovan, Jenny; Mills, Nicola; Smith, Monica; Brindle, Lucy; Jacoby, Ann; Peters, Tim; Frankel, Stephen; Neal, David; Hamdy, Freddie

    2002-01-01

    Problem Recruitment to randomised trials is often difficult, and many important trials are not mounted because recruitment is thought to be “impossible.” Design Controversial ProtecT (prostate testing for cancer and treatment) trial embedded within qualitative research. Background and setting Screening for prostate cancer is hotly debated, and evidence from trials about the effectiveness of treatments (surgery, radiotherapy, and monitoring) is lacking. Mounting a treatment trial is controversial because of past failures and concerns that differences in complications of treatment but not survival make randomisation unacceptable to patients and clinicians, particularly for a trial including monitoring. Strategy for change In-depth interviews explored interpretation of study information. Audiotape recordings of recruitment appointments enabled scrutiny of content and presentation of study information by recruiters. Initial qualitative findings showed that recruiters had difficulty discussing equipoise and presenting treatments equally; they unknowingly used terminology that was misinterpreted by participants. Findings were used to determine changes to content and presentation of information. Effects of change Changes to the order of presenting treatments encouraged emphasis on equivalence, misinterpreted terms were avoided, the non-radical arm was redefined, and randomisation and clinical equipoise were presented more convincingly. The randomisation rate increased from 40% to 70%, all treatments became acceptable, and the three arm trial became the preferred design. Lessons learnt Changes to information and presentation resulted in efficient recruitment acceptable to patients and clinicians. Embedding this controversial trial within qualitative research improved recruitment. Such methods probably have wider applicability and may enable even the most difficult evaluative questions to be tackled. PMID:12364308

  14. The characterization of four gene expression analysis in circulating tumor cells made by Multiplex-PCR from the AdnaTest kit on the lab-on-a-chip Agilent DNA 1000 platform

    PubMed Central

    Škereňová, Markéta; Mikulová, Veronika; Čapoun, Otakar; Zima, Tomáš

    2016-01-01

    Introduction Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing. Materials and methods A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination. Results The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample. Conclusions The characteristics established in our study are in concordance with the manufacturer’s specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization. PMID:26981024

  15. Evaluation of PCR-Reverse Blot Hybridization Assay, REBA Sepsis-ID Test, for Simultaneous Identification of Bacterial Pathogens and mecA and van Genes from Blood Culture Bottles

    PubMed Central

    Park, Soon Deok; Lee, Gyusang; Wang, Hye-young; Park, Min; Kim, Sunghyun; Kim, Hyunjung; Kim, Jungho; Kim, Young Keun; Kim, Hyo Youl; Lee, Hyeyoung

    2014-01-01

    Background The aim of this study was to evaluate a newly developed PCR-based reverse blot hybridization assay (PCR-REBA), REBA Sepsis-ID (M&D, Wonju, Korea), to rapidly detect the presence of bacteremia and antimicrobial resistance gene in blood culture samples. Methods One thousand four hundred consecutive blood culture samples from patients with a delta neutrophil index greater than 2.7% were selected from March to July in 2013. Three hundred positive and 1,100 negative for bacterial growth in blood culture bottles samples were tested by conventional and real-time PCR-REBA, respectively. Results The overall agreement between the conventional identification test and the REBA Sepsis-ID test was 95.3% (286/300). Agreement for gram-positive bacteria, gram-negative bacteria, fungi, and polymicrobials was 94.5% (190/201), 97.3% (71/73), 100% (14/14), and 91.7% (11/12), respectively. The detection rate of the mecA gene from methicillin-resistant Staphylococcus isolates was 97.8% (90/92). The vanA gene was detected in one blood culture sample from which vancomycin-resistant Enterococcus was isolated. When the cycle threshold for real-time PCR was defined as 30.0, 2.4% (26/1,100) of negative blood culture samples tested positive by real-time PCR. Conclusions The REBA Sepsis-ID test is capable of simultaneously and quickly detecting both causative agents and antimicrobial resistance genes, such as mecA and van, in blood culture positive samples. PMID:25368820

  16. Public health implications of molecular point-of-care testing for chlamydia and gonorrhoea in remote primary care services in Australia: a qualitative study

    PubMed Central

    Natoli, L; Guy, R J; Shephard, M; Whiley, D; Tabrizi, S N; Ward, J; Regan, D G; Badman, S G; Anderson, D A; Kaldor, J; Maher, L

    2015-01-01

    Objectives With accurate molecular tests now available for diagnosis of chlamydia and gonorrhoea (Chlamydia trachomatis (CT)/Neisseria gonorrhoeae (NG)) at the point-of-care (POC), we aimed to explore the public health implications (benefits and barriers) of their integration into remote primary care in Australia. Methods Qualitative interviews were conducted with a purposively selected group of 18 key informants reflecting sexual health, primary care, remote Aboriginal health and laboratory expertise. Results Participants believed that POC testing may decrease community prevalence of sexually transmitted infections (STIs), and associated morbidity by reducing the time to treatment and infectious period and expediting partner notification. Also, POC testing could improve acceptability of STI testing, increase testing coverage and result in more targeted prescribing, thereby minimising the risk of antibiotic resistance. Conversely, some felt the immediacy of diagnosis could deter certain young people from being tested. Participants also noted that POC testing may reduce the completeness of communicable disease surveillance data given the current dependence on reporting from pathology laboratories. Others expressed concern about the need to maintain and improve the flow of NG antibiotic sensitivity data, already compromised by the shift to nucleic acid-based testing. This is particularly relevant to remote areas where culture viability is problematic. Conclusions Results indicate a high level of support from clinicians and public health practitioners for wider access to CT/NG POC tests citing potential benefits, including earlier, more accurate treatment decisions and reductions in ongoing transmission. However, the data also highlight the need for new systems to avoid adverse impact on disease surveillance. Trial registration number Australian and New Zealand Clinical Trials Registry: ACTRN12613000808741. PMID:25922100

  17. Sexual risk behaviours and barriers to HIV testing among clients of female sex workers in Guatemala: a qualitative study.

    PubMed

    Lahuerta, Maria; Torrens, Míriam; Sabidó, Meritxell; Batres, Anabella; Casabona, Jordi

    2013-01-01

    Few interventions have targeted clients of female sex workers in Central America, despite their potential role in HIV/STI prevention. Semi-structured interviews were conducted with 30 clients of female sex workers on attitudes towards prevention of HIV/STIs, barriers to condom use and behaviour towards HIV/STI testing and treatment in Escuintla, Guatemala. Despite high knowledge of condoms as an HIV/STI preventive measure, the decision to use them was often based on the client's social judgment of the woman's sexual conduct. Regular clients reported lower condom use. Clients' risk perception diminished with the awareness of the public HIV/STI clinic addressed to female sex workers. Most preferred private clinics to increase confidentiality and were reluctant to take the HIV test for fear of a positive result. Outreach programmes offering HIV/STI counselling and testing to clients of female sex workers could increase their test uptake and health-seeking behaviour and reduce potential transmission to the general population. PMID:23627770

  18. Racial/Ethnic Differences in the Criterion-Related Validity of Cognitive Ability Tests: A Qualitative and Quantitative Review

    ERIC Educational Resources Information Center

    Berry, Christopher M.; Clark, Malissa A.; McClure, Tara K.

    2011-01-01

    The correlation between cognitive ability test scores and performance was separately meta-analyzed for Asian, Black, Hispanic, and White racial/ethnic subgroups. Compared to the average White observed correlation ([image omitted] = 0.33, N = 903,779), average correlations were lower for Black samples ([image omitted] = 0.24, N = 112,194) and…

  19. Analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay, the amplicor CMV test, and a semi-quantitative polymerase chain reaction test after allogeneic marrow transplantation.

    PubMed

    Ksouri, H; Eljed, H; Greco, A; Lakhal, A; Torjman, L; Abdelkefi, A; Ben Othmen, T; Ladeb, S; Slim, A; Zouari, B; Abdeladhim, A; Ben Hassen, A

    2007-03-01

    A pp65 antigenemia assay for polymorphonuclear leukocytes (PMNLs) (CINAkit Rapid Antigenemia), and a qualitative polymerase chain reaction (PCR) test for plasma 'PCR-P qual' (Amplicor cytomegalovirus [CMV] test) were performed for 126 samples (blood and plasma) obtained from 18 bone marrow transplant patients, over a 9-month surveillance period. Among those samples, 92 were assayed with a semi-quantitative PCR test for PMNLs 'PCR-L quant.' The number of samples with a positive CMV test for antigenemia and PCR-P qual assays was 20.63% and 12.7%, respectively, whereas the PCR-L quant assay was positive in 48 of the 92 samples assayed (52.17%). The rates of concordance of the results of PCR-P qual and antigenemia, PCR-P qual and PCR-L quant, antigenemia and PCR-L quant were 92%, 65.2% and 66.8%, respectively. The analysis of the results for the 92 specimens tested by all 3 methods showed a rate of concordance of 63% among all methods. Good agreement (kappa=0.72) was found only between pp65 Ag and PCR-P qual assays. Clinical disease correlates with an antigenemia high viral load. Three patients had CMV disease despite preemptive therapy, and all of them had graft-versus-host-disease (GVHD). PMNLs-based assays are more efficient in monitoring CMV reactivation, but for high-risk patients with GVHD, more sensitive assays (real-time PCR) must be done. PMID:17313466

  20. Collaborative trial for the validation of event-specific PCR detection methods of genetically modified papaya Huanong No.1.

    PubMed

    Wei, Jiaojun; Le, Huangying; Pan, Aihu; Xu, Junfeng; Li, Feiwu; Li, Xiang; Quan, Sheng; Guo, Jinchao; Yang, Litao

    2016-03-01

    For transferring the event-specific PCR methods of genetically modified papaya Huanong No.1 to other laboratories, we validated the previous developed PCR assays of Huanong No.1 according to the international standard organization (ISO) guidelines. A total of 11 laboratories participated and returned their test results in this trial. In qualitative PCR assay, the high specificity and limit of detection as low as 0.1% was confirmed. For the quantitative PCR assay, the limit of quantification was as low as 25 copies. The quantitative biases among ten blind samples were within the range between 0.21% and 10.04%. Furthermore, the measurement uncertainty of the quantitative PCR results was calculated within the range between 0.28% and 2.92% for these ten samples. All results demonstrated that the Huanong No.1 qualitative and quantitative PCR assays were creditable and applicable for identification and quantification of GM papaya Huanong No.1 in further routine lab analysis. PMID:26471522

  1. Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

    PubMed

    Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

    2013-01-01

    Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

  2. Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

    PubMed Central

    Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

    2013-01-01

    Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

  3. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    PubMed

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis. PMID:27316653

  4. Mutations in the rpoB gene of Mycobacterium tuberculosis that interfere with PCR-single-strand conformation polymorphism analysis for rifampin susceptibility testing.

    PubMed Central

    Kim, B J; Kim, S Y; Park, B H; Lyu, M A; Park, I K; Bai, G H; Kim, S J; Cha, C Y; Kook, Y H

    1997-01-01

    Rifampin susceptibility of 32 rifampin-resistant and 26 rifampin-susceptible Mycobacterium tuberculosis strains was analyzed by PCR-single-strand conformation polymorphism (SSCP) and DNA sequencing within the 157-bp region of the rpoB gene (Ala500 to Val550). Two false-positive PCR-SSCP results were observed among the susceptible strains due to the silent mutation Gln513 (CAA-->CAG) and the deletion mutation Thr508 and Ser509. Another silent mutation [Leu511 (CTG-->CTA)], combined with the mutation Ser531-->Leu, was observed in a resistant strain. These results suggest that to rule out false-positive PCR-SSCP results, sequencing of the target DNA is required. PMID:9003625

  5. Comparison of the BD Directigen Flu A+B Kit and the Abbott TestPack RSV with a multiplex RT-PCR ELISA for rapid detection of influenza viruses and respiratory syncytial virus.

    PubMed

    Gröndahl, B; Puppe, W; Weigl, J; Schmitt, H-J

    2005-10-01

    The Directigen Flu A+B enzyme immunoassay and the Abbott TestPack RSV enzyme immunoassay were each compared with a multiplex RT-PCR ELISA by testing 635 nasopharyngeal aspirates collected from children aged < 16 years who had been hospitalised with acute respiratory tract infection during the epidemic season 2002-2003. In this study, the sensitivity of the Directigen Flu A+B assay was unacceptably low (29.3% and 10.0%, respectively) for the detection of influenza A and B viruses. The sensitivity of the Abbott TestPack RSV assay (77.4%) was acceptable and in agreement with the multiplex RT-PCR ELISA. PMID:16153263

  6. Comparing the accuracy of quantitative versus qualitative analyses of interim PET to prognosticate Hodgkin lymphoma: a systematic review protocol of diagnostic test accuracy

    PubMed Central

    Procházka, Vít; Klugar, Miloslav; Bachanova, Veronika; Klugarová, Jitka; Tučková, Dagmar; Papajík, Tomáš

    2016-01-01

    Introduction Hodgkin lymphoma is an effectively treated malignancy, yet 20% of patients relapse or are refractory to front-line treatments with potentially fatal outcomes. Early detection of poor treatment responders is crucial for appropriate application of tailored treatment strategies. Tumour metabolic imaging of Hodgkin lymphoma using visual (qualitative) 18-fluorodeoxyglucose positron emission tomography (FDG-PET) is a gold standard for staging and final outcome assessment, but results gathered during the interim period are less accurate. Analysis of continuous metabolic–morphological data (quantitative) FDG-PET may enhance the robustness of interim disease monitoring, and help to improve treatment decision-making processes. The objective of this review is to compare diagnostic test accuracy of quantitative versus qualitative interim FDG-PET in the prognostication of patients with Hodgkin lymphoma. Methods The literature on this topic will be reviewed in a 3-step strategy that follows methods described by the Joanna Briggs Institute (JBI). First, MEDLINE and EMBASE databases will be searched. Second, listed databases for published literature (MEDLINE, Tripdatabase, Pedro, EMBASE, the Cochrane Central Register of Controlled Trials and WoS) and unpublished literature (Open Grey, Current Controlled Trials, MedNar, ClinicalTrials.gov, Cos Conference Papers Index and International Clinical Trials Registry Platform of the WHO) will be queried. Third, 2 independent reviewers will analyse titles, abstracts and full texts, and perform hand search of relevant studies, and then perform critical appraisal and data extraction from selected studies using the DATARI tool (JBI). If possible, a statistical meta-analysis will be performed on pooled sensitivity and specificity data gathered from the selected studies. Statistical heterogeneity will be assessed. Funnel plots, Begg's rank correlations and Egger's regression tests will be used to detect and/or correct publication

  7. Understanding Treatment Refusal Among Adults Presenting for HIV-Testing in Soweto, South Africa: A Qualitative Study

    PubMed Central

    Dietrich, Janan; Tshabalala, Gugu; Essien, Thandekile; Rough, Kathryn; Wright, Alexi A.; Bangsberg, David R.; Gray, Glenda E.; Ware, Norma C.

    2014-01-01

    HIV treatment initiatives have focused on increasing access to antiretroviral therapy (ART). There is growing evidence, however, that treatment availability alone is insufficient to stop the epidemic. In South Africa, only one third of individuals living with HIV are actually on treatment. Treatment refusal has been identified as a phenomenon among people who are asymptomatic, however, factors driving refusal remain poorly understood. We interviewed 50 purposively sampled participants who presented for voluntary counseling and testing in Soweto to elicit a broad range of detailed perspectives on ART refusal. We then integrated our core findings into an explanatory framework. Participants described feeling “too healthy” to start treatment, despite often having a diagnosis of AIDS. This subjective view of wellness was framed within the context of treatment being reserved for the sick. Taking ART could also lead to unintended disclosure and social isolation. These data provide a novel explanatory model of treatment refusal, recognizing perceived risks and social costs incurred when disclosing one’s status through treatment initiation. Our findings suggest that improving engagement in care for people living with HIV in South Africa will require optimizing social integration and connectivity for those who test positive. PMID:25304330

  8. Understanding treatment refusal among adults presenting for HIV-testing in Soweto, South Africa: a qualitative study.

    PubMed

    Katz, Ingrid T; Dietrich, Janan; Tshabalala, Gugu; Essien, Thandekile; Rough, Kathryn; Wright, Alexi A; Bangsberg, David R; Gray, Glenda E; Ware, Norma C

    2015-04-01

    HIV treatment initiatives have focused on increasing access to antiretroviral therapy (ART). There is growing evidence, however, that treatment availability alone is insufficient to stop the epidemic. In South Africa, only one third of individuals living with HIV are actually on treatment. Treatment refusal has been identified as a phenomenon among people who are asymptomatic, however, factors driving refusal remain poorly understood. We interviewed 50 purposively sampled participants who presented for voluntary counseling and testing in Soweto to elicit a broad range of detailed perspectives on ART refusal. We then integrated our core findings into an explanatory framework. Participants described feeling "too healthy" to start treatment, despite often having a diagnosis of AIDS. This subjective view of wellness was framed within the context of treatment being reserved for the sick. Taking ART could also lead to unintended disclosure and social isolation. These data provide a novel explanatory model of treatment refusal, recognizing perceived risks and social costs incurred when disclosing one's status through treatment initiation. Our findings suggest that improving engagement in care for people living with HIV in South Africa will require optimizing social integration and connectivity for those who test positive. PMID:25304330

  9. Quantitative and Qualitative Responses to Topical Cold in Healthy Caucasians Show Variance between Individuals but High Test-Retest Reliability.

    PubMed

    Moss, Penny; Whitnell, Jasmine; Wright, Anthony

    2016-01-01

    Increased sensitivity to cold may be a predictor of persistent pain, but cold pain threshold is often viewed as unreliable. This study aimed to determine the within-subject reliability and between-subject variance of cold response, measured comprehensively as cold pain threshold plus pain intensity and sensation quality at threshold. A test-retest design was used over three sessions, one day apart. Response to cold was assessed at four sites (thenar eminence, volar forearm, tibialis anterior, plantar foot). Cold pain threshold was measured using a Medoc thermode and standard method of limits. Intensity of pain at threshold was rated using a 10cm visual analogue scale. Quality of sensation at threshold was quantified with indices calculated from subjects' selection of descriptors from a standard McGill Pain Questionnaire. Within-subject reliability for each measure was calculated with intra-class correlation coefficients and between-subject variance was evaluated as group coefficient of variation percentage (CV%). Gender and site comparisons were also made. Forty-five healthy adults participated: 20 male, 25 female; mean age 29 (range 18-56) years. All measures at all four test sites showed high within-subject reliability: cold pain thresholds r = 0.92-0.95; pain rating r = 0.93-0.97; McGill pain quality indices r = 0.87-0.85. In contrast, all measures showed wide between-subject variance (CV% between 51.4% and 92.5%). Upper limb sites were consistently more sensitive than lower limb sites, but equally reliable. Females showed elevated cold pain thresholds, although similar pain intensity and quality to males. Females were also more reliable and showed lower variance for all measures. Thus, although there was clear population variation, response to cold for healthy individuals was found to be highly reliable, whether measured as pain threshold, pain intensity or sensation quality. A comprehensive approach to cold response testing therefore may add validity and

  10. Quantitative and Qualitative Responses to Topical Cold in Healthy Caucasians Show Variance between Individuals but High Test-Retest Reliability

    PubMed Central

    Moss, Penny; Whitnell, Jasmine; Wright, Anthony

    2016-01-01

    Increased sensitivity to cold may be a predictor of persistent pain, but cold pain threshold is often viewed as unreliable. This study aimed to determine the within-subject reliability and between-subject variance of cold response, measured comprehensively as cold pain threshold plus pain intensity and sensation quality at threshold. A test-retest design was used over three sessions, one day apart. Response to cold was assessed at four sites (thenar eminence, volar forearm, tibialis anterior, plantar foot). Cold pain threshold was measured using a Medoc thermode and standard method of limits. Intensity of pain at threshold was rated using a 10cm visual analogue scale. Quality of sensation at threshold was quantified with indices calculated from subjects' selection of descriptors from a standard McGill Pain Questionnaire. Within-subject reliability for each measure was calculated with intra-class correlation coefficients and between-subject variance was evaluated as group coefficient of variation percentage (CV%). Gender and site comparisons were also made. Forty-five healthy adults participated: 20 male, 25 female; mean age 29 (range 18–56) years. All measures at all four test sites showed high within-subject reliability: cold pain thresholds r = 0.92–0.95; pain rating r = 0.93–0.97; McGill pain quality indices r = 0.87–0.85. In contrast, all measures showed wide between-subject variance (CV% between 51.4% and 92.5%). Upper limb sites were consistently more sensitive than lower limb sites, but equally reliable. Females showed elevated cold pain thresholds, although similar pain intensity and quality to males. Females were also more reliable and showed lower variance for all measures. Thus, although there was clear population variation, response to cold for healthy individuals was found to be highly reliable, whether measured as pain threshold, pain intensity or sensation quality. A comprehensive approach to cold response testing therefore may add validity

  11. Multiple qualitative and quantitative methods for free light chain analysis are necessary as first line tests for AL amyloidosis.

    PubMed

    Sečník, Peter; Honsová, Eva; Jabor, Antonín; Lavríková, Petra; Franeková, Janka

    2016-06-01

    The objective of this study was to demonstrate the necessity of using different methods for amyloidogenic light chain detection. Serum and urine agarose gel electrophoresis and immunofixation, as well as serum free light chain (FLC) immunoassay measurements, were evaluated in a patient with verified multiple myeloma and consequent AL amyloidosis confirmed by Congo red staining and immunofluorescence techniques. Conventional chemistry tests [serum and urine electrophoresis (SPE and UPE); serum and urine immunofixation (SIFE and UIFE)] were inconclusive. Only quantitative FLC immunoassay (serum free light chain immunoanalysis, SFLC) provided correct diagnostic information. A combination of gel-based SIFE and UIFE with more novel quantitative FLC immunoassays appears necessary when searching for monoclonal immunoglobulin light chain-related diseases. PMID:26760309

  12. How Do Patients and Health Workers Interact around Malaria Rapid Diagnostic Testing, and How Are the Tests Experienced by Patients in Practice? A Qualitative Study in Western Uganda

    PubMed Central

    Altaras, Robin; Nuwa, Anthony; Agaba, Bosco; Streat, Elizabeth; Tibenderana, James K.; Martin, Sandrine; Strachan, Clare E.

    2016-01-01

    Background Successful scale-up in the use of malaria rapid diagnostic tests (RDTs) requires that patients accept testing and treatment based on RDT results and that healthcare providers treat according to test results. Patient-provider communication is a key component of quality care, and leads to improved patient satisfaction, higher adherence to treatment and better health outcomes. Voiced or perceived patient expectations are also known to influence treatment decision-making among healthcare providers. While there has been a growth in literature on provider practices around rapid testing for malaria, there has been little analysis of inter-personal communication around the testing process. We investigated how healthcare providers and patients interact and engage throughout the diagnostic and treatment process, and how the testing service is experienced by patients in practice. Methods This research was conducted alongside a larger study which explored determinants of provider treatment decision-making following negative RDT results in a rural district (Kibaale) in mid-western Uganda, ten months after RDT introduction. Fifty-five patients presenting with fever were observed during routine outpatient visits at 12 low-level public health facilities. Observation captured communication practices relating to test purpose, results, diagnosis and treatment. All observed patients or caregivers were immediately followed up with in-depth interview. Analysis followed the ‘framework’ approach. A summative approach was also used to analyse observation data. Results Providers failed to consistently communicate the reasons for carrying out the test, and particularly to RDT-negative patients, a diagnostic outcome or the meaning of test results, also leading to confusion over what the test can detect. Patients appeared to value testing, but were frustrated by the lack of communication on outcomes. RDT-negative patients were dissatisfied by the absence of information on an

  13. Experience of sexual violence among women in HIV discordant unions after voluntary HIV counselling and testing: a qualitative critical incident study in Uganda.

    PubMed

    Emusu, Donath; Ivankova, Nataliya; Jolly, Pauline; Kirby, Russell; Foushee, Herman; Wabwire-Mangen, Fred; Katongole, Drake; Ehiri, John

    2009-11-01

    HIV-serodiscordant relationships are those in which one partner is infected with HIV while the other is not. We investigated experiences of sexual violence among women in HIV discordant unions attending HIV post-test club services in Uganda. A volunteer sample of 26 women from three AIDS Information Centres in Uganda who reported having experienced sexual violence in a larger epidemiological study were interviewed, using the qualitative critical incident technique. Data were analysed using TEXTPACK, a software application for computer-assisted content analysis. Incidents of sexual violence narrated by the women included use of physical force and verbal threats. Overall, four themes that characterise the women's experience of sexual violence emerged from the analysis: knowledge of HIV test results, prevalence of sexual violence, vulnerability and proprietary views and reactions to sexual violence. Alcohol abuse by the male partners was an important factor in the experience of sexual violence among the women. Their experiences evoked different reactions and feelings, including concern over the need to have children, fear of infection, desire to separate from their spouses/partners, helplessness, anger and suicidal tendencies. HIV counselling and testing centres should be supported with the capacity to address issues related to sexual violence for couples who are HIV discordant. PMID:20024712

  14. Quality and safety issues highlighted by patients in the handling of laboratory test results by general practices–a qualitative study

    PubMed Central

    2014-01-01

    Background In general practice internationally, many care teams handle large numbers of laboratory test results relating to patients in their care. Related research about safety issues is limited with most of the focus on this workload from secondary care and in North American settings. Little has been published in relation to primary health care in the UK and wider Europe. This study aimed to explore experiences and perceptions of patients with regards to the handling of test results by general practices. Methods A qualitative research approach was used with patients. The setting was west of Scotland general practices from one National Health Service territorial board area. Patients were purposively sampled from practice held lists of patients who received a number of laboratory tests because of chronic medical problems or surveillance of high risk medicines. Focus groups were held and were audio-recorded. Tapes were transcribed and subjected to qualitative analysis. Transcripts were coded and codes merged into themes by two of the researchers. Results 19 participants from four medical practices took part in four focus groups. The main themes identified were: 1. Patients lacked awareness of the results handling process in their practice. 2. Patients usually did not contact their practice for test results, unless they considered themselves to be ill. 3. Patients were concerned about the appropriateness of administrators being involved in results handling. 4. Patients were concerned about breaches of confidentiality when administrators were involved in results handling. 5. Patients valued the use of dedicated results handling staff. 6. Patients welcomed the use of technology to alert them to results being available, and valued the ability to choose how this happened. Conclusions The study confirms the quality and safety of care problems associated with results handling systems and adds to our knowledge of the issues that impact in these areas. Practices need to be

  15. Genetic Test Results and Disclosure to Family Members: Qualitative Interviews of Healthcare Professionals' Perceptions of Ethical and Professional Issues in France.

    PubMed

    D' Audiffret Van Haecke, Diane; de Montgolfier, Sandrine

    2016-06-01

    The benefit of disclosing test results to next of kin is to improve prognosis and-in some cases-even prevent death though earlier monitoring or preventive therapies. Research on this subject has explored the question of intra-familial communication from the standpoint of patients and relatives but rarely, from the standpoint of healthcare professionals. The purpose of this study was to interview relevant healthcare professionals in France, where legislation framing the issue was recently passed. A qualitative study consisting of semi-structured interviews was set up to get a clearer picture of the challenges arising from this issue, its consequences in terms of medical care-service practices, and the positions that frontline professionals have taken in response to this new legal framework. The findings from eight interviews with 7 clinical geneticists and 1 genetic counselor highlight very different patterns of practices among care services and among the genetic diseases involved. It is equally crucial to investigate other issues such as the nature of genetic testing and its consequences in terms of disclosing results to kin, the question of the role of genetic counseling in the disclosure process, the question of prescription by non-geneticist clinicians, and practical questions linked to information content, consent and medical follow-up for patients and their relatives. PMID:26482743

  16. Use of the RFLP-PCR diagnostic test for characterizing MACE and kdr insecticide resistance in the peach potato aphid Myzus persicae.

    PubMed

    Cassanelli, Stefano; Cerchiari, Barbara; Giannini, Sara; Bizzaro, Davide; Mazzoni, Emanuele; Manicardi, Gian Carlo

    2005-01-01

    The peach-potato aphid Myzus persicae (Sulzer) has developed a number of insecticide resistance mechanisms owing to the high selective pressure produced by world-wide insecticide treatments. Knowledge of the geographical distribution and the temporal evolution of these resistant phenotypes helps to develop suitable pest-management programs. Current understanding of the major mechanisms of resistance at the molecular level makes it possible to diagnose the presence of modified acetylcholinesterase (MACE) or knockdown resistance (kdr). This paper describes a rapid method for the identification of both resistance mechanisms in a single molecular assay by using restriction fragment length polymorphism of PCR products (RFLP-PCR) in individual as well as pooled aphids. PMID:15593078

  17. Design and testing of multiplex RT-PCR primers for the rapid detection of influenza A virus genomic segments: Application to equine influenza virus.

    PubMed

    Lee, EunJung; Kim, Eun-Ju; Shin, Yeun-Kyung; Song, Jae-Young

    2016-02-01

    The avian influenza A virus causes respiratory infections in animal species. It can undergo genomic recombination with newly obtained genetic material through an interspecies transmission. However, the process is an unpredictable event, making it difficult to predict the emergence of a new pandemic virus and distinguish its origin, especially when the virus is the result of multiple infections. Therefore, identifying a novel influenza is entirely dependent on sequencing its whole genome. Occasionally, however, it can be time-consuming, costly, and labor-intensive when sequencing many influenza viruses. To compensate for the difficulty, we developed a rapid, cost-effective, and simple multiplex RT-PCR to identify the viral genomic segments. As an example to evaluate its performance, H3N8 equine influenza virus (EIV) was studied for the purpose. In developing this protocol to amplify the EIV eight-segments, a series of processes, including phylogenetic analysis based on different influenza hosts, in silico analyses to estimate primer specificity, coverage, and variation scores, and investigation of host-specific amino acids, were progressively conducted to reduce or eliminate the negative factors that might affect PCR amplification. Selectively, EIV specific primers were synthesized with dual priming oligonucleotides (DPO) system to increase primer specificity. As a result, 16 primer pairs were selected to screen the dominantly circulating H3N8 EIV 8 genome segments: PA (3), PB2 (1), PA (3), NP (3), NA8 (2), HA3 (1), NS (1), and M (2). The diagnostic performance of the primers was evaluated with eight sets composing of four segment combinations using viral samples from various influenza hosts. The PCR results suggest that the multiplex RT-PCR has a wide range of applications in detection and diagnosis of newly emerging EIVs. Further, the proposed procedures of designing multiplex primers are expected to be used for detecting other animal influenza A viruses. PMID

  18. Kinetics of poliovirus shedding following oral vaccination as measured by quantitative reverse transcription-PCR versus culture.

    PubMed

    Taniuchi, Mami; Begum, Sharmin; Uddin, Md Jashim; Platts-Mills, James A; Liu, Jie; Kirkpatrick, Beth D; Chowdhury, Anwarul H; Jamil, Khondoker M; Haque, Rashidul; Petri, William A; Houpt, Eric R

    2015-01-01

    Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], +4, +11, +18, and +25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture(+)/qPCR(+) and culture(-)/qPCR(+) stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day +11 or +18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively. PMID:25378579

  19. Facilitators and Barriers to Linkage to HIV Care among Female Sex Workers Receiving HIV Testing Services at a Community-Based Organization in Periurban Uganda: A Qualitative Study

    PubMed Central

    Kintu, Betty N.

    2016-01-01

    Introduction. While four in ten female sex workers (FSWs) in sub-Saharan Africa are infected with HIV, only a small proportion is enrolled in HIV care. We explored facilitators and barriers to linkage to HIV care among FSWs receiving HIV testing services at a community-based organization in periurban Uganda. Methods. The cross-sectional qualitative study was conducted among 28 HIV positive FSWs from May to July 2014. Key informant interviews were conducted with five project staff and eleven peer educators. Data were collected on facilitators for and barriers to linkage to HIV care and manually analyzed following a thematic framework approach. Results. Facilitators for linkage to HIV care included the perceived good quality of health services with same-day results and immediate initiation of treatment, community peer support systems, individual's need to remain healthy, and having alternative sources of income. Linkage barriers included perceived stigma, fear to be seen at outreach HIV clinics, fear and myths about antiretroviral therapy, lack of time to attend clinic, and financial constraints. Conclusion. Linkage to HIV care among FSWs is influenced by good quality friendly services and peer support. HIV service delivery programs for FSWs should focus on enhancing these and dealing with barriers stemming from stigma and misinformation. PMID:27493826

  20. Facilitators and Barriers to Linkage to HIV Care among Female Sex Workers Receiving HIV Testing Services at a Community-Based Organization in Periurban Uganda: A Qualitative Study.

    PubMed

    Nakanwagi, Sharon; Matovu, Joseph K B; Kintu, Betty N; Kaharuza, Frank; Wanyenze, Rhoda K

    2016-01-01

    Introduction. While four in ten female sex workers (FSWs) in sub-Saharan Africa are infected with HIV, only a small proportion is enrolled in HIV care. We explored facilitators and barriers to linkage to HIV care among FSWs receiving HIV testing services at a community-based organization in periurban Uganda. Methods. The cross-sectional qualitative study was conducted among 28 HIV positive FSWs from May to July 2014. Key informant interviews were conducted with five project staff and eleven peer educators. Data were collected on facilitators for and barriers to linkage to HIV care and manually analyzed following a thematic framework approach. Results. Facilitators for linkage to HIV care included the perceived good quality of health services with same-day results and immediate initiation of treatment, community peer support systems, individual's need to remain healthy, and having alternative sources of income. Linkage barriers included perceived stigma, fear to be seen at outreach HIV clinics, fear and myths about antiretroviral therapy, lack of time to attend clinic, and financial constraints. Conclusion. Linkage to HIV care among FSWs is influenced by good quality friendly services and peer support. HIV service delivery programs for FSWs should focus on enhancing these and dealing with barriers stemming from stigma and misinformation. PMID:27493826

  1. A naked-eye colorimetric "PCR developer"

    NASA Astrophysics Data System (ADS)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated <10 billion reactions per year and a worldwide market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  2. HRP2 and pLDH-Based Rapid Diagnostic Tests, Expert Microscopy, and PCR for Detection of Malaria Infection during Pregnancy and at Delivery in Areas of Varied Transmission: A Prospective Cohort Study in Burkina Faso and Uganda

    PubMed Central

    Cunningham, Jane; Angutoko, Patrick; Ategeka, John; Compaoré, Yves-Daniel; Muehlenbachs, Atis; Somé, Fabrice A.; Ouattara, Aminata; Rouamba, Noél; Ouédraogo, Jean-Bosco; Hopkins, Heidi

    2016-01-01

    Background Intermittent screening and treatment (IST) of malaria during pregnancy has been proposed as an alternative to intermittent preventive treatment in pregnancy (IPTp), where IPTp is failing due to drug resistance. However, the antenatal parasitaemias are frequently very low, and the most appropriate screening test for IST has not been defined. Methodology/Principal Findings We conducted a multi-center prospective study of 990 HIV-uninfected women attending ANC in two different malaria transmission settings at Tororo District Hospital, eastern Uganda and Colsama Health Center in western Burkina Faso. Women were enrolled in the study in the second or third trimester of pregnancy and followed to delivery, generating 2,597 blood samples for analysis. Screening tests included rapid diagnostic tests (RDTs) targeting histidine-rich protein 2 (HRP2) and parasite lactate dehydrogenase (pLDH) and microscopy, compared to nPCR as a reference standard. At enrolment, the proportion of pregnant women who were positive for P. falciparum by HRP2/pan pLDH RDT, Pf pLDH/pan pLDH RDT, microscopy and PCR was 38%, 29%, 36% and 44% in Uganda and 21%, 16%, 15% and 35% in Burkina Faso, respectively. All test positivity rates declined during follow-up. In comparison to PCR, the sensitivity of the HRP2/pan pLDH RDT, Pf pLDH/pan pLDH RDT and microscopy was 75.7%, 60.1% and 69.7% in Uganda, 55.8%, 42.6% and 55.8% in Burkina Faso respectively for all antenatal visits. Specificity was greater than 96% for all three tests. Comparison of accuracy using generalized estimating equation revealed that the HRP2- detecting RDT was the most accurate test in both settings. Conclusions/Significance The study suggests that HRP2-based RDTs are the most appropriate point-of-care test currently available for use during pregnancy especially for symptomatic women, but will still miss some PCR-positive women. The clinical significance of these very low density infections needs to be better defined. PMID

  3. Barriers to and facilitators of hepatitis C testing, management, and treatment among current and former injecting drug users: a qualitative exploration.

    PubMed

    Swan, Davina; Long, Jean; Carr, Olivia; Flanagan, Jean; Irish, Helena; Keating, Shay; Keaveney, Michelle; Lambert, John; McCormick, P Aiden; McKiernan, Susan; Moloney, John; Perry, Nicola; Cullen, Walter

    2010-12-01

    Hepatitis C (HCV) infection is common among injecting drug users (IDUs), yet accessing of HCV care, particularly HCV treatment, is suboptimal. There has been little in-depth study of IDUs experiences of what enables or prevents them engaging at every level of HCV care, including testing, follow-up, management and treatment processes. This qualitative study aimed to explore these issues with current and former IDUs in the greater Dublin area, Ireland. From September 2007 to September 2008 in-depth interviews were conducted with 36 service-users across a range of primary and secondary care services, including: two addiction clinics, a general practice, a community drop-in center, two hepatology clinics, and an infectious diseases clinic. Interviews were analyzed using a grounded theory approach. Barriers to HCV care included perceptions of HCV infection as relatively benign, fear of investigations and treatment, and feeling well. Perceptions were shaped by the discourse about HCV and "horror stories" about the liver biopsy and treatment within their peer networks. Difficulties accessing HCV care included limited knowledge of testing sites, not being referred for specialist investigations and ineligibility for treatment. Employment, education, and addiction were priorities that competed with HCV care. Relationships with health care providers influenced engagement with care: Trust in providers, concern for the service-user, and continuity of care fostered engagement. Education on HCV infection, investigations, and treatment altered perceptions. Becoming symptomatic, responsibilities for children, and wanting to move on from drug use motivated HCV treatment. In conclusion, IDUs face multiple barriers to HCV care. A range of facilitators were identified that could inform future interventions. PMID:21138381

  4. Publishing Qualitative Research.

    ERIC Educational Resources Information Center

    Smith, Mary Lee

    1987-01-01

    Article defines qualitative research and describes the form that an article based on qualitative research might take. Encourages readers to submit articles based on qualitative research to the American Educational Research Journal. (RB)

  5. Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Evrard, A.; Boulle, N.; Lutfalla, G. S.

    Over the past few years there has been a considerable development of DNA amplification by polymerase chain reaction (PCR), and real-time PCR has now superseded conventional PCR techniques in many areas, e.g., the quantification of nucleic acids and genotyping. This new approach is based on the detection and quantification of a fluorescent signal proportional to the amount of amplicons generated by PCR. Real-time detection is achieved by coupling a thermocycler with a fluorimeter. This chapter discusses the general principles of quantitative real-time PCR, the different steps involved in implementing the technique, and some examples of applications in medicine. The polymerase chain reaction (PCR) provides a way of obtaining a large number of copies of a double-stranded DNA fragment of known sequence. This DNA amplification technique, developed in 1985 by K. Mullis (Cetus Corporation), saw a spectacular development over the space of a few years, revolutionising the methods used up to then in molecular biology. Indeed, PCR has many applications, such as the detection of small amounts of DNA, cloning, and quantitative analysis (assaying), each of which will be discussed further below.

  6. SYBR green-based real-time reverse transcription-PCR for typing and subtyping of all hemagglutinin and neuraminidase genes of avian influenza viruses and comparison to standard serological subtyping tests

    USGS Publications Warehouse

    Tsukamoto, K.; Javier, P.C.; Shishido, M.; Noguchi, D.; Pearce, J.; Kang, H.-M.; Jeong, O.M.; Lee, Y.-J.; Nakanishi, K.; Ashizawa, T.

    2012-01-01

    Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10 1.5, 10 2.3, and 10 3.1 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples. Copyright ?? 2012, American Society for Microbiology. All Rights Reserved.

  7. 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

  8. “Men are always scared to test with their partners … it is like taking them to the Police”: Motivations for and barriers to couples’ HIV counselling and testing in Rakai, Uganda: a qualitative study

    PubMed Central

    Matovu, Joseph KB; Wanyenze, Rhoda K; Wabwire-Mangen, Fred; Nakubulwa, Rosette; Sekamwa, Richard; Masika, Annet; Todd, Jim; Serwadda, David

    2014-01-01

    Introduction Uptake of couples’ HIV counselling and testing (couples’ HCT) can positively influence sexual risk behaviours and improve linkage to HIV care among HIV-positive couples. However, less than 30% of married couples have ever tested for HIV together with their partners. We explored the motivations for and barriers to couples’ HCT among married couples in Rakai, Uganda. Methods This was a qualitative study conducted among married individuals and selected key informants between August and October 2013. Married individuals were categorized by prior HCT status as: 1) both partners never tested; 2) only one or both partners ever tested separately; and 3) both partners ever tested together. Data were collected on the motivations for and barriers to couples’ HCT, decision-making processes from tested couples and suggestions for improving couples’ HCT uptake. Eighteen focus group discussions with married individuals, nine key informant interviews with selected key informants and six in-depth interviews with married individuals that had ever tested together were conducted. All interviews were audio-recorded, translated and transcribed verbatim and analyzed using Nvivo (version 9), following a thematic framework approach. Results Motivations for couples’ HCT included the need to know each other's HIV status, to get a treatment companion or seek HIV treatment together – if one or both partners were HIV-positive – and to reduce mistrust between partners. Barriers to couples’ HCT included fears of the negative consequences associated with couples’ HCT (e.g. fear of marital dissolution), mistrust between partners and conflicting work schedules. Couples’ HCT was negotiated through a process that started off with one of the partners testing alone initially and then convincing the other partner to test together. Suggestions for improving couples’ HCT uptake included the need for couple- and male-partner-specific sensitization, and the use of

  9. Diagnostic PCR of dermatophytes--an overview.

    PubMed

    Gräser, Yvonne; Czaika, Viktor; Ohst, Torsten

    2012-10-01

    The prevalence of onychomycosis is increasing steadily, sevenfold alone in the US within the last twenty years. An important aspect in this development is the demographic development of the human population of the industrial countries like Germany. A fast and accurate laboratory diagnosis is essential for successful treatment because 50% of the cases are misdiagnosed when relying on the clinical appearance only. The current diagnosis of dermatophytosis, based on direct microscopy and culture of the clinical specimen, is problematic given the lacking specificity of the former and the length of time needed for the latter. Molecular techniques can help to solve these problems. In recent years, a number of in-house PCR assays have been developed to identify dermatophytes directly from clinical specimens. Based on the "Mikrobiologisch-infektiologischen Qualitätsstandards (MIQ) für Nukleinsäure-Amplifikationstechniken" and the MIQE guideline (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) 11 studies are reviewed which were published between 2007 and 2010. The present article evaluates the quality of the PCR assays regarding false positive and false negative results due to contamination, PCR format, statistical analysis, and diagnostic performance of the studies. It shows that we are only at the beginning of providing high quality PCR diagnosis of dermatophytes. PMID:23013298

  10. Methods for comparing multiple digital PCR experiments.

    PubMed

    Burdukiewicz, Michał; Rödiger, Stefan; Sobczyk, Piotr; Menschikowski, Mario; Schierack, Peter; Mackiewicz, Paweł

    2016-09-01

    The estimated mean copy per partition (λ) is the essential information from a digital PCR (dPCR) experiment because λ can be used to calculate the target concentration in a sample. However, little information is available how to statistically compare dPCR runs of multiple runs or reduplicates. The comparison of λ values from several runs is a multiple comparison problem, which can be solved using the binary structure of dPCR data. We propose and evaluate two novel methods based on Generalized Linear Models (GLM) and Multiple Ratio Tests (MRT) for comparison of digital PCR experiments. We enriched our MRT framework with computation of simultaneous confidence intervals suitable for comparing multiple dPCR runs. The evaluation of both statistical methods support that MRT is faster and more robust for dPCR experiments performed in large scale. Our theoretical results were confirmed by the analysis of dPCR measurements of dilution series. Both methods were implemented in the dpcR package (v. 0.2) for the open source R statistical computing environment. PMID:27551672

  11. Validation of nested PCR and a selective biochemical method as alternatives for mycoplasma detection.

    PubMed

    Cheong, Kyung Ah; Agrawal, Santosh Rani; Lee, Ai-Young

    2011-04-01

    Direct culture is the most common way to reliably detect mycoplasma, but it is not practical for the qualitative control of cell therapeutics because of the elaborate culture medium, the prolonged incubation time, and the large sample volumes. Here, we chose two alternative methods using commercial detection kits, the PCR mycoplasma detection kit with nested PCR and the selective biochemical method, MycoAlert(®), and validated them with the direct culture method as a reference. We tested eight mycoplasma species and five validation parameters: specificity, detection limit, robustness, repeatability, and ruggedness, based on the regulatory guidelines in the US Pharmacopoeia. All experiments were performed using fibroblasts spiked with mycoplasma. Specificity tests for both methods included all mycoplasma species, except Mycoplasma pneumonia and M. genitalium for the nested PCR and Ureaplasma urealyticum for the MycoAlert(®) assay. Regarding the detection limit, the nested PCR proved to be as sensitive as the direct culture method and more sensitive than the MycoAlert(®) assay. The predicted median for probit = 0.9 was 54 (44-76) CFU/ml for M. hyorhinis and 16 (13-23) CFU/ml for M. hominis by the nested PCR, but 431 (346-593) CFU/ml and 105 (87-142) CFU/ml, respectively, with MycoAlert(®). Changes in the concentration of reagents, reagent lot, or individual analysts did not influence the results of the examined methods. The results of this study support nested PCR as a valuable alternative for mycoplasma detection. PMID:20806253

  12. Introduction of Syphilis Point-of-Care Tests, from Pilot Study to National Programme Implementation in Zambia: A Qualitative Study of Healthcare Workers’ Perspectives on Testing, Training and Quality Assurance

    PubMed Central

    Ansbro, Éimhín M.; Gill, Michelle M.; Reynolds, Joanna; Shelley, Katharine D.; Strasser, Susan; Sripipatana, Tabitha; Ncube, Alexander Tshaka; Tembo Mumba, Grace; Terris-Prestholt, Fern; Peeling, Rosanna W.; Mabey, David

    2015-01-01

    Syphilis affects 1.4 million pregnant women globally each year. Maternal syphilis causes congenital syphilis in over half of affected pregnancies, leading to early foetal loss, pregnancy complications, stillbirth and neonatal death. Syphilis is under-diagnosed in pregnant women. Point-of-care rapid syphilis tests (RST) allow for same-day treatment and address logistical barriers to testing encountered with standard Rapid Plasma Reagin testing. Recent literature emphasises successful introduction of new health technologies requires healthcare worker (HCW) acceptance, effective training, quality monitoring and robust health systems. Following a successful pilot, the Zambian Ministry of Health (MoH) adopted RST into policy, integrating them into prevention of mother-to-child transmission of HIV clinics in four underserved Zambian districts. We compare HCW experiences, including challenges encountered in scaling up from a highly supported NGO-led pilot to a large-scale MoH-led national programme. Questionnaires were administered through structured interviews of 16 HCWs in two pilot districts and 24 HCWs in two different rollout districts. Supplementary data were gathered via stakeholder interviews, clinic registers and supervisory visits. Using a conceptual framework adapted from health technology literature, we explored RST acceptance and usability. Quantitative data were analysed using descriptive statistics. Key themes in qualitative data were explored using template analysis. Overall, HCWs accepted RST as learnable, suitable, effective tools to improve antenatal services, which were usable in diverse clinical settings. Changes in training, supervision and quality monitoring models between pilot and rollout may have influenced rollout HCW acceptance and compromised testing quality. While quality monitoring was integrated into national policy and training, implementation was limited during rollout despite financial support and mentorship. We illustrate that new

  13. Introduction of Syphilis Point-of-Care Tests, from Pilot Study to National Programme Implementation in Zambia: A Qualitative Study of Healthcare Workers' Perspectives on Testing, Training and Quality Assurance.

    PubMed

    Ansbro, Éimhín M; Gill, Michelle M; Reynolds, Joanna; Shelley, Katharine D; Strasser, Susan; Sripipatana, Tabitha; Tshaka Ncube, Alexander; Tembo Mumba, Grace; Terris-Prestholt, Fern; Peeling, Rosanna W; Mabey, David

    2015-01-01

    Syphilis affects 1.4 million pregnant women globally each year. Maternal syphilis causes congenital syphilis in over half of affected pregnancies, leading to early foetal loss, pregnancy complications, stillbirth and neonatal death. Syphilis is under-diagnosed in pregnant women. Point-of-care rapid syphilis tests (RST) allow for same-day treatment and address logistical barriers to testing encountered with standard Rapid Plasma Reagin testing. Recent literature emphasises successful introduction of new health technologies requires healthcare worker (HCW) acceptance, effective training, quality monitoring and robust health systems. Following a successful pilot, the Zambian Ministry of Health (MoH) adopted RST into policy, integrating them into prevention of mother-to-child transmission of HIV clinics in four underserved Zambian districts. We compare HCW experiences, including challenges encountered in scaling up from a highly supported NGO-led pilot to a large-scale MoH-led national programme. Questionnaires were administered through structured interviews of 16 HCWs in two pilot districts and 24 HCWs in two different rollout districts. Supplementary data were gathered via stakeholder interviews, clinic registers and supervisory visits. Using a conceptual framework adapted from health technology literature, we explored RST acceptance and usability. Quantitative data were analysed using descriptive statistics. Key themes in qualitative data were explored using template analysis. Overall, HCWs accepted RST as learnable, suitable, effective tools to improve antenatal services, which were usable in diverse clinical settings. Changes in training, supervision and quality monitoring models between pilot and rollout may have influenced rollout HCW acceptance and compromised testing quality. While quality monitoring was integrated into national policy and training, implementation was limited during rollout despite financial support and mentorship. We illustrate that new

  14. Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species

    PubMed Central

    Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-01-01

    Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management. PMID:27562455

  15. Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species.

    PubMed

    Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-01-01

    Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management. PMID:27562455

  16. Assessment Accommodations on Tests of Academic Achievement for Students Who Are Deaf or Hard of Hearing: A Qualitative Meta-Analysis of the Research Literature

    ERIC Educational Resources Information Center

    Cawthon, Stephanie; Leppo, Rachel

    2013-01-01

    The authors conducted a qualitative meta-analysis of the research on assessment accommodations for students who are deaf or hard of hearing. There were 16 identified studies that analyzed the impact of factors related to student performance on academic assessments across different educational settings, content areas, and types of assessment…

  17. Overlap extension PCR cloning.

    PubMed

    Bryksin, Anton; Matsumura, Ichiro

    2013-01-01

    Rising demand for recombinant proteins has motivated the development of efficient and reliable cloning methods. Here we show how a beginner can clone virtually any DNA insert into a plasmid of choice without the use of restriction endonucleases or T4 DNA ligase. Chimeric primers encoding plasmid sequence at the 5' ends and insert sequence at the 3' ends are designed and synthesized. Phusion(®) DNA polymerase is utilized to amplify the desired insert by PCR. The double-stranded product is subsequently employed as a pair of mega-primers in a PCR-like reaction with circular plasmids. The original plasmids are then destroyed in restriction digests with Dpn I. The product of the overlap extension PCR is used to transform competent Escherichia coli cells. Phusion(®) DNA polymerase is used for both the amplification and fusion reactions, so both steps can be monitored and optimized in the same way. PMID:23996437

  18. Single Nucleotide Polymorphism (SNP)-Based Loss of Heterozygosity (LOH) Testing by Real Time PCR in Patients Suspect of Myeloproliferative Disease

    PubMed Central

    Huijsmans, Cornelis J. J.; Poodt, Jeroen; Damen, Jan; van der Linden, Johannes C.; Savelkoul, Paul H. M.; Pruijt, Johannes F. M.; Hilbink, Mirrian; Hermans, Mirjam H. A.

    2012-01-01

    During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n = 6 25–50% and n = 6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25–50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci. PMID:22768290

  19. Development and validation of real-time PCR tests for the identification of four Spodoptera species: Spodoptera eridania, Spodoptera frugiperda, Spodoptera littoralis, and Spodoptera litura (Lepidoptera: Noctuidae).

    PubMed

    Van de Vossenberg, B T L H; Van der Straten, M J

    2014-08-01

    The genus Spodoptera comprises 31 species, 4 of which are listed as quarantine pests for the European Union: Spodoptera eridania (Cramer), Spodoptera frugiperda (Smith), Spodoptera littoralis (Boisduval), and Spodoptera litura (F.). In international trade, the earlier life stages (eggs and larvae) are being intercepted at point of inspection most frequently, challenging the possibilities of morphological identification. To realize a rapid and reliable identification for all stages, we developed and validated four simplex real-time polymerase chain reaction identification tests based on the mitochondrial cytochrome b gene using dual-labeled hydrolysis probes. Method validation on dilutions of extracted DNA of the target organisms showed that low levels of template (up to 0.2-100 pg) can reliably be identified. No cross-reactivity was observed with 14 nontarget Spodoptera and 5 non-Spodoptera species in the specific Spodoptera tests. The tests showed to be repeatable, reproducible (both 100%), and robust. The new Spodoptera tests have proven to be suitable tools for routine identification of all life stages of S. eridania, S. frugiperda, S. littoralis, and S. litura. PMID:25195458

  20. QUALITY CONTROLS FOR PCR

    EPA Science Inventory

    The purpose of this presentation is to present an overview of the quality control (QC) sections of a draft EPA document entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples." This document has been prepared by th...

  1. QUALITY ASSURANCE FOR PCR

    EPA Science Inventory

    The U.S. Environmental Protection Agency (EPA) held a workshop in January 2003 on the detection of viruses in water using polymerase chain reaction (PCR)-based methods. Speakers were asked to address a series of specific questions, including whether a single standard method coul...

  2. Effectively Communicating Qualitative Research

    ERIC Educational Resources Information Center

    Ponterotto, Joseph G.; Grieger, Ingrid

    2007-01-01

    This article is a guide for counseling researchers wishing to communicate the methods and results of their qualitative research to varied audiences. The authors posit that the first step in effectively communicating qualitative research is the development of strong qualitative research skills. To this end, the authors review a process model for…

  3. Qualitative Student Models.

    ERIC Educational Resources Information Center

    Clancey, William J.

    The concept of a qualitative model is used as the focus of this review of qualitative student models in order to compare alternative computational models and to contrast domain requirements. The report is divided into eight sections: (1) Origins and Goals (adaptive instruction, qualitative models of processes, components of an artificial…

  4. Requiem for Qualitative Analysis.

    ERIC Educational Resources Information Center

    Journal of Chemical Education, 1983

    1983-01-01

    Five papers presented at the Seventh Biennial Conference on Chemical Education (Stillwater, Oklahoma 1982) focused on qualitative analysis curricula and instruction. Topics included benefits of qualitative analysis, use of iodo/bromo-complexes in qualitative analysis schemes, lecture demonstrations, and brief descriptions of three courses. (JN)

  5. Evaluation of the Xpert Flu test and comparison with in-house real-time RT-PCR assays for detection of influenza virus from 2008 to 2011 in Marseille, France.

    PubMed

    Salez, N; Ninove, L; Thirion, L; Gazin, C; Zandotti, C; de Lamballerie, X; Charrel, R N

    2012-04-01

    Rapid documentation of respiratory specimens can have an impact on the management of patients and their relatives in terms of preventive and curative measures. We compared the results of the Xpert(®) Flu assay (Cepheid) with three real-time RT-PCR assays using 127 nasopharyngeal samples, of which 75 were positive for influenza A (with 52 identified as A/H1N1-2009) and 52 were positive for influenza B. The Xpert(®) Flu assay presented a quasi-absence of non-interpretable tests, and showed sensitivity and specificity of 100% and 100% for Flu A, 98.4% and 100% for A/H1N1-2009, and 80.7% and 100% for Flu B. PMID:22360446

  6. Quantification of fungal abundance on cultural heritage using real time PCR targeting the β-actin gene.

    PubMed

    Ettenauer, Jörg; Piñar, Guadalupe; Tafer, Hakim; Sterflinger, Katja

    2014-01-01

    The traditional methodology used for the identification of microbes colonizing our cultural heritage was the application of cultivation methods and/or microscopy. This approach has many advantages, as living microorganisms may be obtained for physiological investigations. In addition, these techniques allow the quantitative and qualitative assessment of the investigated environment. Quantitative analyses are done by plate count and the determination of abundance by the colony forming unit (CFU). Nevertheless, these techniques have many drawbacks that lead to an underestimation of the cell numbers and do not provide a comprehensive overview of the composition of the inhabiting microbiota. In the last decades, several molecular techniques have been developed enabling many advantages over the cultivation approach. Mainly PCR-based, fingerprinting techniques allow a qualitative detection and identification of the microbiota. In this study, we developed a real time PCR method as a simple, rapid and reliable tool to detect and quantify fungal abundance using the β-actin gene, which is known to appear as a single-copy gene in fungi. To this end, five different indoor thermal insulation materials applied for historical buildings that were previously tested for their bio-susceptibility against various fungi were subjected to qPCR analyses. The obtained results were compared with those obtained from a previous study investigating the bio-susceptibility of the insulation materials using classical cultivation experiments. Both results correlated well, revealing that Perlite plaster was the most suitable insulation material, showing the lowest fungal CFU and qPCR values. In contrast, insulations made of wood showed to be not recommendable from the microbiological point of view. In addition, the potential of qPCR was tested in other materials of cultural heritage, as old parchments, showing to be a suitable method for measuring fungal abundance in these delicate materials. PMID

  7. Quantification of fungal abundance on cultural heritage using real time PCR targeting the β-actin gene

    PubMed Central

    Ettenauer, Jörg; Piñar, Guadalupe; Tafer, Hakim; Sterflinger, Katja

    2014-01-01

    The traditional methodology used for the identification of microbes colonizing our cultural heritage was the application of cultivation methods and/or microscopy. This approach has many advantages, as living microorganisms may be obtained for physiological investigations. In addition, these techniques allow the quantitative and qualitative assessment of the investigated environment. Quantitative analyses are done by plate count and the determination of abundance by the colony forming unit (CFU). Nevertheless, these techniques have many drawbacks that lead to an underestimation of the cell numbers and do not provide a comprehensive overview of the composition of the inhabiting microbiota. In the last decades, several molecular techniques have been developed enabling many advantages over the cultivation approach. Mainly PCR-based, fingerprinting techniques allow a qualitative detection and identification of the microbiota. In this study, we developed a real time PCR method as a simple, rapid and reliable tool to detect and quantify fungal abundance using the β-actin gene, which is known to appear as a single-copy gene in fungi. To this end, five different indoor thermal insulation materials applied for historical buildings that were previously tested for their bio-susceptibility against various fungi were subjected to qPCR analyses. The obtained results were compared with those obtained from a previous study investigating the bio-susceptibility of the insulation materials using classical cultivation experiments. Both results correlated well, revealing that Perlite plaster was the most suitable insulation material, showing the lowest fungal CFU and qPCR values. In contrast, insulations made of wood showed to be not recommendable from the microbiological point of view. In addition, the potential of qPCR was tested in other materials of cultural heritage, as old parchments, showing to be a suitable method for measuring fungal abundance in these delicate materials. PMID

  8. An improved, PCR-based strategy for the detection of Trypanosoma cruzi in human blood samples.

    PubMed

    Ribeiro-dos-Santos, G; Nishiya, A S; Sabino, E C; Chamone, D F; Saez-Alquézar, A

    1999-10-01

    Attempts were made to improve the PCR-based detection of Trypanosoma cruzi in blood samples, primarily for screening blood donors. Samples were obtained from candidate donors who were reactive in one or two of three serological tests for Chagas disease (and therefore considered 'indeterminate') or in all three tests (3+). Each sample was then examined using three different, PCR-based techniques: 'PCR-I' (in which the target DNA is a nuclear repetitive sequence); 'PCR-II' [amplifying a conserved region of the T. cruzi kinetoplast DNA (kDNA)]; and 'PCR-III' (a new strategy in which the target kDNA is amplified by 'nested' PCR). Among the samples from 3+ individuals, PCR-I, PCR-II and PCR-III amplified two (3.8%) out of 52, four (4.5%) out of 88, and 27 (25.7%) out of 105 samples tested, respectively. Seven, 69 and 70 samples from 'indeterminate' subjects were tested by PCR-I, PCR-II and PCR-III, respectively; there was not a single positive result by PCR-I or PCR-II, but three (4.3%) of the samples tested by PCR-III were positive. In a reconstruction experiment, in conditions in which PCR-I and PCR-II could not detect 10,000 parasites/ml, PCR-III was able to detect one parasite/ml. Although all three PCR-based strategies examined had rather poor sensitivities, PCR-III was far more sensitive than PCR-I or PCR-II. PMID:10715696

  9. Qualitative Research Designs: Selection and Implementation

    ERIC Educational Resources Information Center

    Creswell, John W.; Hanson, William E.; Plano Clark, Vicki L.; Morales, Alejandro

    2007-01-01

    Counseling psychologists face many approaches from which to choose when they conduct a qualitative research study. This article focuses on the processes of selecting, contrasting, and implementing five different qualitative approaches. Based on an extended example related to test interpretation by counselors, clients, and communities, this article…

  10. Rocket engine diagnostics using qualitative modeling techniques

    NASA Technical Reports Server (NTRS)

    Binder, Michael; Maul, William; Meyer, Claudia; Sovie, Amy

    1992-01-01

    Researchers at NASA Lewis Research Center are presently developing qualitative modeling techniques for automated rocket engine diagnostics. A qualitative model of a turbopump interpropellant seal system was created. The qualitative model describes the effects of seal failures on the system steady state behavior. This model is able to diagnose the failure of particular seals in the system based on anomalous temperature and pressure values. The anomalous values input to the qualitative model are generated using numerical simulations. Diagnostic test cases include both single and multiple seal failures.

  11. Rocket engine diagnostics using qualitative modeling techniques

    NASA Technical Reports Server (NTRS)

    Binder, Michael; Maul, William; Meyer, Claudia; Sovie, Amy

    1992-01-01

    Researchers at NASA Lewis Research Center are presently developing qualitative modeling techniques for automated rocket engine diagnostics. A qualitative model of a turbopump interpropellant seal system has been created. The qualitative model describes the effects of seal failures on the system steady-state behavior. This model is able to diagnose the failure of particular seals in the system based on anomalous temperature and pressure values. The anomalous values input to the qualitative model are generated using numerical simulations. Diagnostic test cases include both single and multiple seal failures.

  12. Comparison of the Xpert MTB/RIF Test with an IS6110-TaqMan Real-Time PCR Assay for Direct Detection of Mycobacterium tuberculosis in Respiratory and Nonrespiratory Specimens▿

    PubMed Central

    Armand, Sylvie; Vanhuls, Pascale; Delcroix, Guy; Courcol, René; Lemaître, Nadine

    2011-01-01

    The sensitivities of the Xpert MTB/RIF test and an in-house IS6110-based real-time PCR using TaqMan probes (IS6110-TaqMan assay) for the detection of Mycobacterium tuberculosis complex (MTBC) DNA were compared by use of 117 clinical specimens (97 culture positive and 20 culture negative for MTBC) that were frozen in sediment. The 97 clinical specimens included 60 respiratory and 37 nonrespiratory specimens distributed into 36 smear-positive and 61 smear-negative specimens. Among the 97 culture-positive specimens, 4 had rifampin-resistant isolates. Both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens. The sensitivity of the Xpert MTB/RIF test with the whole smear-negative specimens was more reduced than that of the IS6110-TaqMan assay (48 versus 69%, P = 0.005). Both methods exhibited similar sensitivities with smear-negative respiratory specimens, but the Xpert MTB/RIF test had lower sensitivity with smear-negative nonrespiratory specimens than the IS6110-TaqMan assay (37 versus 71%, P = 0.013). Finally, the sensitivities of the Xpert MTB/RIF test and the IS6110-TaqMan assay were 79% and 84%, respectively, with respiratory specimens and 53% and 78%, respectively (P = 0.013), with nonrespiratory specimens. The Xpert MTB/RIF test correctly detected the rifampin resistance in smear-positive specimens but not in the one smear-negative specimen. The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens. PMID:21411592

  13. Improving Laboratory Efficiency by Automation of Preanalytic Processing of ThinPrep Specimens for Real-Time PCR High-Risk HPV Testing.

    PubMed

    Barbieri, Daniela; Venturoli, Simona; Costa, Silvano; Landini, Maria Paola

    2016-06-01

    Cervical specimens collected in liquid-based cytology (LBC) media are the most common sample type used for high-risk human papillomavirus (HPV) testing. Since preanalytic steps such as vortexing and decapping vials, liquid transfer to a sample input tube with matching unique identifier, and recapping the original vials are required for processing LBC samples prior to running the Abbott RealTime High Risk HPV assay (Abbott, Wiesbaden, Germany), a full manual execution can be complicated, especially in high-throughput diagnostic contexts. Here, a custom-configured worktable setup for the Tecan Freedom EVO (Tecan, Männedorf, Switzerland) designed to automate and control preanalytic steps for ThinPrep (Hologic, Marlborough, MA) samples was used to evaluate the impact of automated versus manual preanalytics. Archival results for manual processing of 226 samples were compared with those obtained with the Tecan protocol, observing a very good overall concordance for final assay interpretation (95.6%). High overall agreement (100%) resulted also from retesting 99 samples by both the preanalytical protocols. High reproducibility was observed analyzing 23 randomly selected samples by automated preprocessing in triplicate. Hence, the new configuration of the Tecan platform translates the manual steps required to process ThinPrep specimens into automated operations, controls sample identification, and allows for saving hands-on time, while maintaining assay reproducibility and ensuring reliability of results, making it suitable for screening settings. PMID:25673634

  14. Evaluation of PCR based coprodiagnosis of human opisthorchiasis.

    PubMed

    Stensvold, C R; Saijuntha, W; Sithithaworn, P; Wongratanacheewin, S; Strandgaard, H; Ornbjerg, N; Johansen, M V

    2006-01-01

    In this study, a recently developed PCR test for the detection of Opisthorchis viverrini in human faecal samples was evaluated using two parasitological methods as references. During a survey of foodborne trematodes (FBT) in the Vientiane Province, Lao PDR, 85 samples were collected and evaluated for FBT eggs by the Kato Katz (KK) technique, the formalin ethyl acetate concentration technique (FECT) and a PCR analysis for the distinction between O. viverrini and other FBT. The two parasitological methods did not differ in the ability of detecting FBT eggs, and a single KK reading was characterized by a sensitivity of 85% when compared to two FECT readings. The PCR tested positive only in cases where eggs had been demonstrated by parasitological examination. However, the PCR tested negative in some samples with very high egg counts. Demonstrating a PCR sensitivity of approximately 50% in samples with faecal egg counts>1000, the previously reported PCR sensitivity based on in vitro studies was not supported. It is believed that technical problems rather than diagnostic reference related issues were responsible for the relatively low PCR performance. Further studies should aim at optimizing DNA extraction and amplification, and future PCR evaluation should include specificity control such as the scanning electron microscopy of eggs in test samples or the expulsion of adult trematodes from PCR tested individuals. PMID:16253202

  15. Comprehensive GMO detection using real-time PCR array: single-laboratory validation.

    PubMed

    Mano, Junichi; Harada, Mioko; Takabatake, Reona; Furui, Satoshi; Kitta, Kazumi; Nakamura, Kosuke; Akiyama, Hiroshi; Teshima, Reiko; Noritake, Hiromichi; Hatano, Shuko; Futo, Satoshi; Minegishi, Yasutaka; Iizuka, Tayoshi

    2012-01-01

    We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports. PMID:22649939

  16. Qualitative Research in Organizational and Vocational Psychology, 1979-1999.

    ERIC Educational Resources Information Center

    Lee, Thomas W.; Mitchell, Terence R.; Sablynski, Chris J.

    1999-01-01

    Describes qualitative techniques and their use in industrial and vocational psychology for theory generation, elaboration, and testing. Discusses research design, data analysis, and best practices using qualitative methods. Contains 54 references. (SK)

  17. Multiplex real-time quantitative PCR, microscopy and rapid diagnostic immuno-chromatographic tests for the detection of Plasmodium spp: performance, limit of detection analysis and quality assurance

    PubMed Central

    2009-01-01

    Background Accurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease. Materials and methods A total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR), and two rapid diagnostic immuno-chromatographic tests (ICT) in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD) analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated. Results QPCR is the most analytically sensitive method (sensitivity 99.41%), followed by CARESTART (sensitivity 88.24%), and BINAXNOW (sensitivity 86.47%) for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R2 = 0.9746) in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/μl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more costly than reference

  18. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  19. Testing a cycle of family violence model in conflict-affected, low-income countries: a qualitative study from Timor-Leste.

    PubMed

    Rees, Susan; Thorpe, Rosamund; Tol, Wietse; Fonseca, Mira; Silove, Derrick

    2015-04-01

    The present study examines key aspects of an emerging cycle of violence model as applied to conflict-affected countries. We focus specifically on the roles of intimate partner violence (IPV), consequent experiences of explosive anger amongst women, and associated patterns of harsh parenting. Between 2010 and 2011, we conducted a women-centred and culturally sensitive qualitative inquiry with 77 mothers drawn consecutively from a data-base of all adults residing in two villages in Timor-Leste. We over-sampled women who in the preceding whole of household survey met criteria for Intermittent Explosive Disorder (IED). Our methodology included in-depth qualitative interviews followed by a focus group with a comprehensive array of service providers. We used the NVivo software package to manage and analyse data. Our findings provide support for a link between IPV and experiences of explosive anger amongst Timorese mothers. Furthermore, women commonly reported that experiences of explosive anger were accompanied by harsh parenting directed at their children. Women identified the role of patriarchy in legitimizing and perpetuating IPV. Our findings suggest that empowering women to address IPV and poverty may allow them to overcome or manage feelings of anger in a manner that will reduce risk of associated harsh parenting. A fuller examination of the cycle of violence model will need to take into account wider contributing factors at the macro-level (historical, conflict-related, political), the meso-level (community-wide adherence to patriarchal norms affecting the rights and roles of women), and the micro-level (family interactions and gendered role expectations, individual psychological responses, and parenting). Longitudinal studies in post-conflict settings are needed to examine whether the sequence of male violence against women, mothers experience of explosive anger, and consequent harsh parenting contributes to risk of aggression and mental disorder in offspring

  20. Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

    PubMed

    Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

    2015-05-01

    Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors. PMID:25850372

  1. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    PubMed

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number. PMID:27451195

  2. Motivating Men Who Have Sex with Men to Get Tested for HIV through the Internet and Mobile Phones: A Qualitative Study

    PubMed Central

    Blas, Magaly M.; Menacho, Luis A.; Alva, Isaac E.; Cabello, Robinson; Orellana, E. Roberto

    2013-01-01

    Background Men who have sex with men (MSM) have the highest HIV prevalence in Peru, yet they are underserved by traditional preventive programs. In Peru, the Internet and mobile phones have emerged as an effective and convenient tool to reach this population. Methods and Findings From October 2010 to February 2011, we conducted eight focus groups with gay identified MSM (closeted and out-of-the-closet) and with self-identified heterosexual MSM in order to identify key features and preferences to be used to tailor culturally-appropriate messages that could be delivered through Internet and mobile phones to motivate MSM to get tested for HIV. Participants reported that in order to motivate HIV testing among MSM, interventions need to be based on motivational messages that encourage participants to overcome the fear of getting tested. Messages should increase the HIV risk perception (of participants who do not consider themselves at risk) by eliciting risky situations experienced by MSM. Messages should emphasize confidentiality, respect and the professionalism of the personnel conducting the counseling and testing. A thorough explanation of the process of HIV testing and the steps to follow after receiving the results should be provided. Messages should also contain information about the venue where the test will be conducted in terms of client characteristics, location, hours of operation and personnel. Finally, stigmatizing and stereotyping messages or images about “being gay” should not be included, as they act as deterrents for getting tested. Conclusions Interventions aimed at motivating HIV testing among MSM should include motivational messages that reduce the fear of getting tested and increase the risk perception of participants. They should also market the venue where the testing will be conducted, the professionals who will perform the tests, and the type of tests available. Stigmatizing messages or images should be avoided. PMID:23320116

  3. Teaching Qualitative Research

    ERIC Educational Resources Information Center

    Delyser, Dydia

    2008-01-01

    Explicitly qualitative research has never before been so popular in human geography, and this article hopes to encourage more graduate students and faculty members to undertake the teaching of qualitative geography. The article describes one such course for graduate students, highlighting its challenges and rewards, and focusing on exercises…

  4. Qualitative Studies: Historiographical Antecedents.

    ERIC Educational Resources Information Center

    Mills, Rilla Dean

    This paper provides an overview of qualitative studies' antecedents among historiographers and of the positivist tide which nearly engulfed them. Humans live by interpretations. The task of social science--the basic task of qualitative studies--is to study these interpretations so that we can better understand the meanings which people use to…

  5. Visualizing Qualitative Information

    ERIC Educational Resources Information Center

    Slone, Debra J.

    2009-01-01

    The abundance of qualitative data in today's society and the need to easily scrutinize, digest, and share this information calls for effective visualization and analysis tools. Yet, no existing qualitative tools have the analytic power, visual effectiveness, and universality of familiar quantitative instruments like bar charts, scatter-plots, and…

  6. Sampling in Qualitative Research

    PubMed Central

    LUBORSKY, MARK R.; RUBINSTEIN, ROBERT L.

    2011-01-01

    In gerontology the most recognized and elaborate discourse about sampling is generally thought to be in quantitative research associated with survey research and medical research. But sampling has long been a central concern in the social and humanistic inquiry, albeit in a different guise suited to the different goals. There is a need for more explicit discussion of qualitative sampling issues. This article will outline the guiding principles and rationales, features, and practices of sampling in qualitative research. It then describes common questions about sampling in qualitative research. In conclusion it proposes the concept of qualitative clarity as a set of principles (analogous to statistical power) to guide assessments of qualitative sampling in a particular study or proposal. PMID:22058580

  7. PCR+ In Diesel Fuels and Emissions Research

    SciTech Connect

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  8. Further improvement and validation of MagMAX-96 AI/ND viral RNA isolation for efficient removal of RT-PCR inhibitors from cloacal swabs and tissues for rapid diagnosis of avian influenza virus by RT reverse transcription PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time RT-PCR (RRT-PCR) is a high throughput molecular diagnostic test used for rapid detection of avian influenza virus (AIV) in clinical samples. However the performance of RRT-PCR can be adversely affected by RT-PCR inhibitors present in the sample. The tested commercial RNA extraction kits ...

  9. International collaborative study of the endogenous reference gene, sucrose phosphate synthase (SPS), used for qualitative and quantitative analysis of genetically modified rice.

    PubMed

    Jiang, Lingxi; Yang, Litao; Zhang, Haibo; Guo, Jinchao; Mazzara, Marco; Van den Eede, Guy; Zhang, Dabing

    2009-05-13

    One rice ( Oryza sativa ) gene, sucrose phosphate synthase (SPS), has been proven to be a suitable endogenous reference gene for genetically modified (GM) rice detection in a previous study. Herein are the reported results of an international collaborative ring trial for validation of the SPS gene as an endogenous reference gene and its optimized qualitative and quantitative polymerase chain reaction (PCR) systems. A total of 12 genetically modified organism (GMO) detection laboratories from seven countries participated in the ring trial and returned their results. The validated results confirmed the species specificity of the method through testing 10 plant genomic DNAs, low heterogeneity, and a stable single-copy number of the rice SPS gene among 7 indica varieties and 5 japonica varieties. The SPS qualitative PCR assay was validated with a limit of detection (LOD) of 0.1%, which corresponded to about 230 copies of haploid rice genomic DNA, while the limit of quantification (LOQ) for the quantitative PCR system was about 23 copies of haploid rice genomic DNA, with acceptable PCR efficiency and linearity. Furthermore, the bias between the test and true values of eight blind samples ranged from 5.22 to 26.53%. Thus, we believe that the SPS gene is suitable for use as an endogenous reference gene for the identification and quantification of GM rice and its derivates. PMID:19326953

  10. The friends and family test: a qualitative study of concerns that influence the willingness of English National Health Service staff to recommend their organisation

    PubMed Central

    Minion, Joel T; McKee, Lorna; Willars, Janet; Martin, Graham

    2014-01-01

    Objectives The views of practitioners at the ‘sharp end’ of care provision are increasingly recognised as important indicators of quality of care. The National Health Service (NHS) Staff Survey in England has quantified employees' views on how far they would be happy with the standard of care provided by their organisation if a friend or family member needed treatment. We aimed to characterise the concerns that might affect the willingness of staff to recommend their own organisations. Design Qualitative study involving semi-structured interviews. Data analysis based on the constant comparative method. Participants Members of clinical and managerial staff in four NHS organisations (n = 70), and senior stakeholders across the NHS including clinicians, managers and others with a strategic or senior-level perspective (n = 98). Setting One hundred and sixty-eight interviews were conducted: 70 in four case study organisations and 98 across the wider English NHS. Main outcome measures Not applicable. Results Asking study participants the ‘if a friend…’ question offered insider views on the quality of care. Some staff had no concerns, but others, identified significant problems with consistency, reliability and behaviour of staff. Participants identified reasons for poor care that included inadequate organisational systems; structural problems of understaffing and under-resourcing; weaknesses in professional cultures and professional competence and failure to deal with problems such as unacceptable conduct. Participants emphasised that staff were not always able to deliver high-quality care because they worked in difficult conditions. Conclusions Asking staff to give accounts of their willingness to recommend their organisation to family and friends elicits important insights into quality and safety of care. Such accounts might be able to provide warning signs that could signal organisational decline and avert healthcare scandals, but use outside a

  11. A Qualitative Analysis of Provider Barriers and Solutions to HIV Testing for Substance Users in a Small, Largely Rural Southern State

    PubMed Central

    Wright, Patricia B.; Curran, Geoffrey M.; Stewart, Katharine E.; Booth, Brenda M.

    2013-01-01

    Purpose Integrating HIV testing programs into substance use treatment is a promising avenue to help increase access to HIV testing for rural drug users. Yet few outpatient substance abuse treatment facilities in the United States provide HIV testing. The purpose of this study was to identify barriers to incorporating HIV testing with substance use treatment from the perspectives of treatment and testing providers in Arkansas. Methods We used purposive sampling from state directories to recruit providers at state, organization, and individual levels to participate in this exploratory study. Using an interview guide, the first and second authors conducted semi-structured individual interviews in each provider’s office or by telephone. All interviews were recorded, transcribed verbatim, and entered into ATLAS.ti software (ATLAS.ti Scientific Sofware Development GmbH, Berlin, Germany). We used constant comparison and content analysis techniques to identify codes, categories, and primary patterns in the data. Findings The sample consisted of 28 providers throughout the state, 18 from the substance use system and 10 from the public/ community health system. We identified 7 categories of barriers: environmental constraints, policy constraints, funding constraints, organizational structure, limited inter- and intra-agency communication, burden of responsibility, and client fragility. Conclusions This study presents the practice-based realities of barriers to integrating HIV testing with substance use treatment in a small, largely rural state. Some system and/or organization leaders were either unaware of or not actively pursuing external funds available to them specifically for engaging substance users in HIV testing. However, funding does not address the system-level need for coordination of resources and services at the state level. PMID:24088216

  12. Mistrust in marriage-Reasons why men do not accept couple HIV testing during antenatal care- a qualitative study in eastern Uganda

    PubMed Central

    2010-01-01

    Background A policy for couple HIV counseling and testing was introduced in 2006 in Uganda, urging pregnant women and their spouses to be HIV tested together during antenatal care (ANC). The policy aims to identify HIV-infected pregnant women to prevent mother-to-child transmission of HIV through prophylactic antiretroviral treatment, to provide counseling, and to link HIV-infected persons to care. However, the uptake of couple testing remains low. This study explores men's views on, and experiences of couple HIV testing during ANC. Methods The study was conducted at two time points, in 2008 and 2009, in the rural Iganga and Mayuge districts of eastern Uganda. We carried out nine focus group discussions, about 10 participants in each, and in-depth interviews with 13 men, all of whom were fathers. Data were collected in the local language, Lusoga, audio-recorded and thereafter translated and transcribed into English and analyzed using content analysis. Results Men were fully aware of the availability of couple HIV testing, but cited several barriers to their use of these services. The men perceived their marriages as unstable and distrustful, making the idea of couple testing unappealing because of the conflicts it could give rise to. Further, they did not understand why they should be tested if they did not have symptoms. Finally, the perceived stigmatizing nature of HIV care and rude attitudes among health workers at the health facilities led them to view the health facilities providing ANC as unwelcoming. The men in our study had several suggestions for how to improve the current policy: peer sensitization of men, make health facilities less stigmatizing and more male-friendly, train health workers to meet men's needs, and hold discussions between health workers and community members. Conclusions In summary, pursuing couple HIV testing as a main avenue for making men more willing to test and support PMTCT for their wives, does not seem to work in its current form

  13. PCR identification of Mycobacterium bovis BCG.

    PubMed Central

    Talbot, E A; Williams, D L; Frothingham, R

    1997-01-01

    The attenuated bacillus Calmette-Guérin (BCG) vaccine strain is derived from a virulent strain of Mycobacterium bovis. BCG is difficult to differentiate from other strains of M. bovis and other members of the M. tuberculosis complex by conventional methods. Recently, a genomic region designated RD1 was found to be present in all virulent M. bovis and M. tuberculosis strains tested but deleted from all BCG strains tested. With this information, a multiplex PCR method was developed to detect the RD1 deletion. A large collection of BCG and other M. tuberculosis complex strains from diverse host and geographic origins was tested. RD1 was deleted in 23 of 23 BCG strains. RD1 was present in 129 of 129 other M. tuberculosis complex strains. This multiplex PCR method can be used as a tool for the rapid and specific identification of BCG. PMID:9041390

  14. Identification of Actinobacillus actinomycetemcomitans in subgingival plaque by PCR.

    PubMed Central

    Flemmig, T F; Rüdiger, S; Hofmann, U; Schmidt, H; Plaschke, B; Strätz, A; Klaiber, B; Karch, H

    1995-01-01

    The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the

  15. Perceptions of risk and predictive testing held by the first-degree relatives of patients with rheumatoid arthritis in England, Austria and Germany: a qualitative study

    PubMed Central

    Stack, Rebecca J; Stoffer, Michaela; Englbrecht, Mathias; Mosor, Erika; Falahee, Marie; Simons, Gwenda; Smolen, Josef; Schett, Georg; Buckley, Chris D; Kumar, Kanta; Hansson, Mats; Hueber, Axel; Stamm, Tanja; Raza, Karim

    2016-01-01

    Objectives The family members of patients with rheumatoid arthritis (RA) are at increased risk of developing RA and are potential candidates for predictive testing. This study explored the perceptions of first-degree relatives of people with RA about being at risk of RA and engaging in predictive testing. Methods 34 first-degree relatives (siblings and offspring) of patients with RA from the UK, Germany and Austria participated in semistructured interviews about their perceptions of RA risk and the prospect of predictive testing. Interviews were audio-recorded, transcribed verbatim and analysed using thematic analysis. Results First-degree relatives were aware of their susceptibility to RA, but were unsure of the extent of their risk. When considering their future risk, some relatives were concerned about the potential impact that RA would have on their lives. Relatives were concerned that knowing their actual risk would increase their anxiety and would affect decisions about their future. Also, relatives were concerned about the levels of uncertainty associated with predictive testing. Those in favour of knowing their future risk felt that they would need additional support to understand the risk information and cope with the emotional impact of this information. Conclusions Identifying individuals at risk of RA may allow targeted interventions to reduce the risk and consequence of future disease; however, relatives have concerns about predictive testing and risk information. The development of strategies to quantify and communicate risk needs to take these views into account and incorporate approaches to mitigate concerns and minimise the psychological impact of risk information. PMID:27357193

  16. Computers and Qualitative Research.

    ERIC Educational Resources Information Center

    Willis, Jerry; Jost, Muktha

    1999-01-01

    Discusses the use of computers in qualitative research, including sources of information; collaboration; electronic discussion groups; Web sites; Internet search engines; electronic sources of data; data collection; communicating research results; desktop publishing; hypermedia and multimedia documents; electronic publishing; holistic and…

  17. A Specific Qualitative Detection Method for Peanut (Arachis Hypogaea) in Foods Using Polymerase Chain Reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed a qualitative detection method for peanuts in foods using polymerase chain reaction (PCR). We designed a universal primer pair CP 03-5’/ CP 03-3’ to confirm the validity of the DNAs for PCR. The plant specific amplified fragments were detected from 13 kinds of plants using the universal...

  18. Detection of Treponema pallidum in the vitreous by PCR

    PubMed Central

    Müller, M; Ewert, I; Hansmann, F; Tiemann, C; Hagedorn, H J; Solbach, W; Roider, J; Nölle, B; Laqua, H; Hoerauf, H

    2007-01-01

    Background Ocular involvement of syphilis still poses a clinical challenge due to the chameleonic behaviour of the disease. As the serodiagnosis has significant limitations, the direct detection of Treponema pallidum (TP) in the vitreous represents a desirable diagnostic tool. Methods Real‐time polymerase chain reaction (PCR) for the detection of TP was applied in diagnostic vitrectomies of two patients with acute chorioretinitis. Qualitative verification of TP by real‐time PCR and melting point analysis according to a modified protocol was ruled out. Patients underwent complete ophthalmological examination with fundus photographs, fluorescein angiography, serological examination, antibiotic treatment and follow‐up. Results In two cases of acute chorioretinitis of unknown origin, real‐time PCR of vitreous specimens of both patients provided evidence of TP and was 100% specific. Initial diagnosis of presumed viral retinitis was ruled out by PCR of vitreous specimen. Patients were treated with systemic antibiotics and showed prompt improvement in visual function and resolution of fundus lesions. Conclusions With real‐time PCR, detection of TP in the vitreous was possible and delivered a sensitive, quick and inexpensive answer to a disease rather difficult to assess. In cases of acute chorioretinitis, the use of PCR‐based assays of vitreous specimens in the diagnostic evaluation of patients is advisable. Although syphilitic chorioretinitis is a rare disease, PCR should include search for TP, as diagnostic dilemmas prolong definitive treatment in a sight‐threatening disease. PMID:17108014

  19. HETEROSEXUAL PARTNERSHIPS AND THE NEED FOR HIV PREVENTION AND TESTING FOR MEN WHO HAVE SEX WITH MEN AND WOMEN IN CHINA: A QUALITATIVE STUDY

    PubMed Central

    Wang, Sijia; Song, Dandan; Huang, Wen; He, Huan; Wang, Min; Manning, David; Zaller, Nickolas; Zhang, Hongbo; Operario, Don

    2016-01-01

    Previous studies have reported that approximately 30% of men who have sex with men (MSM) in China have concurrent female partners. Men who have sex with men and women (MSMW) might “bridge” HIV transmission to their female sex partners. This study aimed to explore (a) motivations for why MSMW in China engage in relationships and sexual behaviors with female partners; (b) patterns of sexual behaviors and condom use between MSMW and their female partners; and (c) barriers to and strategies for encouraging MSMW and their female partners to undergo HIV testing. The authors conducted in-depth interviews with 30 MSMW in two urban cities in China, Guangzhou and Chengdu, and used thematic analysis methods to code and interpret the data. MSMW described family, social, and workplace pressures to have a female partner, and expressed futility about their ability to form stable same-sex relationships. Although participants reported concern about the risk of personally acquiring and transmitting HIV or other sexually transmitted infections (STIs) to their female partners, they described the challenges to using condoms with female partners. HIV-positive participants described how stigma restricted their ability to disclose their HIV status to female partners, and HIV-negative participants displayed less immediate concern about the need for female partners to undergo HIV testing. Participants described a range of possible strategies to encourage HIV testing among female partners. These findings highlight the urgent need for HIV risk reduction and testing interventions for Chinese MSMW in the context of heterosexual partnerships, and they also under-score the additional need for privacy and cultural sensitivity when designing future studies. PMID:25915698

  20. Investigation of vesicular rashes for HSV and VZV by PCR.

    PubMed

    Beards, G; Graham, C; Pillay, D

    1998-03-01

    Vesicular fluid from rashes of 132 patients was tested by a multiplex PCR shown to be specific for herpes simplex virus (HSV) type 1 and 2, and varicella zoster virus (VZV) genomic DNA. The results were compared with those obtained by examination by electron microscopy and virus isolation by cell culture. The PCR did not differentiate between HSV 1 and 2. By PCR, 64 HSV infections and 53 VZV infections were identified, with presumed 100% sensitivity and specificity. Fifteen specimens tested negative by PCR, electron microscopy, and virus isolation for herpes viruses. The sensitivities of virus isolation and electron microscopy for detection of herpes simplex virus were 56% and 80%. For varicella zoster virus, the sensitivities of virus isolation and electron microscopy were 47% and 60%. These data illustrate the advantage of rapid PCR diagnosis of herpes simplex virus and varicella zoster virus in vesicle fluids. PMID:9515761

  1. Antigenic typing of canine parvovirus using differential PCR.

    PubMed

    Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Sharma, N S

    2014-12-01

    Canine parvovirus (CPV) is an enteric pathogen causing hemorrhagic enteritis in pups of 3-6 months of age and is mainly transmitted via feco-oral route. In the present study, a total of 85 animals rectal swabs suspected of CPV were tested using a PCR, nested PCR and a newly designed differential PCR. Using PCR 7 (8.23 %) animals were positive whereas 39 (45.88 %) were positive by using nested PCR and 40 (47.05 %) were positive for either one or more than one antigenic types of CPV using differential PCR. Using differential PCR it was found that CPV-2a and CPV-2b were the most prevailing antigenic types. Also it was found that dogs that were vaccinated too yielded positive CPV indicating a possible presence of additional CPV antigenic types. Thus, the primers used in differential PCR can be used in a single PCR reaction to detect various antigenic types of CPV. PMID:25674626

  2. Incorporating information about pre-implantation genetic diagnosis into discussions about testing and risk-management for BRCA1/2 mutations: A qualitative study of patient preferences

    PubMed Central

    Hurley, Karen; Rubin, Lisa; Werner-Lin, Allison; Sagi, Michal; Kemel, Yelena; Stern, Rikki; Phillips, Aliza; Cholst, Ina; Kauff, Noah; Offit, Kenneth

    2016-01-01

    Background Studies show that BRCA1/2 mutation carriers are interested in learning about reproductive options such as pre-implantation genetic diagnosis (PGD) to prevent passing their risk onto their children. However, attitudes vary widely, and the procedure raises complex ethical and psychosocial issues. This complexity, plus the highly technical nature of PGD, makes it difficult to integrate PGD information into genetic counseling sessions that already cover probabilistic, emotionally-charged risk information. Method Thirty-three reproductive age BRCA1/2 mutation carriers who had previously undergone genetic counseling viewed a tutorial about PGD and were interviewed about attitudes towards PGD, and preferences about how to include PGD information in genetic counseling. Results Most participants preferred to be briefly informed of availability of PGD information, and to receive written materials about PGD, but with the option of deferring detailed discussion if they already feel overloaded or perceive that PGD is not immediately relevant to their risk management and/or childbearing plans. For some, the stress of testing temporarily interfered with information processing, producing states of cognitive avoidance (“in a fog,” “tuning out”). Some preferred to discuss PGD with a physician with whom they had an ongoing relationship (e.g., OB/GYN, primary care provider, oncologist). Conclusions Providers offering cancer genetic testing can consider indicating availability of PGD information, while attending to patients’ level of interest and ability to absorb information. Research is needed to link patient responses to information overload to psychosocial outcomes (e.g., distress, decision quality). Continuing medical education is needed to support providers in facilitating informed decisions about PGD. PMID:22736296

  3. Performance Testing of PCR Assay in Blood Samples for the Diagnosis of Toxoplasmic Encephalitis in AIDS Patients from the French Departments of America and Genetic Diversity of Toxoplasma gondii: A Prospective and Multicentric Study

    PubMed Central

    Ajzenberg, Daniel; Lamaury, Isabelle; Demar, Magalie; Vautrin, Cyrille; Cabié, André; Simon, Stéphane; Nicolas, Muriel; Desbois-Nogard, Nicole; Boukhari, Rachida; Riahi, Homayoun; Dardé, Marie-Laure; Massip, Patrice; Dupon, Michel; Preux, Pierre-Marie; Labrunie, Anaïs; Boncoeur, Marie-Paule

    2016-01-01

    Background Toxoplasmic encephalitis in patients with AIDS is a life-threatening disease mostly due to reactivation of Toxoplasma gondii cysts in the brain. The main objective of this study was to evaluate the performance of real-time PCR assay in peripheral blood samples for the diagnosis of toxoplasmic encephalitis in AIDS patients in the French West Indies and Guiana. Methodology/Principal Findings Adult patients with HIV and suspicion of toxoplasmic encephalitis with start of specific antitoxoplasmic therapy were included in this study during 40 months. The real-time PCR assay targeting the 529 bp repeat region of T. gondii was performed in two different centers for all blood samples. A Neighbor-Joining tree was reconstructed from microsatellite data to examine the relationships between strains from human cases of toxoplasmosis in South America and the Caribbean. A total of 44 cases were validated by a committee of experts, including 36 cases with toxoplasmic encephalitis. The specificity of the PCR assay in blood samples was 100% but the sensitivity was only 25% with moderate agreement between the two centers. Altered level of consciousness and being born in the French West Indies and Guiana were the only two variables that were associated with significantly decreased risk of false negative results with the PCR assay. Conclusion/Significance Our results showed that PCR sensitivity in blood samples increased with severity of toxoplasmic encephalitis in AIDS patients. Geographic origin of patients was likely to influence PCR sensitivity but there was little evidence that it was caused by differences in T. gondii strains. Trial Registration ClinicalTrials.gov NCT00803621 PMID:27355620

  4. "Smoking in Children's Environment Test": a qualitative study of experiences of a new instrument applied in preventive work in child health care

    PubMed Central

    2011-01-01

    Background Despite knowledge of the adverse health effects of passive smoking, children are still being exposed. Children's nurses play an important role in tobacco preventive work through dialogue with parents aimed at identifying how children can be protected from environmental tobacco smoke (ETS) exposure. The study describes the experiences of Child Health Care (CHC) nurses when using the validated instrument SiCET (Smoking in Children's Environment Test) in dialogue with parents. Method In an intervention in CHC centres in south-eastern Sweden nurses were invited to use the SiCET. Eighteen nurses participated in focus group interviews. Transcripts were reviewed and their contents were coded into categories by three investigators using the method described for focus groups interviews. Results The SiCET was used in dialogue with parents in tobacco preventive work and resulted in focused discussions on smoking and support for behavioural changes among parents. The instrument had both strengths and limitations. The nurses experienced that the SiCET facilitated dialogue with parents and gave a comprehensive view of the child's ETS exposure. This gave nurses the possibility of taking on a supportive role by offering parents long-term help in protecting their child from ETS exposure and in considering smoking cessation. Conclusion Our findings indicate that the SiCET supports nurses in their dialogue with parents on children's ETS exposure at CHC. There is a need for more clinical use and evaluation of the SiCET to determine its usefulness in clinical practice under varying circumstances. PMID:22172056

  5. Qualitative Assertions as Prescriptive Statements

    ERIC Educational Resources Information Center

    Nolen, Amanda; Talbert, Tony

    2011-01-01

    The primary question regarding prescriptive appropriateness is a difficult one to answer for the qualitative researcher. While there are certainly qualitative researchers who have offered prescriptive protocols to better define and describe the terrain of qualitative research design and there are qualitative researchers who offer research…

  6. “I Do Feel Like a Scientist at Times”: A Qualitative Study of the Acceptability of Molecular Point-Of-Care Testing for Chlamydia and Gonorrhoea to Primary Care Professionals in a Remote High STI Burden Setting

    PubMed Central

    Natoli, Lisa; Guy, Rebecca J.; Shephard, Mark; Causer, Louise; Badman, Steven G.; Hengel, Belinda; Tangey, Annie; Ward, James; Coburn, Tony; Anderson, David; Kaldor, John; Maher, Lisa

    2015-01-01

    Background Point-of-care tests for chlamydia (CT) and gonorrhoea (NG) could increase the uptake and timeliness of testing and treatment, contribute to improved disease control and reduce reproductive morbidity. The GeneXpert (Xpert CT/NG assay), suited to use at the point-of-care, is being used in the TTANGO randomised controlled trial (RCT) in 12 remote Australian health services with a high burden of sexually transmissible infections (STIs). This represents the first ever routine use of a molecular point-of-care diagnostic for STIs in primary care. The purpose of this study was to explore the acceptability of the GeneXpert to primary care staff in remote Australia. Methods In-depth qualitative interviews were conducted with 16 staff (registered or enrolled nurses and Aboriginal Health Workers/Practitioners) trained and experienced with GeneXpert testing. Interviews were digitally-recorded and transcribed verbatim prior to content analysis. Results Most participants displayed positive attitudes, indicating the test was both easy to use and useful in their clinical context. Participants indicated that point-of-care testing had improved management of STIs, resulting in more timely and targeted treatment, earlier commencement of partner notification, and reduced follow up efforts associated with client recall. Staff expressed confidence in point-of-care test results and treating patients on this basis, and reported greater job satisfaction. While point-of-care testing did not negatively impact on client flow, several found the manual documentation processes time consuming, suggesting that improved electronic connectivity and test result transfer between the GeneXpert and patient management systems could overcome this. Managing positive test results in a shorter time frame was challenging for some but most found it satisfying to complete episodes of care more quickly. Conclusions In the context of a RCT, health professionals working in remote primary care in Australia

  7. Engineered DNA polymerase improves PCR results for plastid DNA1

    PubMed Central

    Schori, Melanie; Appel, Maryke; Kitko, AlexaRae; Showalter, Allan M.

    2013-01-01

    • Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. • Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. • Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase. PMID:25202519

  8. A thermally baffled device for highly stabilized convective PCR.

    PubMed

    Chang, Hsiao-Fen Grace; Tsai, Yun-Long; Tsai, Chuan-Fu; Lin, Ching-Ko; Lee, Pei-Yu; Teng, Ping-Hua; Su, Chen; Jeng, Chien-Chung

    2012-05-01

    Rayleigh-Bénard convective PCR is a simple and effective design for amplification of DNA. Convective PCR is, however, extremely sensitive to environmental temperature fluctuations, especially when using small- diameter test tubes. Therefore, this method is inherently unstable with limited applications. Here, we present a convective PCR device that has been modified by adding thermal baffles. With this thermally baffled device the influence from fluctuations in environmental temperature were significantly reduced, even in a wind tunnel (1 m/s). The thermally baffled PCR instrument described here has the potential to be used as a low-cost, point-of-care device for PCR-based molecular diagnostics in the field. PMID:22241586

  9. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    NASA Astrophysics Data System (ADS)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  10. Pertussis Tests

    MedlinePlus

    ... as: Whooping Cough Tests Formal name: Bordetella pertussis Culture; Bordetella pertussis by PCR; Bordetella pertussis Antibodies (IgA, ... outbreak, at least one case be confirmed using culture. Culture – this test was the "gold standard" for ...

  11. Sporulation properties and antimicrobial susceptibility in endemic and rare Clostridium difficile PCR ribotypes.

    PubMed

    Zidaric, Valerija; Rupnik, Maja

    2016-06-01

    Increased sporulation and antibiotic resistance have been proposed to be associated with certain Clostridium difficile epidemic strains such as PCR ribotype 027. In this study we examined these properties in another widespread PCR ribotype, 014/020, in comparison to prevalent PCR ribotype 002 and a group of rarely represented PCR ribotypes. Highest sporulation was observed in 014/020 strains at 24 h, while after 72 h PCR ribotype 002 and rare PCR ribotypes formed higher total number of spores. PCR ribotype 014/020 strains exhibited slightly higher resistance to tested antimicrobials, followed by group of rare PCR ribotypes and less common PCR ribotype 002. Neither sporulation properties nor antibiotic resistance clearly differed in endemic and rare strains. PMID:27095618

  12. Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

  13. [Rapid PCR authentication Lonicera japanica].

    PubMed

    Jiang, Chao; Hou, Jing-Yi; Huang, Lu-Qi; Yuan, Yuan; Chen, Min; Jin, Yan

    2014-10-01

    To simply and rapid authenticate Lonicera japanica. Rapid allele-specific PCR primer was designed base on trnL-trnF 625 G/T Single nucleotide polymorphism and the PCR reaction systems including annealing temperature was optimized; optimized results were performed to authenticate L. japanica and its 9 adulterants. When 100 x SYBR Green I was added in the PCR product of 87 degrees C initial denatured 1 min; 87 degrees C denatured 5 s, 68 degrees C annealing 5 s, 30 cycle; L. japanica visualize strong green fluorescence under 365 nm UV lamp whereas adulterants without. The results indicate rapid allele-specific PCR could authenticate L. japanica and its adulterants rapidly and simply. PMID:25612418

  14. Disciplining Qualitative Research

    ERIC Educational Resources Information Center

    Denzin, Norman K.; Lincoln, Yvonna S.; Giardina, Michael D.

    2006-01-01

    Qualitative research exists in a time of global uncertainty. Around the world, governments are attempting to regulate scientific inquiry by defining what counts as "good" science. These regulatory activities raise fundamental, philosophical epistemological, political and pedagogical issues for scholarship and freedom of speech in the academy. This…

  15. Entropy Is Simple, Qualitatively.

    ERIC Educational Resources Information Center

    Lambert, Frank L.

    2002-01-01

    Suggests that qualitatively, entropy is simple. Entropy increase from a macro viewpoint is a measure of the dispersal of energy from localized to spread out at a temperature T. Fundamentally based on statistical and quantum mechanics, this approach is superior to the non-fundamental "disorder" as a descriptor of entropy change. (MM)

  16. Demystifying Interdisciplinary Qualitative Research

    ERIC Educational Resources Information Center

    Greckhamer, Thomas; Koro-Ljungberg, Mirka; Cilesiz, Sebnem; Hayes, Sharon

    2008-01-01

    This article seeks to demystify, through deconstruction, the concept of "interdisciplinarity" in the context of qualitative research to contribute to a new praxis of knowledge production through reflection on the possibilities and impossibilities of interdisciplinarity. A review and discussion of disciplinarity and interdisciplinarity leads the…

  17. [Qualitative case study].

    PubMed

    Debout, Christophe

    2016-06-01

    The qualitative case study is a research method which enables a complex phenomenon to be explored through the identification of different factors interacting with each other. The case observed is a real situation. In the field of nursing science, it may be a clinical decision-making process. The study thereby enables the patient or health professional experience to be conceptualised. PMID:27338694

  18. Bookstart: A Qualitative Evaluation.

    ERIC Educational Resources Information Center

    Moore, Maggie; Wade, Barrie

    2003-01-01

    A qualitative study of Bookstart, which gave free children's books and support to British inner-city families, collected data from librarians, health visitors, and nursery school staff. Bookstart increased positive attitudes toward and interest in books. Health visitors' support was crucial in helping parents use the books to support children's…

  19. The Qualitative Similarity Hypothesis

    ERIC Educational Resources Information Center

    Paul, Peter V.; Lee, Chongmin

    2010-01-01

    Evidence is presented for the qualitative similarity hypothesis (QSH) with respect to children and adolescents who are d/Deaf or hard of hearing. The primary focus is on the development of English language and literacy skills, and some information is provided on the acquisition of English as a second language. The QSH is briefly discussed within…

  20. Characterization of the exogenous insert and development of event-specific PCR detection methods for genetically modified Huanong No. 1 papaya.

    PubMed

    Guo, Jinchao; Yang, Litao; Liu, Xin; Guan, Xiaoyan; Jiang, Lingxi; Zhang, Dabing

    2009-08-26

    Genetically modified (GM) papaya (Carica papaya L.), Huanong No. 1, was approved for commercialization in Guangdong province, China in 2006, and the development of the Huanong No. 1 papaya detection method is necessary for implementing genetically modified organism (GMO) labeling regulations. In this study, we reported the characterization of the exogenous integration of GM Huanong No. 1 papaya by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. The results suggested that one intact copy of the initial construction was integrated in the papaya genome and which probably resulted in one deletion (38 bp in size) of the host genomic DNA. Also, one unintended insertion of a 92 bp truncated NptII fragment was observed at the 5' end of the exogenous insert. Furthermore, we revealed its 5' and 3' flanking sequences between the insert DNA and the papaya genomic DNA, and developed the event-specific qualitative and quantitative PCR assays for GM Huanong No. 1 papaya based on the 5' integration flanking sequence. The relative limit of detection (LOD) of the qualitative PCR assay was about 0.01% in 100 ng of total papaya genomic DNA, corresponding to about 25 copies of papaya haploid genome. In the quantitative PCR, the limits of detection and quantification (LOD and LOQ) were as low as 12.5 and 25 copies of papaya haploid genome, respectively. In practical sample quantification, the quantified biases between the test and true values of three samples ranged from 0.44% to 4.41%. Collectively, we proposed that all of these results are useful for the identification and quantification of Huanong No. 1 papaya and its derivates. PMID:19645503

  1. Ligation-independent cloning of PCR products (LIC-PCR).

    PubMed Central

    Aslanidis, C; de Jong, P J

    1990-01-01

    A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones. Images PMID:2235490

  2. A study of PCR inhibition mechanisms using real time PCR.

    PubMed

    Opel, Kerry L; Chung, Denise; McCord, Bruce R

    2010-01-01

    In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co-extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance. PMID:20015162

  3. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments.

    PubMed

    Svec, David; Tichopad, Ales; Novosadova, Vendula; Pfaffl, Michael W; Kubista, Mikael

    2015-03-01

    We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range. PMID:27077029

  4. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  5. A qualitative study of GP, NP and patient views about the use of rapid streptococcal antigen detection tests (RADTs) in primary care: ‘swamped with sore throats?’

    PubMed Central

    Leydon, Gerry M; McDermott, Lisa; Moore, Mike; Williamson, Ian; Hobbs, F D Richard; Lambton, Tessa; Cooper, Rebecca; Henderson, Hugo; Little, Paul

    2013-01-01

    Objective To explore patient and healthcare professionals’ (HCP) views of clinical scores and rapid streptococcal antigen detection tests (RADTs) for acute sore throat. Design Qualitative semistructured interview study. Setting UK primary care. Participants General practitioners (GPs), nurse practitioners (NPs) and patients from general practices across Hampshire, Oxfordshire and the West Midlands who were participating in the Primary Care Streptococcal Management (PRISM) study. Method Semistructured, face-to-face and phone interviews were conducted with GPs, NPs and patients from general practices across Hampshire, Oxfordshire and the West Midlands. Results 51 participants took part in the study. Of these, 42 were HCPs (29 GPs and 13 NPs) and 9 were patients. HCPs could see a positive role for RADTs in terms of reassurance, as an educational tool for patients, and for aiding inexperienced practitioners, but also had major concerns about RADT use in clinical practice. Particular concerns included the validity of the tests (the role of other bacteria, and carrier states), the tension and possible disconnect with clinical assessment and intuition, the issues of time and resource use and the potential for medicalisation of self-limiting illness. In contrast, however, experience of using RADTs over time seemed to make some participants more positive about using the tests. Moreover, patients were much more positive about the place of RADTs in providing reassurance and in limiting their antibiotic use. Conclusions It is unlikely that RADTs will have a (comfortable) place in clinical practice in the near future until health professionals’ concerns are met, and they have direct experience of using them. The routine use of clinical scoring systems for acute upper respiratory illness also face important barriers related to clinicians’ perceptions of their utility in the face of clinician experience and intuition. PMID:23558734

  6. Development and validation of a real-time PCR assay for detection and quantification of Tuber magnatum in soil

    PubMed Central

    2012-01-01

    Background Tuber magnatum, the Italian white truffle, is the most sought-after edible ectomycorrhizal mushroom. Previous studies report the difficulties of detecting its mycorrhizas and the widespread presence of its mycelium in natural production areas, suggesting that the soil mycelium could be a good indicator to evaluate its presence in the soil. In this study a specific real-time PCR assay using TaqMan chemistry was developed to detect and quantify T. magnatum in soil. This technique was then applied to four natural T. magnatum truffières located in different regions of Italy to validate the method under different environmental conditions. Results The primer/probe sets for the detection and quantification of T. magnatum were selected from the ITS rDNA regions. Their specificity was tested in silico and using qualitative PCR on DNA extracted from 25 different fungal species. The T. magnatum DNA concentration was different in the four experimental truffières and higher in the productive plots. T. magnatum mycelium was however also detected in most of the non-productive plots. Ascoma production during the three years of the study was correlated with the concentration of T. magnatum DNA. Conclusions Taken together, these results suggest that the specific real-time PCR assay perfected in this study could be an useful tool to evaluate the presence and dynamics of this precious truffle in natural and cultivated truffières. PMID:22672347

  7. Clostridium difficile PCR Ribotypes in Calves, Canada

    PubMed Central

    Stämpfli, Henry R.; Duffield, Todd; Peregrine, Andrew S.; Trotz-Williams, Lise A.; Arroyo, Luis G.; Brazier, Jon S.; Weese, J. Scott

    2006-01-01

    We investigated Clostridium difficile in calves and the similarity between bovine and human C. difficile PCR ribotypes by conducting a case-control study of calves from 102 dairy farms in Canada. Fecal samples from 144 calves with diarrhea and 134 control calves were cultured for C. difficile and tested with an ELISA for C. difficile toxins A and B. C. difficile was isolated from 31 of 278 calves: 11 (7.6%) of 144 with diarrhea and 20 (14.9%) of 134 controls (p = 0.009). Toxins were detected in calf feces from 58 (56.8%) of 102 farms, 57 (39.6%) of 144 calves with diarrhea, and 28 (20.9%) of 134 controls (p = 0.0002). PCR ribotyping of 31 isolates showed 8 distinct patterns; 7 have been identified in humans, 2 of which have been associated with outbreaks of severe disease (PCR types 017 and 027). C. difficile may be associated with calf diarrhea, and cattle may be reservoirs of C. difficile for humans. PMID:17283624

  8. Digital PCR analysis of circulating nucleic acids.

    PubMed

    Hudecova, Irena

    2015-10-01

    Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date. PMID:25828047

  9. Qualitative Research: Comments and Controversies.

    ERIC Educational Resources Information Center

    Schutz, Robert W.

    1989-01-01

    This article comments upon the use of qualitative research in physical education, exercise, and sport science. Topics include unresolved methodological problems, data analysis, and the scope of qualitative research. (IAH)

  10. Dengue Fever Testing

    MedlinePlus

    ... Dengue Virus by PCR Related tests: Arbovirus Testing , West Nile Virus Testing , Zika Virus Testing , Complete Blood Count , Electrolytes ... person is infected with another arbovirus such as West Nile virus . A health practitioner will consider a person's test ...

  11. PCR in laboratory diagnosis of human Borrelia burgdorferi infections.

    PubMed Central

    Schmidt, B L

    1997-01-01

    The laboratory diagnosis of Lyme borreliosis, the most prevalent vector-borne disease in the United States and endemic in parts of Europe and Asia, is currently based on serology with known limitations. Direct demonstration of Borrelia burgdorferi by culture may require weeks, while enzyme-linked immunosorbent assays for antigen detection often lack sensitivity. The development of the PCR has offered a new dimension in the diagnosis. Capable of amplifying minute amounts of DNA into billions of copies in just a few hours, PCR facilitates the sensitive and specific detection of DNA or RNA of pathogenic organisms. This review is restricted to applications of PCR methods in the diagnosis of human B. burgdorferi infections. In the first section, methodological aspects, e.g., sample preparation, target selection, primers and PCR methods, and detection and control of inhibition and contamination, are highlighted. In the second part, emphasis is placed on diagnostic aspects, where PCR results in patients with dermatological, neurological, joint, and ocular manifestations of the disease are discussed. Here, special attention is given to monitoring treatment efficacy by PCR tests. Last, specific guidelines on how to interpret PCR results, together with the advantages and limitations of these new techniques, are presented. PMID:8993863

  12. Interaction of quantitative PCR components with polymeric surfaces.

    PubMed

    Gonzalez, Asensio; Grimes, Ronan; Walsh, Edmond J; Dalton, Tara; Davies, Mark

    2007-04-01

    This study investigated the effect of exposing a polymerase chain reaction (PCR) mixture to capillary tubing of different materials and lengths, at different contact times and flow rates and the adsorption of major reaction components into the tubing wall. Using 0.5 mm ID tubing, lengths of 40 cm and residence times up to 45 min, none of the tested polymeric materials was found to affect subsequent PCR amplification. However, after exposure of the mixture to tubing lengths of 3 m or reduction of sample volume, PCR inhibition occurred, increasing with the volume to length ratio. Different flow velocities did not affect PCR yield. When the adsorption of individual PCR components was studied, significant DNA adsorption and even more significant adsorption of the fluorescent dye Sybr Green I was found. The results indicate that PCR inhibition in polymeric tubing results from adsorption of reaction components to wall surfaces, increasing substantially with tubing length or sample volume reduction, but not with contact time or flow velocities typical in dynamic PCR amplification. The data also highlight that chemical compatibility of polymeric capillaries with DNA dyes should be carefully considered for the design of quantitative microfluidic devices. PMID:17180709

  13. Testing.

    ERIC Educational Resources Information Center

    Killoran, James, Ed.

    1984-01-01

    This journal issue addresses the issue of testing in the social studies classroom. The first article, "The Role of Testing" (Bragaw), focuses on the need for tests to reflect the objectives of the study completed. The varying functions of pop quizzes, weekly tests, and unit tests are explored. "Testing Thinking Processes" (Killoran, Zimmer, and…

  14. International collaborative study of the endogenous reference gene LAT52 used for qualitative and quantitative analyses of genetically modified tomato.

    PubMed

    Yang, Litao; Zhang, Haibo; Guo, Jinchao; Pan, Liangwen; Zhang, Dabing

    2008-05-28

    One tomato ( Lycopersicon esculentum) gene, LAT52, has been proved to be a suitable endogenous reference gene for genetically modified (GM) tomato detection in a previous study. Herein are reported the results of a collaborative ring trial for international validation of the LAT52 gene as endogenous reference gene and its analytical systems; 14 GMO detection laboratories from 8 countries were invited, and results were finally received from 13. These data confirmed the species specificity by testing 10 plant genomic DNAs, less allelic variation and stable single copy number of the LAT52 gene, among 12 different tomato cultivars. Furthermore, the limit of detection of LAT52 qualitative PCR was proved to be 0.1%, which corresponded to 11 copies of haploid tomato genomic DNA, and the limit of quantification for the quantitative PCR system was about 10 copies of haploid tomato genomic DNA with acceptable PCR efficiency and linearity. Additionally, the bias between the test and true values of 8 blind samples ranged from 1.94 to 10.64%. All of these validated results indicated that the LAT52 gene is suitable for use as an endogenous reference gene for the identification and quantification of GM tomato and its derivates. PMID:18442244

  15. The effect of multiplex-PCR-assessed major pathogens causing subclinical mastitis on somatic cell profiles.

    PubMed

    Goli, Mohammad; Ezzatpanah, Hamid; Ghavami, Mehrdad; Chamani, Mohammad; Aminafshar, Mehdi; Toghiani, Majid; Eghbalsaied, Shahin

    2012-10-01

    The major pathogens causing mastitis were evaluated by multiplex-polymerase chain reaction (M-PCR) with self-designed primers in four quarters of the first, third, and fifth parities in industrial, semi-industrial, and traditional dairy cattle farms in Iran. With the incidence of infection in the quarters by Staphylococcus aureus and Streptococcus agalactiae, the mean log somatic cell count (log SCC) increased from 5.06 to 5.77. The smallest changes occurred with Escherichia coli. Contagious pathogens, when compared with environmental pathogens, were more prevalent and common and created more profound quantitative and qualitative changes in SCC profiles. The second part of the study surveyed the diversity of contaminating pathogens and their effect on quantitative and qualitative profiles of somatic cells. M-PCR was used to determine the absence (M-PCR(-)) and presence of one (M-PCR(+1)), two (M-PCR(+2)), and three (M-PCR(+3)) major pathogens in raw milk samples. Quarter log SCC increased from 5.06 (for M-PCR(-1)) to 5.5 (for M-PCR(+1)), 5.7 (for M-PCR(+2)), and 6 (for M-PCR(+3)). Percent changes in polymorphonuclears (PMNs) were not significant between different quarters and parities but were significant between different farms in terms of pathogen diversity (P < 0.05). Therefore, by increasing the number of types of major pathogens involved in subclinical mastitis, SCC of udder quarters and the proportion of PMNs significantly increased, whereas the proportion of lymphocytes significantly decreased. This subject is very important in increasing the shelf life of dairy products, because PMNs are introduced to the enzymatic pools. PMID:22535149

  16. Quantification of Bacterial Transcripts during Infection Using Competitive Reverse Transcription-PCR (RT-PCR) and LightCycler RT-PCR

    PubMed Central

    Goerke, Christiane; Bayer, Manfred G.; Wolz, Christiane

    2001-01-01

    Bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments. In order to monitor bacterial gene expression and regulation in the host, methods for direct transcript analysis from clinical specimens are needed. For most bacterial infections, amplification of the mRNAs of interest is necessary due to the low numbers of cells present and the low levels of specific transcripts. Here we compare two methods of quantitative reverse transcription-PCR (RT-PCR)—competitive RT-PCR using a one-tube system followed by standard gel analysis and the real-time detection of PCR product formation by fluorescence resonance energy transfer technology using the LightCycler unit. We isolated Staphylococcus aureus RNA directly from clinical specimens obtained from cystic fibrosis patients with chronic S. aureus lung infection and from an animal model of foreign-body infection with no further cultivation of the bacteria. Competitive RT-PCR and LightCycler RT-PCR were tested for their ability to quantify the transcription of a constitutively expressed gyrase gene (gyr) and a highly regulated α-toxin gene (hla) of S. aureus. Reproducible results were obtained with both methods. A sensitivity of 104 (gyr) and 103 (hla) copies, respectively, was reached, which was sufficient for the quantification of transcripts during bacterial infection. Overall, the competitive RT-PCR is a robust technique which does not need special RNA purification. On the negative side, it is labor intensive and time consuming, thus limiting the numbers of samples which can be analyzed at a given time. LightCycler RT-PCR is very susceptible to even traces of inhibitors, but it allows high-throughput processing of samples. PMID:11238208

  17. Short communication: comparing real-time PCR and bacteriological cultures for Streptococcus agalactiae and Staphylococcus aureus in bulk-tank milk samples.

    PubMed

    Zanardi, G; Caminiti, A; Delle Donne, G; Moroni, P; Santi, A; Galletti, G; Tamba, M; Bolzoni, G; Bertocchi, L

    2014-09-01

    For more than 30 yr, a control plan for Streptococcus agalactiae and Staphylococcus aureus has been carried out in more than 1,500 dairy herds of the province of Brescia (northern Italy). From 2010 to 2011, the apparent prevalence of Strep. agalactiae has been relatively stable around 10%, but the apparent prevalence of Staph. aureus has been greater than 40% with an increasing trend. The aim of this paper was to estimate and compare the diagnostic accuracy of 3 assays for the detection of Strep. agalactiae and Staph. aureus in bulk-tank milk samples (BTMS) in field conditions. The assays were a qualitative and a quantitative bacteriological culture (BC) for each pathogen and a homemade multiplex real-time PCR (rt-PCR). Because a gold standard was not available, the sensitivities (Se) and specificities (Sp) were evaluated using a Bayesian latent class approach. In 2012 we collected one BTMS from 165 dairy herds that were found positive for Strep. agalactiae in the previous 2-yr campaigns of eradication plan. In most cases, BTMS collected in these herds were positive for Staph. aureus as well, confirming the wide spread of this pathogen. At the same time we also collected composite milk samples from all the 8,624 lactating cows to evaluate the within-herd prevalence of Strep. agalactiae. Streptococcus agalactiae samples were cultured using a selective medium Tallium Kristalviolette Tossin, whereas for Staph. aureus, we used Baird Parker modified medium with added Rabbit Plasma Fibrinogen ISO-Formulation. In parallel, BTMS were tested using the rt-PCR. Regarding Strep. agalactiae, the posterior median of Se and Sp of the 2 BC was similar [qualitative BC: Se=98%, posterior credible interval (95%PCI): 94-100%, and Sp=99%, 95%PCI: 96-100%; quantitative BC: Se=99%, 95%PCI: 96-100%, and Sp=99%, 95%PCI: 95-100%] and higher than those of the rt-PCR (at 40 cycle threshold, Se=92%, 95%PCI: 85-97%; Sp=94%, 95%PCI: 88-98%). Also in case of Staph. aureus, the posterior medians

  18. Qualitative Methods in Mental Health Services Research

    PubMed Central

    Palinkas, Lawrence A.

    2014-01-01

    Qualitative and mixed methods play a prominent role in mental health services research. However, the standards for their use are not always evident, especially for those not trained in such methods. This paper reviews the rationale and common approaches to using qualitative and mixed methods in mental health services and implementation research based on a review of the papers included in this special series along with representative examples from the literature. Qualitative methods are used to provide a “thick description” or depth of understanding to complement breadth of understanding afforded by quantitative methods, elicit the perspective of those being studied, explore issues that have not been well studied, develop conceptual theories or test hypotheses, or evaluate the process of a phenomenon or intervention. Qualitative methods adhere to many of the same principles of scientific rigor as quantitative methods, but often differ with respect to study design, data collection and data analysis strategies. For instance, participants for qualitative studies are usually sampled purposefully rather than at random and the design usually reflects an iterative process alternating between data collection and analysis. The most common techniques for data collection are individual semi-structured interviews, focus groups, document reviews, and participant observation. Strategies for analysis are usually inductive, based on principles of grounded theory or phenomenology. Qualitative methods are also used in combination with quantitative methods in mixed method designs for convergence, complementarity, expansion, development, and sampling. Rigorously applied qualitative methods offer great potential in contributing to the scientific foundation of mental health services research. PMID:25350675

  19. Sensitive detection of sample interference in environmental qPCR.

    PubMed

    Green, Hyatt C; Field, Katharine G

    2012-06-15

    Sample interference in environmental applications of quantitative PCR (qPCR) can prevent accurate estimations of molecular markers in the environment. We developed a spike-and-recovery approach using a mutant strain of Escherichia coli that contains a chromosomal insertion of a mutant GFP gene. The method was tested in water samples by separately reducing extraction efficiency or adding humic acids and ethanol, compounds that often contaminate environmental DNA extracts, and analyzing qPCR amplification of the spiked E. coli control and human fecal Bacteroides markers (HF183 and HF134). This approach, coupled with previously developed kinetic outlier detection (KOD) methods, allowed sensitive detection of PCR inhibition at much lower inhibitor concentrations than alternative approaches using Cq values or amplification efficiencies. Although HF183 was more sensitive to the effects of qPCR inhibitors than the E. coli control assay, KOD methods correctly identified inhibition of both control and HF183 assays in samples containing as little as 0.1 ng humic acids per reaction or 5% ethanol. Because sigmoidal modeling methods allow distinction of qPCR inhibition from poor DNA recovery, we were able to simultaneously identify qPCR-inhibited reactions and estimate recovery of nucleic acids in environmental samples using a single control assay. Since qPCR is currently used to estimate important water quality parameters that have serious economic and human health outcomes, these results are timely. While we demonstrate the methods in the context of water quality regulation, they will be useful in all areas of environmental research that use qPCR. PMID:22560896

  20. Control for stochastic sampling variation and qualitative sequencing error in next generation sequencing

    PubMed Central

    Blomquist, Thomas; Crawford, Erin L.; Yeo, Jiyoun; Zhang, Xiaolu; Willey, James C.

    2015-01-01

    Background Clinical implementation of Next-Generation Sequencing (NGS) is challenged by poor control for stochastic sampling, library preparation biases and qualitative sequencing error. To address these challenges we developed and tested two hypotheses. Methods Hypothesis 1: Analytical variation in quantification is predicted by stochastic sampling effects at input of (a) amplifiable nucleic acid target molecules into the library preparation, (b) amplicons from library into sequencer, or (c) both. We derived equations using Monte Carlo simulation to predict assay coefficient of variation (CV) based on these three working models and tested them against NGS data from specimens with well characterized molecule inputs and sequence counts prepared using competitive multiplex-PCR amplicon-based NGS library preparation method comprising synthetic internal standards (IS). Hypothesis 2: Frequencies of technically-derived qualitative sequencing errors (i.e., base substitution, insertion and deletion) observed at each base position in each target native template (NT) are concordant with those observed in respective competitive synthetic IS present in the same reaction. We measured error frequencies at each base position within amplicons from each of 30 target NT, then tested whether they correspond to those within the 30 respective IS. Results For hypothesis 1, the Monte Carlo model derived from both sampling events best predicted CV and explained 74% of observed assay variance. For hypothesis 2, observed frequency and type of sequence variation at each base position within each IS was concordant with that observed in respective NTs (R2 = 0.93). Conclusion In targeted NGS, synthetic competitive IS control for stochastic sampling at input of both target into library preparation and of target library product into sequencer, and control for qualitative errors generated during library preparation and sequencing. These controls enable accurate clinical diagnostic reporting of

  1. Qualitative Variation in Constructive Alignment in Curriculum Design

    ERIC Educational Resources Information Center

    Trigwell, Keith; Prosser, Michael

    2014-01-01

    Constructive alignment has emerged as a powerful curriculum design idea, but little is known of the extent to which the effectiveness of this idea is a function of qualitative variation. This article introduces a model of qualitative variation in constructive alignment, and uses the results from known alignment studies to test the model. The…

  2. Toxoplasmosis Testing

    MedlinePlus

    ... Toxoplasma gondii Molecular Detection by PCR Related tests: TORCH ; CSF Analysis ; Amniotic Fluid Analysis At a Glance ... may sometimes be performed as part of a TORCH panel . TORCH is an acronym for several infections ...

  3. On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform.

    PubMed

    Tachibana, Hiroaki; Saito, Masato; Shibuya, Shogo; Tsuji, Koji; Miyagawa, Nobuyuki; Yamanaka, Keiichiro; Tamiya, Eiichi

    2015-12-15

    Polymerase chain reaction (PCR)-based genetic testing has become a routine part of clinical diagnoses and food testing. In these fields, rapid, easy-to-use, and cost-efficient PCR chips are expected to be appeared for providing such testing on-site. In this study, a new autonomous disposable plastic microfluidic PCR chip was created, and was utilized for quantitative detection of pathogenic microorganisms. To control the capillary flow of the following solution in the PCR microchannel, a driving microchannel was newly designed behind the PCR microchannel. This allowed the effective PCR by simply dropping the PCR solution onto the inlet without any external pumps. In order to achieve disposability, injection-molded cyclo-olefin polymer (COP) of a cost-competitive plastic was used for the PCR chip. We discovered that coating the microchannel walls with non-ionic surfactant produced a suitable hydrophilic surface for driving the capillary flow through the 1250-mm long microchannel. As a result, quantitative real-time PCR with the lowest initial concentration of human, Escherichia coli (E. coli), and pathogenic E. coli O157 genomic DNA of 4, 0.0019, 0.031 pg/μl, respectively, was successfully achieved in less than 18 min. Our results indicate that the platform presented in this study provided a rapid, easy-to-use, and low-cost real-time PCR system that could be potentially used for on-site gene testing. PMID:26210470

  4. Guidelines for the qualitative detection of viral genomes in dried blood spots.

    PubMed

    Gibellini, Davide; De Crignis, Elisa; Re, Maria Carla

    2012-01-01

    Dried blood spots (DBSs) are a useful alternative to blood sampling especially in children or for screening high-risk populations in developing countries. DBS blood collection can be employed in the diagnosis of viral infections by PCR or RT-PCR and also in viral genome sequencing. In addition, the advent of multiplex PCR approaches has led to further diagnostic and methodological improvements allowing simultaneous detection of two or more different viral genomes in the same sample and amplification reaction. This chapter describes general guidelines for the qualitative viral genome amplification and detection in DBS providing an example application of a qualitative real-time SYBR Green-based multiplex RT-PCR assay targeting two major viral pathogens, HIV-1 and HCV. PMID:22782809

  5. Comparing Rapid and Specific Detection of Brucella in Clinical Samples by PCR-ELISA and Multiplex-PCR Method

    PubMed Central

    Mohammad Hasani, Sharareh; Mirnejad, Reza; Amani, Jafar; Vafadar, Mohamad javad

    2016-01-01

    Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests. Methods: In this experimental and sectional study, during March 2014 to Sep 2015, 52 blood samples of suspected patients with clinical symptoms of brucellosis were evaluated in medical centers all over Iran with serum titers higher than 1:80. Using two pairs of specific primers of Brucella abortus, B. melitensis and DIG-dUTP, Fragment IS711 (The common gene fragment in B. melitensis and B. abortus) was amplified. DIG-ELISA was performed using specific probes of these 2 species of Brucella and patterns were subsequently analyzed, then positive responses were compared by detecting gel electrophoresis. Results: PCR-ELISA method detected all 28 samples from 52 positive samples. Its sensitivity was 6.0 pg concentration of genomic DNA of Brucella. In gel electrophoresis method, 22 samples of all positive samples were detected. PCR-ELISA was more efficient than PCR and bacterial culture method at P-value <0.05. Conclusion: PCR-ELISA molecular method is more sensitive than other molecular methods, lack of mutagenic color and also a semi-quantitative ability. This method is more effective and more accurate compared to PCR, serology and culture of bacteria. PCR-ELISA does not have false responses. The limitation of this method is detection of bacteria in the genus compared to Multiplex PCR and Gel Electrophoresis. PMID:27499776

  6. Detection and quantification of Bacillus cereus group in milk by droplet digital PCR.

    PubMed

    Porcellato, Davide; Narvhus, Judith; Skeie, Siv Borghild

    2016-08-01

    Droplet digital PCR (ddPCR) is one of the newest and most promising methods for the detection and quantification of molecular targets by PCR. Here, we optimized and used a new ddPCR assay for the detection and quantification of the Bacillus cereus group in milk. We also compared the ddPCR to a standard qPCR assay. The new ddPCR assay showed a similar coefficient of determination and a better limit of detection compared to the qPCR assay during quantification of the target molecules in the samples. However, the ddPCR assay has a limitation during quantification of a high number of target molecules. This new assay was then tested for the quantification of the B. cereus group in 90 milk samples obtained over three months from two different dairies and the milk was stored at different temperatures before sampling. The ddPCR assay showed good agreement with the qPCR assay for the quantification of the B. cereus group in milk, and due to its lower detection limit more samples were detected as positive. The new ddPCR assay is a promising method for the quantification of target bacteria in low concentration in milk. PMID:27211508

  7. A proline racemase based PCR for identification of Trypanosoma vivax in cattle blood.

    PubMed

    Fikru, Regassa; Hagos, Ashenafi; Rogé, Stijn; Reyna-Bello, Armando; Gonzatti, Mary Isabel; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe

    2014-01-01

    A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764-783 bp and 983-1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide. PMID:24416292

  8. The qualitative similarity hypothesis.

    PubMed

    Paul, Peter V; Lee, Chongmin

    2010-01-01

    Evidence is presented for the qualitative similarity hypothesis (QSH) with respect to children and adolescents who are d/Deaf or hard of hearing. The primary focus is on the development of English language and literacy skills, and some information is provided on the acquisition of English as a second language. The QSH is briefly discussed within the purview of two groups of cognitive models: those that emphasize the cognitive development of individuals and those that pertain to disciplinary or knowledge structures. It is argued that the QSH has scientific merit with implications for classroom instruction. Future research should examine the validity of the QSH in other disciplines such as mathematics and science and should include perspectives from social as well as cognitive models. PMID:20415280

  9. Multiplex PCR: Optimization and Application in Diagnostic Virology

    PubMed Central

    Elnifro, Elfath M.; Ashshi, Ahmed M.; Cooper, Robert J.; Klapper, Paul E.

    2000-01-01

    PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance. PMID:11023957

  10. Distinguishing human and possum faeces using PCR markers.

    PubMed

    Devane, M; Robson, B; Nourozi, F; Wood, D; Gilpin, B J

    2013-09-01

    Specificity testing of two published polymerase chain reaction (PCR) markers for the detection of human faecal pollution, revealed 100% false-positive rates to brush-tailed possum faeces (n = 10), but low false-positive rates against other potential pollution sources. Cross-reaction with possums could be a problem with other human-specific markers; therefore, a possum PCR marker was developed for use in conjunction with human PCR markers. The possum PCR marker was based on Bacteroidales 16S ribosomal ribonucleic acid sequences, and was tested on 233 individual faecal samples from 11 other animal species. Sensitivity of the possum marker in possum faeces (n = 36) was high at 83.3%. Cross-reactivity of the possum marker was limited to black swan (7/20 samples), human (2/48 samples) and rabbit (1/10) faecal samples, all at marker concentrations at least four orders of magnitude lower than possum faeces. The possum marker was not detected in human sewage or the faeces of other animal species. Specificity of the possum PCR marker, therefore, was high at 95.7%. To exclude the possibility that only possum pollution is being detected, additional testing by other faecal source tracking methods is required where the water sample is positive for both human and possum markers. PMID:23981869

  11. Structured Qualitative Research: Organizing “Mountains of Words” for Data Analysis, both Qualitative and Quantitative

    PubMed Central

    Johnson, Bruce D.; Dunlap, Eloise; Benoit, Ellen

    2008-01-01

    Qualitative research creates mountains of words. U.S. federal funding supports mostly structured qualitative research, which is designed to test hypotheses using semi-quantitative coding and analysis. The authors have 30 years of experience in designing and completing major qualitative research projects, mainly funded by the US National Institute on Drug Abuse [NIDA]. This article reports on strategies for planning, organizing, collecting, managing, storing, retrieving, analyzing, and writing about qualitative data so as to most efficiently manage the mountains of words collected in large-scale ethnographic projects. Multiple benefits accrue from this approach. Several different staff members can contribute to the data collection, even when working from remote locations. Field expenditures are linked to units of work so productivity is measured, many staff in various locations have access to use and analyze the data, quantitative data can be derived from data that is primarily qualitative, and improved efficiencies of resources are developed. The major difficulties involve a need for staff who can program and manage large databases, and who can be skillful analysts of both qualitative and quantitative data. PMID:20222777

  12. Improved PCR Amplification of Broad Spectrum GC DNA Templates

    PubMed Central

    Guido, Nicholas; Starostina, Elena; Leake, Devin; Saaem, Ishtiaq

    2016-01-01

    Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10–90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content. PMID:27271574

  13. Rapid diagnosis of goose viral infections by multiplex PCR.

    PubMed

    Chen, Zongyan; Li, Chuanfeng; Li, Guoxin; Yu, Hai; Jiang, Yifeng; Yan, Liping; Meng, Chunchun; Zhou, Yanjun; Tong, Guangzhi; Liu, Guangqing

    2013-08-01

    Goose parvovirus (GPV), newcastle disease virus (NDV), goose herpesvirus (GHV) and goose adenovirus (GAV) are considered collectively to be four of the most important and widespread viruses of geese. Because all of these viruses cause similar pathological changes, histological differentiation among these viruses is difficult. A reliable, specific and sensitive multiplex PCR (mPCR) assay was developed for the combined detection of GPV, NDV, GHV and GAV in clinical samples of geese. Using the mPCR technique, single infections with GPV (28/76; 36.8%), NDV (9/76; 11.8%), GHV (3/76; 3.9%) and GAV (12/76; 15.8%) were identified in the samples; co-infections with GAV and either GPV or NDV (31.6%; 24/76) were also identified with this approach. The results for all of the samples tested were the same in both the uPCR and mPCR systems. The mPCR approach is considered to be useful for routine molecular diagnosis and epidemiological applications in geese. PMID:23518397

  14. Common Perspectives in Qualitative Research.

    PubMed

    Flannery, Marie

    2016-07-01

    The primary purpose of this column is to focus on several common core concepts that are foundational to qualitative research. Discussion of these concepts is at an introductory level and is designed to raise awareness and understanding of several conceptual foundations that undergird qualitative research. Because of the variety of qualitative approaches, not all concepts are relevant to every design and tradition. However, foundational aspects were selected for highlighting. PMID:27314194

  15. DNA Microarray-Based PCR Ribotyping of Clostridium difficile

    PubMed Central

    Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2014-01-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. PMID:25411174

  16. Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations.

    PubMed

    Taylor, Sean C; Carbonneau, Julie; Shelton, Dawne N; Boivin, Guy

    2015-11-01

    The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and ΔCq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery. PMID:26315318

  17. EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

    EPA Science Inventory

    EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. The viral ribonucleic acid (RNA) from water sample concentrates is extracted and tested for enterovirus and norovirus RNA using reverse transcription-quantitative PCR (RT-qPCR). V...

  18. PCR detection of Helicobacter pylori in clinical samples.

    PubMed

    Rimbara, Emiko; Sasatsu, Masanori; Graham, David Y

    2013-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etiologically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental plaque, and stools as well as environmental samples. Whenever possibly, noninvasive tests are preferred over invasive tests. H. pylori are excreted in the stool. Culture from stool is variable whereas stool antigen testing is widely used. Stool consists of a complicated mixture of commensal bacteria and chemicals and often includes inhibitors of PCR. Nevertheless, simple extraction methods are available to efficiently extract DNA from human stools and nested-PCR targeting the 23S rRNA gene have proven to be highly sensitive for the detection of H. pylori. Detection of clarithromycin susceptibility/resistance is important clinically and the mutation of the 23S rRNA gene responsible for resistance can also be detected using stool. This described method can be modified for other clinical samples such as gastric juice or biopsy material. PMID:23104297

  19. Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.

    PubMed Central

    Vilcek, S; Elvander, M; Ballagi-Pordány, A; Belák, S

    1994-01-01

    Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and

  20. DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus)

    PubMed Central

    Li, Xiaolei; Zeng, Weiping; Liao, Jing; Liang, Zhenbiao; Huang, Shuhua

    2015-01-01

    We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes of Bungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites of SpeI and BstEII in the COI sequence of B. multicinctus to allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion with SpeI and BstEII (except for that of Zaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA of B. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS. PMID:26078770

  1. DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus).

    PubMed

    Li, Xiaolei; Zeng, Weiping; Liao, Jing; Liang, Zhenbiao; Huang, Shuhua; Chao, Zhi

    2015-01-01

    We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes of Bungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites of SpeI and BstEII in the COI sequence of B. multicinctus to allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion with SpeI and BstEII (except for that of Zaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA of B. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS. PMID:26078770

  2. Qualitative interviewing as measurement.

    PubMed

    Paley, John

    2010-04-01

    The attribution of beliefs and other propositional attitudes is best understood as a form of measurement, however counter-intuitive this may seem. Measurement theory does not require that the thing measured should be a magnitude, or that the calibration of the measuring instrument should be numerical. It only requires a homomorphism between the represented domain and the representing domain. On this basis, maps measure parts of the world, usually geographical locations, and 'belief' statements measure other parts of the world, namely people's aptitudes. Having outlined an argument for this view, I deal with an obvious objection to it: that self-attribution of belief cannot be an exercise in measurement, because we are all aware, from introspection, that our beliefs have an intrinsically semantic form. Subsequently, I turn to the philosophical and methodological ramifications of the measurement theoretic view. I argue, first, that it undermines at least one version of constructivism and, second, that it provides an effective alternative to the residually Cartesian philosophy that underpins much qualitative research. Like other anti-Cartesian strategies, belief-attribution-as-measurement implies that the objective world is far more knowable than the subjective one, and that reality is ontologically prior to meaning. I regard this result as both plausible and welcome. PMID:20415963

  3. Optimizing methods for PCR-based analysis of predation

    PubMed Central

    Sint, Daniela; Raso, Lorna; Kaufmann, Rüdiger; Traugott, Michael

    2011-01-01

    Molecular methods have become an important tool for studying feeding interactions under natural conditions. Despite their growing importance, many methodological aspects have not yet been evaluated but need to be considered to fully exploit the potential of this approach. Using feeding experiments with high alpine carabid beetles and lycosid spiders, we investigated how PCR annealing temperature affects prey DNA detection success and how post-PCR visualization methods differ in their sensitivity. Moreover, the replicability of prey DNA detection among individual PCR assays was tested using beetles and spiders that had digested their prey for extended times postfeeding. By screening all predators for three differently sized prey DNA fragments (range 116–612 bp), we found that only in the longest PCR product, a marked decrease in prey detection success occurred. Lowering maximum annealing temperatures by 4 °C resulted in significantly increased prey DNA detection rates in both predator taxa. Among the three post-PCR visualization methods, an eightfold difference in sensitivity was observed. Repeated screening of predators increased the total number of samples scoring positive, although the proportion of samples testing positive did not vary significantly between different PCRs. The present findings demonstrate that assay sensitivity, in combination with other methodological factors, plays a crucial role to obtain robust trophic interaction data. Future work employing molecular prey detection should thus consider and minimize the methodologically induced variation that would also allow for better cross-study comparisons. PMID:21507208

  4. PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

    EPA Science Inventory

    ABSTRACT

    Palatal Dysmorphogenesis : Quantitative RT-PCR

    Gary A. Held and Barbara D. Abbott

    Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

  5. Value of PCR in surgically treated patients with staphylococcal infective endocarditis: a 4-year retrospective study.

    PubMed

    Zaloudíková, B; Němcová, E; Pol, J; Sorm, Z; Wurmová, S; Novotná, K; Vaněrková, M; Holá, V; Růžička, F; Dušek, L; Němec, P; Freiberger, T

    2012-06-01

    The aim of the study was to establish a diagnostic value for broad-range polymerase chain reaction (br-PCR) and staphylococci-specific multiplex PCR (ssm-PCR) performed on surgical material from patients with staphylococcal infective endocarditis (IE). Data were analysed retrospectively from 60 patients with suspected staphylococcal IE and 59 controls who were surgically treated at three cardiosurgery centres over 4 years. Both PCR tests showed high agreement and could be aggregated. In patients with definite and rejected IE, the clinical sensitivity and specificity of PCR reached 89 and 95%, respectively. Tissue culture (TC) and PCR agreed with blood culture (BC) in 29% and 67% of IE cases. TC helped to determine aetiology in five BC negative cases while PCR aided in nine cases. Out of 52 patients with conclusive staphylococcal IE, 40 were diagnosed with S. aureus and 12 with coagulase-negative staphylococci. PCR was shown to be highly superior to TC in confirming preoperative diagnosis of IE. In addition to aid in culture negative patients, PCR helped to establish or refine aetiology in inconclusive cases. We suggest that simultaneous br-PCR and ssm-PCR performed on surgical material together with histopathology could significantly increase the performance of current Duke criteria. PMID:21964590

  6. Real time PCR on disposable PDMS chip with a miniaturized thermal cycler.

    PubMed

    Xiang, Q; Xu, B; Fu, R; Li, D

    2005-12-01

    This paper presents the design and implementation of a low-cost miniature PCR device consisting of a disposable reactor chip and a miniature thermal cycler. The simple fabrication of the PCR chip by PDMS (Polydimethylsiloxane) does not need micro-machining or photolithography processes. The thermal cycler was built with a thin film heater for heating and a fan for rapid cooling. This device can perform PCR tests in a single well chip or a multiple-well chip. It can run PCR reactions of different volumes to meet specific application requirements. The smallest reaction volume tested in this work is 0.9 microL. In addition, this device fits any standard fluorescence microscope for real time detection, which makes real time PCR affordable for most research labs and clinics with a fluorescence microscope. Real-time PCR of E. coli stx1 has been demonstrated with the device described. PMID:16404505

  7. Using Numbers in Qualitative Research

    ERIC Educational Resources Information Center

    Maxwell, Joseph A.

    2010-01-01

    The use of numerical/quantitative data in qualitative research studies and reports has been controversial. Prominent qualitative researchers such as Howard Becker and Martyn Hammersley have supported the inclusion of what Becker called "quasi-statistics": simple counts of things to make statements such as "some," "usually," and "most" more…

  8. Qualitative Science in Experimental Time

    ERIC Educational Resources Information Center

    Eisenhart, Margaret

    2006-01-01

    This article addresses the "state of qualitative inquiry" in the sense of how that inquiry is being positioned in the current construction of a US national policy agenda for "scientifically based" education research. In the author's view, qualitative inquiry is being drowned out in the national agenda despite its ability to provide the kinds of…

  9. Modified Real-Time PCR for Detecting, Differentiating, and Quantifying Ureaplasma urealyticum and Ureaplasma parvum

    PubMed Central

    Vancutsem, Ellen; Soetens, Oriane; Breugelmans, Maria; Foulon, Walter; Naessens, Anne

    2011-01-01

    We evaluated a previously described quantitative real-time PCR (qPCR) for quantifying and differentiating Ureaplasma parvum and U. urealyticum. Because of nonspecific reactions with Staphylococcus aureus DNA in the U. parvum PCR, we developed a modified qPCR and designed new primers. These oligonucleotides eradicated cross-reactions, indicating higher specificity. The detection limits of the qPCR were determined at 1 and 3 colony-forming units/ml for U. parvum and U. urealyticum, respectively. The quantification limits of the assay for both Ureaplasma species ranged from 2.106 to 2.101 copy numbers per PCR. A total of 300 patient samples obtained from the lower genital tract were tested with this newly designed qPCR assay and compared with culture results. Of the samples, 132 (44.0%) were culture positive, whereas 151 (50.3%) tested positive using qPCR. The U. parvum and U. urealyticum species were present in 79.5% and 12.6% of the qPCR-positive samples, respectively. Both species were found in 7.9% of those samples. Quantification of U. parvum and U. urealyticum in the samples ranged from less than 2.5 × 103 to 7.4 × 107 copies per specimen. In conclusion, the modified qPCR is a suitable method for rapid detection, differentiation, and quantification of U. parvum and U. urealyticum. PMID:21354056

  10. Development and validation of a quantitative PCR assay for Ichthyophonus spp.

    PubMed

    White, Vanessa C; Morado, J Frank; Crosson, Lisa M; Vadopalas, Brent; Friedman, Carolyn S

    2013-04-29

    Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus. PMID:23670081

  11. Comparative Evaluation of Four Real-Time PCR Methods for the Quantitative Detection of Epstein-Barr Virus from Whole Blood Specimens.

    PubMed

    Buelow, Daelynn; Sun, Yilun; Tang, Li; Gu, Zhengming; Pounds, Stanley; Hayden, Randall

    2016-07-01

    Monitoring of Epstein-Barr virus (EBV) load in immunocompromised patients has become integral to their care. An increasing number of reagents are available for quantitative detection of EBV; however, there are little published comparative data. Four real-time PCR systems (one using laboratory-developed reagents and three using analyte-specific reagents) were compared with one another for detection of EBV from whole blood. Whole blood specimens seeded with EBV were used to determine quantitative linearity, analytical measurement range, lower limit of detection, and CV for each assay. Retrospective testing of 198 clinical samples was performed in parallel with all methods; results were compared to determine relative quantitative and qualitative performance. All assays showed similar performance. No significant difference was found in limit of detection (3.12-3.49 log10 copies/mL; P = 0.37). A strong qualitative correlation was seen with all assays that used clinical samples (positive detection rates of 89.5%-95.8%). Quantitative correlation of clinical samples across assays was also seen in pairwise regression analysis, with R(2) ranging from 0.83 to 0.95. Normalizing clinical sample results to IU/mL did not alter the quantitative correlation between assays. Quantitative EBV detection by real-time PCR can be performed over a wide linear dynamic range, using three different commercially available reagents and laboratory-developed methods. EBV was detected with comparable sensitivity and quantitative correlation for all assays. PMID:27157323

  12. PCR detection of bovine herpesviruses from nonbovine ruminants in Hungary.

    PubMed

    Kálmán, Dóra; Egyed, László

    2005-07-01

    Polymerase chain reaction (PCR) was used to test six different nonbovine ruminant species for five bovine herpesviruses including infectious bovine rhinotracheitis virus (BoHV-1), bovine herpes mammillitis virus (BoHV-2), Movar-type herpesvirus (BoHV-4), bovine herpesvirus type 5 (BoHV-5), and alcelaphine herpesvirus 1 (AlHV-1). Species tested included 56 roe deer (Capreolus capreolus), 66 red deer (Cervus elaphus), 20 fallow deer (Dama dama), 16 mouflon (Ovis musimon), 34 domestic sheep, and 44 domestic goats, which were sampled in Hungary in 2003. Tracheal and popliteal lymph nodes collected from these animals were tested for the presence of the five bovine herpesviruses using three nested (two of which were duplex) PCR assays. Three bovine herpesviruses (BoHV-1, -2, and -4) were detected, whereas no evidence of AlHV-1 or BoHV-5 was observed. Prevalence of BoHV-1 ranged from 12% to 47%, and PCR-positive results were observed in all species tested. BoHV-2 was detected from roe deer, red deer, fallow deer, mouflon, and domestic sheep, and the prevalence in these species ranged from 3% to 50%. BoHV-4 was detected in all species, with prevalence ranging from 12% to 69%. Sequenced PCR products were 99-100% identical to bovine herpesviral sequences deposited in the GenBank. PMID:16244057

  13. Real-time PCR for Strongyloides stercoralis-associated meningitis.

    PubMed

    Nadir, Eyal; Grossman, Tamar; Ciobotaro, Pnina; Attali, Malka; Barkan, Daniel; Bardenstein, Rita; Zimhony, Oren

    2016-03-01

    Four immunocompromised patients, immigrants from Ethiopia, presented with diverse clinical manifestations of meningitis associated with Strongyloides stercoralis dissemination as determined by identification of intestinal larvae. The cerebrospinal fluid of 3 patients was tested by a validated (for stool) real-time PCR for S. stercoralis and was found positive, establishing this association. PMID:26704620

  14. Detection of Treponema pallidum by a sensitive reverse transcriptase PCR.

    PubMed Central

    Centurion-Lara, A; Castro, C; Shaffer, J M; Van Voorhis, W C; Marra, C M; Lukehart, S A

    1997-01-01

    Syphilis is diagnosed by serologic testing or by identification of the causative agent, Treponema pallidum. The bacterium has historically been detected in clinical specimens by dark-field microscopy, immunostaining with polyclonal or monoclonal antibodies, or the rabbit inoculation test (RIT). RIT is considered to be very sensitive and specific, although it is available only in research settings and is not clinically useful due to the length of time required to obtain a result. In recent years, several PCR methods have been developed for the detection of T. pallidum, but none of these has shown a clear advantage in sensitivity over RIT. We have developed a specific and highly sensitive reverse transcriptase PCR (RT-PCR) that targets a 366 bp region of the 16S rRNA of T. pallidum. This RT-PCR can detect a single organism by Southern analysis when whole organisms are diluted and 10(-2) to 10(-3) T. pallidum organisms when RNA equivalents are used to make cDNA. The test was demonstrated to detect 10(-2) T. pallidum RNA equivalents in cerebrospinal fluid. Twenty different strains of T. pallidum, isolated from cerebrospinal fluids, aqueous humor, blood, and chancres, were shown to be detectable by this test. This efficient and sensitive technique could be more useful than existing methods for detecting very low numbers of organisms in clinical samples. PMID:9163442

  15. Detection of carbapenemases in Enterobacteriaceae by a commercial multiplex PCR.

    PubMed

    Kaase, Martin; Szabados, Florian; Wassill, Lars; Gatermann, Sören G

    2012-09-01

    A commercial multiplex PCR (hyplex SuperBug ID) was tested with a collection of 132 clinical Enterobacteriaceae strains producing different carbapenemases. The sensitivity for the detection of KPC-, VIM-, NDM-, and OXA-48-encoding genes was 100%, whereas two IMP variants were missed. PMID:22785190

  16. QUANTITATIVE PCR OF SELECTED ASPERGILLUS, PENICILLIUM AND PAECILOMYCES SPECIES

    EPA Science Inventory

    A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan®) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of Aspergillus, Penicillium and ...

  17. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    ERIC Educational Resources Information Center

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  18. Real-time PCR in Food Science: PCR Diagnostics.

    PubMed

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control. PMID:23513039

  19. Electrothermal modeling of silicon PCR chips

    NASA Astrophysics Data System (ADS)

    Cui, Zheng; Zhao, Zhan; Xia, Shanhong

    2001-04-01

    Polymerase chain reaction (PCR) on a microchip has drawn considerable attention in recent years. Although a microchip can have must fast heating and cooling rate, the delicacy in its structure makes the PCR experiment difficult and cracks often occurs particularly for the thin membrane type of PCR chips. Electrothermal modeling of PCR chips is presented using commercial MEMS software tool IntelliSuiteTM, with the aim of identifying the problems encountered in experiment and finding an optimum chip structure. Heating characteristics of four different heater designs have been compared, so have the PCR chambers with fixed frame and with suspended frame. The thermal stress analysis has shown that the structure and heater design can make significant difference in heating characteristics and in reducing the failure of PCR chips. The computer simulation has confirmed what has been found in experiment the reason of membrane cracks. Improvement in PCR chip design has been proposed.

  20. [Do Multiplex PCR techniques displace classical cultures in microbiology?].

    PubMed

    Auckenthaler, Raymond; Risch, Martin

    2015-02-01

    Multiplex PCR technologies progressively find their way in clinical microbiology. This technique allows the simultaneous amplification of multiple DNA targets in a single test run for the identification of pathogens up to the species level. Various pathogens of infectious diseases can be detected by a symptom-oriented approach clearly and quickly with high reliability. Essentially multiplex PCR panels are available for clarification of gastrointestinal, respiratory, sexually transmitted infections and meningitis. Today's offer from industry, university hospitals and large private laboratories of Switzerland is tabulated and commented. PMID:25630288

  1. Early diagnosis of Lassa fever by reverse transcription-PCR.

    PubMed Central

    Demby, A H; Chamberlain, J; Brown, D W; Clegg, C S

    1994-01-01

    We developed a method based on a coupled reverse transcription-PCR (RT-PCR) for the detection of Lassa virus using primers specific for regions of the S RNA segment which are well conserved between isolates from Sierra Leone, Liberia, and Nigeria. The specificity of the assay was confirmed by Southern blotting with a chemiluminescent probe. The assay was able to detect 1 to 10 copies of a plasmid or an RNA transcript containing the target sequence. There was complete concordance between RT-PCR and virus culture for the detection of Lassa virus in a set of 29 positive and 32 negative serum samples obtained on admission to the hospital from patients suspected of having Lassa fever in Sierra Leone. Specificity was confirmed by the failure of amplification of specific products from serum samples collected from 129 healthy blood donors in Sierra Leone or from tissue culture supernatants from cells infected with related arenaviruses (Mopeia, lymphocytic choriomeningitis, Tacaribe, and Pichinde viruses). Sequential serum samples from 29 hospitalized patients confirmed to have Lassa fever were tested by RT-PCR and for Lassa virus-specific antibodies by indirect immunofluorescence (IF). RT-PCR detected virus RNA in 79% of the patients at the time of admission, comparing favorably with IF, which detected antibodies in only 21% of the patients. Lassa virus RNA was detected by RT-PCR in all 29 patients by the third day of admission, whereas antibody was detectable by IF in only 52% of the patients. These results point to an important role for RT-PCR in the management of suspected cases of Lassa fever. Images PMID:7883875

  2. Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach

    PubMed Central

    Hořínek, Aleš; Novotná, Michaela; Calda, Pavel; Korabečná, Marie

    2015-01-01

    Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods’ performance parameters—standard curve linearity, detection limit and measurement precision—were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438). PMID:26562517

  3. Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

    PubMed

    Odongo, Steven; Delespaux, Vincent; Ngotho, Maina; Bekkele, Serkalem Mindaye; Magez, Stefan

    2016-09-01

    We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05). PMID:27423733

  4. Qualitative versus Quantitative Results: An Experimental Introduction to Data Interpretation.

    ERIC Educational Resources Information Center

    Johnson, Eric R.; Alter, Paula

    1989-01-01

    Described is an experiment in which the student can ascertain the meaning of a negative result from a qualitative test by performing a more sensitive quantitative test on the same sample. Methodology for testing urinary glucose with a spectrophotometer at 630 nm and with commercial assaying glucose strips is presented. (MVL)

  5. Positivism and qualitative nursing research.

    PubMed

    Paley, J

    2001-01-01

    Despite the hostility to positivism shown by qualitative methodologists in nursing, as in other disciplines, the epistemological and ontological instincts of qualitative researchers seem to coincide with those of the positivists, especially Bayesian positivists. This article suggests that positivists and qualitative researchers alike are pro-observation, proinduction, pro-plausibility and pro-subjectivity. They are also anti-cause, anti-realist, anti-explanation, anti-correspondence, anti-truth. In only one respect is there a significant difference between positivist and qualitative methodologists: most positivists have believed that, methodologically, the natural sciences and the social sciences are the same; most qualitative researchers are adamant that they are not. However, if positivism fails as a philosophy of the natural sciences (which it probably does), it might well succeed as a philosophy of the social sciences, just because there is a methodological watershed between the two. Reflex antagonism to positivism might therefore be a major obstacle to understanding the real reasons why qualitative research and the natural sciences are methodologically divergent; and less hostility on the part of qualitative nurse researchers might bring certain advantages in its wake. PMID:11885869

  6. Understanding Qualitative Research: A Strategic Approach to Qualitative Methodology.

    ERIC Educational Resources Information Center

    Husband, Robert; Foster, William

    1987-01-01

    Discusses the basic character of qualitative, humanistic research, identifying its philosophical and theoretical commitments. Provides a taxonomy of investigative strategies employed, including naturalistic inquiry, contextualization, maximized comparisons, sensitizing concepts, and analytic induction. Classifies methods employed as participant…

  7. Real-Time PCR Improves Helicobacter pylori Detection in Patients with Peptic Ulcer Bleeding

    PubMed Central

    Casalots, Alex; Sanfeliu, Esther; Boix, Loreto; García-Iglesias, Pilar; Sánchez-Delgado, Jordi; Montserrat, Antònia; Bella-Cueto, Maria Rosa; Gallach, Marta; Sanfeliu, Isabel; Segura, Ferran; Calvet, Xavier

    2011-01-01

    Background and Aims Histological and rapid urease tests to detect H. pylori in biopsy specimens obtained during peptic ulcer bleeding episodes (PUB) often produce false-negative results. We aimed to examine whether immunohistochemistry and real-time PCR can improve the sensitivity of these biopsies. Patients and Methods We selected 52 histology-negative formalin-fixed paraffin-embedded biopsy specimens obtained during PUB episodes. Additional tests showed 10 were true negatives and 42 were false negatives. We also selected 17 histology-positive biopsy specimens obtained during PUB to use as controls. We performed immunohistochemistry staining and real-time PCR for 16S rRNA, ureA, and 23S rRNA for H. pylori genes on all specimens. Results All controls were positive for H. pylori on all PCR assays and immunohistochemical staining. Regarding the 52 initially negative biopsies, all PCR tests were significantly more sensitive than immunohistochemical staining (p<0.01). Sensitivity and specificity were 55% and 80% for 16S rRNA PCR, 43% and 90% for ureA PCR, 41% and 80% for 23S rRNA PCR, and 7% and 100% for immunohistochemical staining, respectively. Combined analysis of PCR assays for two genes were significantly more sensitive than ureA or 23S rRNA PCR tests alone (p<0.05) and marginally better than 16S rRNA PCR alone. The best combination was 16S rRNA+ureA, with a sensitivity of 64% and a specificity of 80%. Conclusions Real-time PCR improves the detection of H. pylori infection in histology-negative formalin-fixed paraffin-embedded biopsy samples obtained during PUB episodes. The low reported prevalence of H. pylori in PUB may be due to the failure of conventional tests to detect infection. PMID:21625499

  8. Using PCR to Target Misconceptions about Gene Expression †

    PubMed Central

    Wright, Leslie K.; Newman, Dina L.

    2013-01-01

    We present a PCR-based laboratory exercise that can be used with first- or second-year biology students to help overcome common misconceptions about gene expression. Biology students typically do not have a clear understanding of the difference between genes (DNA) and gene expression (mRNA/protein) and often believe that genes exist in an organism or cell only when they are expressed. This laboratory exercise allows students to carry out a PCR-based experiment designed to challenge their misunderstanding of the difference between genes and gene expression. Students first transform E. coli with an inducible GFP gene containing plasmid and observe induced and un-induced colonies. The following exercise creates cognitive dissonance when actual PCR results contradict their initial (incorrect) predictions of the presence of the GFP gene in transformed cells. Field testing of this laboratory exercise resulted in learning gains on both knowledge and application questions on concepts related to genes and gene expression. PMID:23858358

  9. A Specific Qualitative Detection Method for Peanut (Arachis Hypogagea) in Foods Using Polymerase Chain Reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A qualitative method for detection of peanuts in foods using polymerase chain reaction was developed. A universal primer pair CP 03-5 /CP 03-3 was designed to confirm the validity of the DNAs for PCR. The plant-specific amplified fragments were detected from 13 kinds of plants using the universal pr...

  10. Trajectory constraints in qualitative simulation

    SciTech Connect

    Brajnik, G.; Clancy, D.J.

    1996-12-31

    We present a method for specifying temporal constraints on trajectories of dynamical systems and enforcing them during qualitative simulation. This capability can be used to focus a simulation, simulate non-autonomous and piecewise-continuous systems, reason about boundary condition problems and incorporate observations into the simulation. The method has been implemented in TeQSIM, a qualitative simulator that combines the expressive power of qualitative differential equations with temporal logic. It interleaves temporal logic model checking with the simulation to constrain and refine the resulting predicted behaviors and to inject discontinuous changes into the simulation.

  11. Direct Fluorescence Detection of Allele-Specific PCR Products Using Novel Energy-Transfer Labeled Primers.

    PubMed

    Winn-Deen

    1998-12-01

    Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the PCR product liberated during the detection step. The author has combined the PCR amplification and detection steps into a single procedure suitable for closed-tube analysis. Methods and Results: Allele-specific PCR primers were designed as Sunrise energy-transfer primers and contained a 3' terminal mismatch to distinguish between normal and mutant DNA. Cloned normal (W64) and mutant (R64) templates of the beta3-adrenergic receptor gene were tested to verify amplification specificity and yield. A no-target negative control was also run with each reaction. After PCR, each reaction was tested for fluorescence yield by measuring fluorescence on a spectrofluorimeter or fluorescent microtitreplate reader. The cloned controls and 24 patient samples were tested for the W64R mutation by two methods. The direct fluorescence results with the Sunrise allele-specific PCR method gave comparable genotypes to those obtained with the PCR/ restriction digest/gel electrophoresis control method. No PCR artifacts were observed in the negative controls or in the PCR reactions run with the mismatched target. Conclusions: The results of this pilot study indicate good PCR product and fluorescence yield from allele-specific energy-transfer labeled primers, and the capability of distinguishing between normal and mutant alleles based on fluorescence alone, without the need for restriction digestion, gel electrophoresis, or hybridization with an allele-specific probe. PMID:10089280

  12. Collaborative trial validation studies of real-time PCR-based GMO screening methods for detection of the bar gene and the ctp2-cp4epsps construct.

    PubMed

    Grohmann, Lutz; Brünen-Nieweler, Claudia; Nemeth, Anne; Waiblinger, Hans-Ulrich

    2009-10-14

    Polymerase Chain Reaction (PCR)-based screening methods targeting genetic elements commonly used in genetically modified (GM) plants are important tools for the detection of GM materials in food, feed, and seed samples. To expand and harmonize the screening capability of enforcement laboratories, the German Federal Office of Consumer Protection and Food Safety conducted collaborative trials for interlaboratory validation of real-time PCR methods for detection of the phosphinothricin acetyltransferase (bar) gene from Streptomyces hygroscopicus and a construct containing the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens sp. strain CP4 (ctp2-cp4epsps), respectively. To assess the limit of detection, precision, and accuracy of the methods, laboratories had to analyze two sets of 18 coded genomic DNA samples of events LLRice62 and MS8 with the bar method and NK603 and GT73 with the ctp2-cp4epsps method at analyte levels of 0, 0.02, and 0.1% GM content, respectively. In addition, standard DNAs were provided to the laboratories to generate calibration curves for copy number quantification of the bar and ctp2-cp4epsps target sequences present in the test samples. The study design and the results obtained are discussed with respect to the difficult issue of developing general guidelines and concepts for the collaborative trial validation of qualitative PCR screening methods. PMID:19807158

  13. Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

    PubMed

    Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

    2013-10-30

    Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis. PMID:23971699

  14. Specific primer sets used to amplify by PCR the hepatitis B virus overlapping S/Pol region select different viral variants.

    PubMed

    Cuestas, M L; Mathet, V L; Oubiña, J R

    2012-10-01

    PCR detection of viral genomes has provided new insights into viral diagnosis. Nowadays, it is the most frequently used nucleic acid testing (qualitative and quantitative) technique. The aim of this study was to analyse the major circulating hepatitis B virus (HBV) variants PCR-amplified by three sets of primers in a patient infected with genotype E. The HBV S/Pol overlapping genomic region was amplified from the serum of an infected child using three primer sets previously described. Sequence analysis corresponding to the HBV S/Pol region revealed the presence of different viral populations depending on the set of primers used. D144A S-escape mutant was detected with two of the primer sets, while the rtL217R mutant within the Pol - conferring resistance to Adefovir - could be picked up with a different pair of primer sets. This study undoubtedly implies that the description of viral polymorphisms should be stated together with the sequence of the primers used for PCR amplification when studies of escape and/or antiviral-resistant HBV mutants are carried out. PMID:22967107

  15. Multiplexed Primer Prediction for PCR

    Energy Science and Technology Software Center (ESTSC)

    2007-07-23

    MPP predicts sets of multiplex-compatible primers for Polymerase Chain Reaction (PCR), finding a near minimal set of primers such that at least one amplicon will be generated from every target sequence in the input file. The code finds highly conserved oligos that are suitable as primers, according to user-specified desired primer characteristics such as length, melting temperature, and amplicon length. The primers are predicted not to form unwanted dimer or hairpin structures. The target sequencesmore » used as input can be diverse, since no multiple sequence alighment is required. The code is scalable, taking up to tens of thousands of sequences as input, and works, for example, to find a "universal primer set" for all viral genomes provided as a single input file. The code generates a periodic check-point file, thus in the event of premature execution termination, the application can be restarted from the last check-point file.« less

  16. A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methods.

    PubMed Central

    Cobb, B D; Clarkson, J M

    1994-01-01

    Taguchi methods are used widely as the basis for development trials during industrial process design. Here, we describe their suitability for optimisation of the PCR. Unlike conventional strategies, these arrays revealed the effects and interactions of specific reaction components simultaneously using just a few reactions, negating the need for extensive experimental investigation. Reaction components which effected product yield were easily determined. In addition, this technique was applied to the qualitative investigation of RAPD-PCR profiles, where optimisation of the size and distribution of a number of products was determined. Images PMID:7937094

  17. Development of a PCR Assay for Rapid Detection of Enterococci

    PubMed Central

    Ke, Danbing; Picard, François J.; Martineau, Francis; Ménard, Christian; Roy, Paul H.; Ouellette, Marc; Bergeron, Michel G.

    1999-01-01

    Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR

  18. Surmounting a PCR challenge using a Contradictory matrix from the Theory of Inventive Problem Solving (TRIZ).

    PubMed

    Drábek, Jiří

    2016-01-01

    In this paper I tested whether Contradictory Matrix with 40 Inventive Principles, the simplest instrument from the Theory of Inventive Problem Solving (TRIZ), is a useful approach to a real-life PCR scenario. The PCR challenge consisted of standardization of fluorescence melting curve measurements in Competitive Amplification of Differentially Melting Amplicons (CADMA) PCR for multiple targets. Here I describe my way of using the TRIZ Matrix to generate seven alternative solutions from which I can choose the successful solution, consisting of repeated cycles of amplification and melting in a single PCR run. PMID:26835236

  19. PCR-based study of conserved and variable DNA sequences of Tritrichomonas foetus isolates from Saskatchewan, Canada.

    PubMed Central

    Riley, D E; Wagner, B; Polley, L; Krieger, J N

    1995-01-01

    The protozoan parasite Tritrichomonas foetus causes infertility and spontaneous abortion in cattle. In Saskatchewan, Canada, the culture prevalence of trichomonads was 65 of 1,048 (6%) among 1,048 bulls tested within a 1-year period ending in April 1994. Saskatchewan was previously thought to be free of the parasite. To confirm the culture results, possible T. foetus DNA presence was determined by the PCR. All of the 16 culture-positive isolates tested were PCR positive by a single-band test, but one PCR product was weak. DNA fingerprinting by both T17 PCR and randomly amplified polymorphic DNA PCR revealed genetic variation or polymorphism among the T. foetus isolates. T17 PCR also revealed conserved loci that distinguished these T. foetus isolates from Trichomonas vaginalis, from a variety of other protozoa, and from prokaryotes. TCO-1 PCR, a PCR test designed to sample DNA sequence homologous to the 5' flank of a highly conserved cell division control gene, detected genetic polymorphism at low stringency and a conserved, single locus at higher stringency. These findings suggested that T. foetus isolates exhibit both conserved genetic loci and polymorphic loci detectable by independent PCR methods. Both conserved and polymorphic genetic loci may prove useful for improved clinical diagnosis of T. foetus. The polymorphic loci detected by PCR suggested either a long history of infection or multiple lines of T. foetus infection in Saskatchewan. Polymorphic loci detected by PCR may provide data for epidemiologic studies of T. foetus. PMID:7615746

  20. Molecular detection and identification of intimin alleles in pathogenic Escherichia coli by multiplex PCR.

    PubMed

    Reid, S D; Betting, D J; Whittam, T S

    1999-08-01

    A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenic Escherichia coli. The method was tested on 87 strains representing the diarrheagenic E. coli clones. The results show that the PCR assay accurately detects eae and resolves alleles encoding the alpha, beta, and gamma intimin variants. PMID:10405431

  1. Molecular Detection and Identification of Intimin Alleles in Pathogenic Escherichia coli by Multiplex PCR

    PubMed Central

    Reid, Sean D.; Betting, David J.; Whittam, Thomas S.

    1999-01-01

    A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenic Escherichia coli. The method was tested on 87 strains representing the diarrheagenic E. coli clones. The results show that the PCR assay accurately detects eae and resolves alleles encoding the α, β, and γ intimin variants. PMID:10405431

  2. Real time RT-PCR for the H5 HA subtype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (RRT-PCR) for type A influenza detection is widely used with subsequent tests for subtype identification. Due to the importance of the H5 HA subtype, rapid and sensitive detection of the H5 HA subtype is particularly useful due to the importance of the recent Asian H5N1 HPAI viruse...

  3. Joint association analysis of bivariate quantitative and qualitative traits.

    PubMed

    Yuan, Mengdie; Diao, Guoqing

    2011-01-01

    Univariate genome-wide association analysis of quantitative and qualitative traits has been investigated extensively in the literature. In the presence of correlated phenotypes, it is more intuitive to analyze all phenotypes simultaneously. We describe an efficient likelihood-based approach for the joint association analysis of quantitative and qualitative traits in unrelated individuals. We assume a probit model for the qualitative trait, under which an unobserved latent variable and a prespecified threshold determine the value of the qualitative trait. To jointly model the quantitative and qualitative traits, we assume that the quantitative trait and the latent variable follow a bivariate normal distribution. The latent variable is allowed to be correlated with the quantitative phenotype. Simultaneous modeling of the quantitative and qualitative traits allows us to make more precise inference on the pleiotropic genetic effects. We derive likelihood ratio tests for the testing of genetic effects. An application to the Genetic Analysis Workshop 17 data is provided. The new method yields reasonable power and meaningful results for the joint association analysis of the quantitative trait Q1 and the qualitative trait disease status at SNPs with not too small MAF. PMID:22373162

  4. Qualitative perspectives in translational research.

    PubMed

    Tripp-Reimer, Toni; Doebbeling, Bradley

    2004-01-01

    The rapid uptake of qualitative approaches in translational research can be best understood in the context of recent innovations in health services research, as well as an overarching concern with improving the quality of health care. Qualitative approaches highlight the human dimension in health care by foregrounding the perceptions, experiences, and behaviors of both consumers and providers of care. As such, these methods are particularly useful for addressing the complex issues related to improving health care quality and implementing system change. This overview traces a brief history of the factors contributing to the recent and rapid growth of qualitative methods in health research in general and translational research in particular; describes the varieties of qualitative approaches employed in this research; and illustrates the utility of these approaches for variable identification, instrument development, description/explanation of patient/provider perceptions and behaviors, individual/organizational change, and theory refinement. PMID:17129338

  5. A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System.

    PubMed

    Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

    2016-01-01

    Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit

  6. A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

    PubMed Central

    Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

    2016-01-01

    Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit

  7. Qualitative research: comments and controversies.

    PubMed

    Schutz, R W

    1989-03-01

    Larry Locke's timely and well-written introduction to qualitative research procedures will undoubtedly serve its purpose. It makes us reassess our traditional beliefs and practices, educates us on the rudiments of qualitative methodology, and, hopefully, makes us more tolerant and appreciative of alternate ways of conducting research. Although Locke focuses his paper on pedagogical research issues, it is important to realize that many other sub-disciplines within the general field of physical education also utilize qualitative procedures. For example, 10 years ago Martens (1979) called for a paradigm shift in sport psychology by appealing to researchers to abandon their labs and to embark on naturalistic field studies. While North American sport psychologists, and psychologists in general, have been slow to formalize qualitative techniques, the European psychology community has been much more active (e.g., Ashworth, Giorgi, & de Koning, 1986). Perhaps Locke's article will encourage researchers in all our sub-disciplines to consider the utility of qualitative research. Hopefully, readers will treat Locke's article as an introduction to the broad area of qualitative research and not as a rigorous set of procedures for conducting participant observation research in school physical education studies. Additionally, it must be recognized that there are other approaches and other applications, that the area has its critics and its unresolved methodological problems, and that qualitative research does not necessarily exclude the application of formalized data analyses. Keeping these issues in mind, the addition of qualitative approaches to our repetoire of research methodologies can only enhance the quality of research in physical education and exercise and sport science. PMID:2489822

  8. Integrated Real-Time PCR for Detection and Monitoring of Legionella pneumophila in Water Systems▿

    PubMed Central

    Yaradou, Diaraf Farba; Hallier-Soulier, Sylvie; Moreau, Sophie; Poty, Florence; Hillion, Yves; Reyrolle, Monique; André, Janine; Festoc, Gabriel; Delabre, Karine; Vandenesch, François; Etienne, Jerome; Jarraud, Sophie

    2007-01-01

    We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers. PMID:17194840

  9. QBone University and Lab Interconnect Testbed (QUALIT). Final report

    SciTech Connect

    Teitelbaum, Benjamin

    2001-10-19

    The QUALIT grant funded two broad categories of work: (1) Project-wide QBone engineering, instrumentation, and integration; (2) Focused workshops and measurement work relating specifically to advanced university/DOE connectivity. Significant progress has been made in both areas and, to both, QUALIT funding has been a key enabling resource. This final report summarizes the accomplishments of the QUALIT project and explains changes to the technical focus of the project that, while significant, remained true to the overall project goal: to research, test, and deploy IP layer traffic differentiation to redress congestion-related end-to-end performance problems on key university-DOE lab paths.

  10. Qualitative model-based diagnostics for rocket systems

    NASA Technical Reports Server (NTRS)

    Maul, William; Meyer, Claudia; Jankovsky, Amy; Fulton, Christopher

    1993-01-01

    A diagnostic software package is currently being developed at NASA LeRC that utilizes qualitative model-based reasoning techniques. These techniques can provide diagnostic information about the operational condition of the modeled rocket engine system or subsystem. The diagnostic package combines a qualitative model solver with a constraint suspension algorithm. The constraint suspension algorithm directs the solver's operation to provide valuable fault isolation information about the modeled system. A qualitative model of the Space Shuttle Main Engine's oxidizer supply components was generated. A diagnostic application based on this qualitative model was constructed to process four test cases: three numerical simulations and one actual test firing. The diagnostic tool's fault isolation output compared favorably with the input fault condition.

  11. Qualitative methods for assessing risk

    SciTech Connect

    Mahn, J.A.; Hannaman, G.W.; Kryska, P.

    1995-04-01

    The Department of Energy`s (DOE) non-nuclear facilities generally require only a qualitative accident analysis to assess facility risks in accordance with DOE Order 5481.1B, Safety Analysis and Review System. Achieving a meaningful qualitative assessment of risk necessarily requires the use of suitable non-numerical assessment criteria. Typically, the methods and criteria for assigning facility-specific accident scenarios to the qualitative severity and likelihood classification system in the DOE order requires significant judgment in many applications. Systematic methods for more consistently assigning the total accident scenario frequency and associated consequences are required to substantiate and enhance future risk ranking between various activities at Sandia National Laboratories (SNL). SNL`s Risk Management and National Environmental Policy Act (NEPA) Department has developed an improved methodology for performing qualitative risk assessments in accordance wi the DOE order requirements. Products of this effort are an improved set of qualitative description that permit (1) definition of the severity for both technical and programmatic consequences that may result from a variety of accident scenarios, and (2) qualitative representation of the likelihood of occurrence. These sets of descriptions are intended to facilitate proper application of DOE criteria for assessing facility risks.

  12. Multiplex nested RT-PCR for detecting avian influenza virus, infectious bronchitis virus and Newcastle disease virus.

    PubMed

    Nguyen, Thanh Trung; Kwon, Hyuk-Joon; Kim, Il-Hwan; Hong, Seung-Min; Seong, Won-Jin; Jang, Jin-Wook; Kim, Jae-Hong

    2013-03-01

    In this study, multiplex nested RT-PCR (mnRT-PCR) was applied to simultaneous detect multiplex PCR with the higher sensitivity of nested PCR that is required for avian influenza, infectious bronchitis and Newcastle disease virus using two steps of amplification. For the first PCR, primers that were specific for each virus were newly designed from the nucleoprotein gene of AIV, the nucleocapsid protein gene of IBV and the fusion protein gene of NDV to amplify products of 665, 386 and 236 nucleotides, respectively. The multiplex PCR step provides mass amplification using common primers, which increased markedly the sensitivity of the test. Non-specific reactions were not observed when other viruses and bacteria were used for evaluating the mnRT-PCR. As a field application, 172 samples were tested by RT-PCR and mnRT-PCR. Among these samples, the concordance rates for mnRT-PCR and the single conventional RT-PCR showed 98.9% (kappa=0.98) and 98.8% (kappa=0.96) similarity for IBV and AIV, respectively. As a result, it is recommended the multiplex nested PCR as an effective tool for detecting and studying the molecular epidemiology of various mixed infections of one or more of these viruses in poultry. PMID:23261801

  13. Evaluation of an Automated Rapid Diagnostic Test for Detection of Clostridium difficile

    PubMed Central

    Tojo, Masayoshi; Nagamatsu, Maki; Hayakawa, Kayoko; Mezaki, Kazuhisa; Kirikae, Teruo; Ohmagari, Norio

    2014-01-01

    The Verigene Clostridium difficile Nucleic Acid Test (Verigene CDF Test) (Nanosphere, Northbrook, IL, USA) is a new multiplex qualitative polymerase chain reaction (PCR) test used to detect C. difficile toxin genes in fecal specimens. To evaluate the performance of the new method, we tested 69 fecal samples from patients with suspected C. difficile infection using the Verigene CDF test, an enzyme immunoassay (EIA) and PCR following anaerobic fecal culture. The sensitivity, specificity, and accuracy of the Verigene CDF test were 96.7% (29/30), 97.4% (38/39), and 97.1% (67/69) respectively, using PCR following fecal culture as a reference method. We also analyzed the potential clinical impact of the Verigene CDF test using chart reviews of the 69 patients with suspected C. difficile infection and found that 11 of the 69 patients were incorrectly diagnosed, and the Verigene CDF test would have led to them receiving more appropriate management including practice of treatment and contact precaution, although, of the 69 patients, there are two whose samples were incorrectly identified with the Verigene CDF test. The Verigene CDF test will have a positive impact on patient care. PMID:25170836

  14. Real-time PCR in microfluidic devices

    NASA Astrophysics Data System (ADS)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

  15. Cost-Effective Pooling of DNA from Nasopharyngeal Swab Samples for Large-Scale Detection of Bacteria by Real-Time PCR

    PubMed Central

    Edouard, Sophie; Prudent, Elsa; Gautret, Philippe; Memish, Ziad A.

    2014-01-01

    We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed. PMID:25552360

  16. Detection of respiratory syncytial virus by reverse transcription-PCR and hybridization with a DNA enzyme immunoassay.

    PubMed Central

    Freymuth, F; Eugene, G; Vabret, A; Petitjean, J; Gennetay, E; Brouard, J; Duhamel, J F; Guillois, B

    1995-01-01

    Nasal aspirates from 238 infants hospitalized with acute respiratory infections during the winter of 1994 and 1995 were tested for respiratory syncytial virus (RSV) by immunofluorescence assay (IFA) and the viral isolation technique (VIT) and by two PCR and hybridization methods: reverse transcription PCR 1 (RT-PCR1), which amplifies the RNAs of all RSV strains, and RT-PCR-2, which allows subgroup classification of RSV. RT-PCR-1 and RT-PCR-2 detected viral sequences in 56.7% (135 of 238) and 48.3% (115 of 238) of the samples, respectively, while only 80 (33.6%) samples were found to be positive by IFA and VIT. Of the PCR-positive specimens, 57 were missed by these routine techniques in RT-PCR-1 and 45 were missed in RT-PCR-2. Although the RSV-PCR-1 and RSV-PCR-2 techniques amplified two different sequences of the RSV genome, they gave similar results for 218 (91.6%) nasal aspirates. Compared with conventional methods, the sensitivity, specificity, and agreement were 97.5, 63.9, and 75.2%, respectively, for RT-PCR-1 and 89.7, 71.9, and 77.7%, respectively, for RT-PCR-2, and for these two RT-PCR assays, the positive predictive value (PPV) and the index of agreement (kappa) were comparable and moderate, respectively: PPV was 57.8% and kappa was 0.52 in RT-PCR-1, and PPV was 60.9% and kappa was 0.54 in RT-PCR-2. However, there was a perfect correlation between the two RT-PCRs, with a PPV of 100% and an excellent index of agreement (kappa = 0.88). Therefore, most RT-PCR results were really true positive, and VIT and IFA, which missed some of them, appeared to be less sensitive. PMID:8586738

  17. Clinical Evaluation of COBAS TaqMan PCR for the Detection of Mycobacterium tuberculosis and M. avium Complex

    PubMed Central

    Ikegame, Satoshi; Sakoda, Yoritake; Fujino, Nao; Taguchi, Kazuhito; Kawasaki, Masayuki; Kajiki, Akira

    2012-01-01

    A retrospective observational study was performed to determine the sensitivity and limitation of PCR test for the detection of Mycobacterium tuberculosis and M. avium complex. We obtained clinical specimens collected from the respiratory tract, cultured M. tuberculosis or M. avium complex, and performed PCR analysis. A total of 299 samples (M. tuberculosis, 177; M. avium, 35; M. intracellulare, 87) were analyzed by COBAS TaqMan PCR from April 2007 to March 2011. The PCR positivity rates were 50–55%, 70–100%, 88–98%, and 100% in smear-negative, smear 1+, 2+, and 3+ groups, respectively. The PCR positivity of tuberculosis in smear 1+ was 80.6%, which was statistically significantly (P < 0.001) lower than that of smear 2+ (97.3%). From January 2005 to March 2007, we collected an additional 138 samples (M. tuberculosis, 74; M. avium, 21; M. intracellulare, 43), which were analyzed by COBAS Amplicor PCR. The PCR positivity rates obtained using COBAS TaqMan PCR and COBAS Amplicor PCR were not significantly different. The sensitivity of PCR test for mycobacteria is not sufficient in case of smear 1+. Careful consideration must be given to the interpretation of negative PCR test results in smear 1+, because smear-positive tuberculosis is the criterion for isolation. PMID:23029612

  18. Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus.

    PubMed

    Karim, Mohammad R; Fout, G Shay; Johnson, Clifford H; White, Karen M; Parshionikar, Sandhya U

    2015-07-01

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay. PMID:25796356

  19. One-PCR-tube approach for in situ DNA isolation and detection.

    PubMed

    Huang, Xin; Hou, Lihua; Xu, Xiaohe; Chen, Hongjun; Ji, Haifeng; Zhu, Shuifang

    2011-10-21

    Traditional real-time polymerase chain reaction (PCR) requires a purified DNA sample for PCR amplification and detection. This requires PCR tests be conducted in clean laboratories, and limits its applications for field tests. This work developed a method that can carry out DNA purification, amplification and detection in a single PCR tube. The polypropylene PCR tube was first treated with chromic acid and peptide nucleic acids (PNA) as DNA-capturer were immobilized on the internal surface of the tube. Cauliflower mosaic virus 35S (CaMV-35S) promoter in the crude extract was hybridized with the PNA on the tube surface, and the inhibitors, interfering agents and irrelevant DNA in the crude extract were effectively removed by rinsing with buffer solutions. The tube that has captured the target DNA can be used for the following real-time PCR (RT-PCR). By using this approach, the detection of less than 2500 copies of 35S plasmids in a complex sample could be completed within 3 hours. Chocolate samples were tested for real sample analysis, and 35S plasmids in genetically modified chocolate samples have been successfully identified with this method in situ. The novel One-PCR-tube method is competitive for commercial kits with the same time and simpler operation procedure. This method may be widely used for identifying food that contains modified DNA and specific pathogens in the field. PMID:21879029

  20. Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

    USGS Publications Warehouse

    Purcell, Maureen K.; Thompson, Rachel L.; Garver, Kyle A.; Hawley, Laura M.; Batts, William N.; Sprague, Laura; Sampson, Corie; Winton, James R.

    2013-01-01

    Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

  1. 7 CFR 301.92-12 - Testing protocols.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... on positive results of any approved test. Positive PCR or other molecular tests do not require confirmatory culture tests, nor do positive culture tests require confirmatory PCR or other molecular tests; however, if culture tests return other than positive results, an APHIS-approved PCR or other...

  2. 7 CFR 301.92-12 - Testing protocols.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... on positive results of any approved test. Positive PCR or other molecular tests do not require confirmatory culture tests, nor do positive culture tests require confirmatory PCR or other molecular tests; however, if culture tests return other than positive results, an APHIS-approved PCR or other...

  3. Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

    PubMed Central

    Barbano, Raffaela; Pasculli, Barbara; Coco, Michelina; Fontana, Andrea; Copetti, Massimiliano; Rendina, Michelina; Valori, Vanna Maria; Graziano, Paolo; Maiello, Evaristo; Fazio, Vito Michele; Parrella, Paola

    2015-01-01

    BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method. PMID:26690267

  4. Analytical Comparison of In Vitro-Spiked Human Serum and Plasma for PCR-Based Detection of Aspergillus fumigatus DNA: a Study by the European Aspergillus PCR Initiative

    PubMed Central

    Mengoli, Carlo; Springer, Jan; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Klingspor, Lena; Lagrou, Katrien; Melchers, Willem J. G.; Morton, C. Oliver; Barnes, Rosemary A.; Donnelly, J. Peter; White, P. Lewis

    2015-01-01

    The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing. PMID:26085614

  5. Qualitative methods for assessing risk

    SciTech Connect

    Mahn, J.A.; Hannaman, G.W.; Kryska, P.

    1995-03-01

    The purpose of this document is to describe a qualitative risk assessment process that supplements the requirements of DOE/AL 5481.1B. Although facility managers have a choice of assessing risk either quantitatively or qualitatively, trade offs are involved in making the most appropriate choice for a given application. The results that can be obtained from a quantitative risk assessment are significantly more robust than those results derived from a qualitative approach. However, the advantages derived from quantitative risk assessment are achieved at a greater expenditure of money, time and convenience. This document provides the elements of a framework for performing a much less costly qualitative risk assessment, while retaining the best attributes of quantitative methods. The approach discussed herein will; (1) provide facility managers with the tools to prepare consistent, site wide assessments, and (2) aid the reviewers who may be tasked to evaluate the assessments. Added cost/benefit measures of the qualitative methodology include the identification of mechanisms for optimally allocating resources for minimizing risk in an expeditious, and fiscally responsible manner.

  6. 9 CFR 145.14 - Testing.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... assay (ELISA) test,3 a polymerase chain reaction (PCR)-based test, or a combination of two or more of... reverse transcriptase/polymerase chain reaction (RRT-PCR) assay. (A) The RRT-PCR tests must be conducted... 0579-0007) Editorial Note: For Federal Register citations affecting § 145.14, see the List of...

  7. Development of a multiplex PCR for identification of vineyard mealybugs.

    PubMed

    Daane, Kent M; Middleton, Mathew C; Sforza, René; Cooper, Monica L; Walton, Vaughn M; Walsh, Douglas B; Zaviezo, Tania; Almeida, Rodrigo P P

    2011-12-01

    A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus ficus (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26±2°C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6-8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs. PMID:22217778

  8. Filtrates and Residues: Qualitative Analysis of Some Transition Metals.

    ERIC Educational Resources Information Center

    Kilner, Cary

    1985-01-01

    Describes a qualitative analysis laboratory in which students examine specific precipitates that can be used to identify copper, cobalt, nickel, and iron cations. The objective of the laboratory is to determine which test or sequence of tests unambiguously identifies each cation and to use the results to identify several unknowns. (JN)

  9. Comparative analysis of conventional PCR and real-time PCR to diagnose shrimp WSD

    PubMed Central

    Leal, C.A.G.; Carvalho-Castro, G.A.; Cottorello, A.C.; Leite, R.C.; Figueiredo, H.C.P.

    2013-01-01

    The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples. PMID:24516428

  10. Digital PCR for detection of citrus pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

  11. A philosophical analysis of the general methodology of qualitative research: a critical rationalist perspective.

    PubMed

    Rudnick, Abraham

    2014-09-01

    Philosophical discussion of the general methodology of qualitative research, such as that used in some health research, has been inductivist or relativist to date, ignoring critical rationalism as a philosophical approach with which to discuss the general methodology of qualitative research. This paper presents a discussion of the general methodology of qualitative research from a critical rationalist perspective (inspired by Popper), using as an example mental health research. The widespread endorsement of induction in qualitative research is positivist and is suspect, if not false, particularly in relation to the context of justification (or rather theory testing) as compared to the context of discovery (or rather theory generation). Relativism is riddled with philosophical weaknesses and hence it is suspect if not false too. Theory testing is compatible with qualitative research, contrary to much writing about and in qualitative research, as theory testing involves learning from trial and error, which is part of qualitative research, and which may be the form of learning most conducive to generalization. Generalization involves comparison, which is a fundamental methodological requirement of any type of research (qualitative or other); hence the traditional grounding of quantitative and experimental research in generalization. Comparison--rather than generalization--is necessary for, and hence compatible with, qualitative research; hence, the common opposition to generalization in qualitative research is misdirected, disregarding whether this opposition's claims are true or false. In conclusion, qualitative research, similar to quantitative and experimental research, assumes comparison as a general methodological requirement, which is necessary for health research. PMID:22592885

  12. Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

    PubMed Central

    Requena, Teresa; Burton, Jeremy; Matsuki, Takahiro; Munro, Karen; Simon, Mary Alice; Tanaka, Ryuichiro; Watanabe, Koichi; Tannock, Gerald W.

    2002-01-01

    Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations. PMID:11976117

  13. Improved thermal cycling durability and PCR compatibility of polymer coated quantum dot

    NASA Astrophysics Data System (ADS)

    Xun, Zhe; Zhao, Xiaoyun; Guan, Yifu

    2013-09-01

    Quantum dots have experienced rapid development in imaging, labeling and sensing in medicine and life science. To be suitable for polymerase chain reaction (PCR) assay, we have tested QD thermal cycling durability and compatibility, which have not been addressed in previous reports. In this study, we synthesized CdSe/ZnS QDs with a surface modification with high-MW amphiphilic copolymers and observed that Mg2+ ions in the PCR reaction could induce the QDs to precipitate and reduce their fluorescence signal significantly after thermal cycling. To overcome this problem, we used mPEG2000 to conjugate the QD surface for further protection, and found that this modification enables QDs to endure 40 thermal cycles in the presence of other components essential for PCR reactions. We have also identified that QDs have different effects on rTaq and Ex Taq polymerization systems. A high QD concentration could apparently reduce the PCR efficiency, but this inhibition was relieved significantly in the Ex PCR system as the concentration of Ex Taq polymerase was increased. Real-time PCR amplification results showed that QDs could provide a sufficiently measurable fluorescence signal without excessively inhibiting the DNA amplification. Based on this improved thermal cycling durability and compatibility with the PCR system, QDs have the potential to be developed as stable fluorescent sensors in PCR and real-time PCR amplification.

  14. Improved thermal cycling durability and PCR compatibility of polymer coated quantum dot.

    PubMed

    Xun, Zhe; Zhao, Xiaoyun; Guan, Yifu

    2013-09-01

    Quantum dots have experienced rapid development in imaging, labeling and sensing in medicine and life science. To be suitable for polymerase chain reaction (PCR) assay, we have tested QD thermal cycling durability and compatibility, which have not been addressed in previous reports. In this study, we synthesized CdSe/ZnS QDs with a surface modification with high-MW amphiphilic copolymers and observed that Mg²⁺ ions in the PCR reaction could induce the QDs to precipitate and reduce their fluorescence signal significantly after thermal cycling. To overcome this problem, we used mPEG2000 to conjugate the QD surface for further protection, and found that this modification enables QDs to endure 40 thermal cycles in the presence of other components essential for PCR reactions. We have also identified that QDs have different effects on rTaq and Ex Taq polymerization systems. A high QD concentration could apparently reduce the PCR efficiency, but this inhibition was relieved significantly in the Ex PCR system as the concentration of Ex Taq polymerase was increased. Real-time PCR amplification results showed that QDs could provide a sufficiently measurable fluorescence signal without excessively inhibiting the DNA amplification. Based on this improved thermal cycling durability and compatibility with the PCR system, QDs have the potential to be developed as stable fluorescent sensors in PCR and real-time PCR amplification. PMID:23924819

  15. Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.

    PubMed

    Soltan, Mohamed A; Tsai, Yun-Long; Lee, Pei-Yu A; Tsai, Chuan-Fu; Chang, Hsiao-Fen G; Wang, Hwa-Tang T; Wilkes, Rebecca P

    2016-09-01

    There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA. PMID:27180038

  16. OPPORTUNISTIC ASPERGILLUS PATHOGENS MEASURED IN HOME AND HOSPITAL TAP WATER BY MOLD SPECIFIC QUANTITATIVE PCR (MSQPCR)

    EPA Science Inventory

    Opportunistic fungal pathogens are a concern because of the increasing number of immunocompromised patients. The goal of this research was to test a simple extraction method and rapid quantitative PCR (QPCR) measurement of the occurrence of potential pathogens, Aspergillus fumiga...

  17. Evaluation of the use of PCR and reverse transcriptase PCR for detection of pathogenic bacteria in biosolids from anaerobic digestors and aerobic composters.

    PubMed

    Burtscher, Carola; Wuertz, Stefan

    2003-08-01

    A PCR-based method and a reverse transcriptase PCR (RT-PCR)-based method were developed for the detection of pathogenic bacteria in organic waste, using Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and Staphylococcus aureus as model organisms. In seeded organic waste samples, detection limits of less than 10 cells per g of organic waste were achieved after one-step enrichment of bacteria, isolation, and purification of DNA or RNA before PCR or RT-PCR amplification. To test the reproducibility and reliability of the newly developed methods, 46 unseeded samples were collected from diverse aerobic (composting) facilities and anaerobic digestors and analyzed by both culture-based classical and newly developed PCR-based procedures. No false-positive but some false-negative results were generated by the PCR- or RT-PCR-based methods after one-step enrichment when compared to the classical detection methods. The results indicated that the level of activity of the tested bacteria in unseeded samples was very low compared to that of freshly inoculated cells, preventing samples from reaching the cell density required for PCR-based detection after one-step enrichment. However, for Salmonella spp., a distinct PCR product could be obtained for all 22 nonamended samples that tested positive for Salmonella spp. by the classical detection procedure when a selective two-step enrichment (20 h in peptone water at 37 degrees C and 24 h in Rappaport Vassiliadis medium at 43 degrees C) was performed prior to nucleic acid extraction and PCR. Hence, the classical procedure was shortened, since cell plating and further differentiation of isolated colonies can be omitted, substituted for by highly sensitive and reliable detection based on nucleic acid extraction and PCR. Similarly, 2 of the 22 samples in which Salmonella spp. were detected also tested positive for Listeria monocytogenes according to a two-step enrichment procedure followed by PCR, compared to 3 samples

  18. Comparison of real-time PCR techniques to cytotoxigenic culture methods for diagnosing Clostridium difficile infection.

    PubMed

    Knetsch, C W; Bakker, D; de Boer, R F; Sanders, I; Hofs, S; Kooistra-Smid, A M D; Corver, J; Eastwood, K; Wilcox, M H; Kuijper, E J

    2011-01-01

    In the past decade, the incidence of Clostridium difficile infections (CDI) with a more severe course has increased in Europe and North America. Assays that are capable of rapidly diagnosing CDI are essential. Two real-time PCRs (LUMC and LvI) targeting C. difficile toxin genes (tcdB, and tcdA and tcdB, respectively) were compared with the BD GeneOhm PCR (targeting the tcdB gene), using cytotoxigenic culture as a gold standard. In addition, a real-time PCR targeting the tcdC frameshift mutation at position 117 (Δ117 PCR) was evaluated for detecting toxigenic C. difficile and the presence of PCR ribotype 027 in stool samples. In total, 526 diarrheal samples were prospectively collected and included in the study. Compared with those for cytotoxigenic culture, sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were for PCR LUMC 96.0%, 88.0%, 66.0%, and 98.9%, for PCR LvI 100.0%, 89.4%, 69.7%, and 100.0%, for PCR Δ117 98.0%, 90.7%, 71.9%, and 99.5%, and for PCR BD GeneOhm 88.3%, 96.9%, 86.5%, and 97.4%. Compared to those with feces samples cultured positive for C. difficile type 027, the sensitivity, specificity, PPV, and NPV of the Δ117 PCR were 95.2%, 96.2%, 87.0%, and 98.7%. We conclude that all real-time PCRs can be applied as a first screening test in an algorithm for diagnosing CDI. However, the low PPVs hinder the use of the assays as stand-alone tests. Furthermore, the Δ117 PCR may provide valuable information for minimizing the spread of the epidemic C. difficile PCR ribotype 027. PMID:20980562

  19. PCR und ELISA - Alternativen zum Maustest für die Analyse des Botulismus-Neurotoxin-C1 Giftbildungspotentiales in Umweltproben? [PCR and ELISA - in vitro alternatives to the mouse-bioassay for assessing the botulinum-neurotoxin-C1 production potential in environmental samples?

    USGS Publications Warehouse

    Zechmeister, T.C.; Farnleitner, A.H.; Rocke, T.E.; Pittner, F.; Rosengarten, R.; Mach, R.L.; Herzig, A.; Kirschner, A.K.T.

    2002-01-01

    Botulism is one of the most important bird diseases world-wide and is caused by the intoxication with Botulinum-Neurotoxin-C1 (BoNt-C1), which is produced by toxigenic clostridia under appropriate conditions. Avian botulism leads regularly to large losses among the migrating bird populations breeding and resting at the saltwater pools of the Austrian national park Neusiedler See-Seewinkel. Despite of its ethical dubiousness and its high technical expense the mouse-bioassay is still used as the routine standard method for the detection of BoNt-C1. According to the 3R-concept, in vitro alternative methods for the qualitative detection of BoNt-C1 (immunostick-ELISA) and a corresponding BoNt-C1 gene fragment (nested-PCR) were established. In order to estimate the BoNt-C1 production potential the methods were tested with sediment samples from different saltwater pools subjected to cultivation conditions appropriate for in vitro BoNt-C1-production. With the mouse-bioassay, 52 out of 77 samples were found to have a positive toxin production potential. The immunostick-ELISA showed a similar sensitivity as the mouse-bioassay and exhibited a highly significant positive correlation (r=0.94; p<0.001) with the mouse-bioassay in detecting BoNt-C1. The nested-PCR approach revealed higher numbers of positive BoNt-C1 gene fragment detections as compared to the direct toxin analysis approaches. A weak correlation (r=0.21; p=0.07) with the mouse-bioassay was discernible, no correlation was found with the immunostick-ELISA (r=0.09; p=0.46). Obviously, the PCR approach detected the BoNt-C1 gene fragment in some of the samples where no toxin expression has occurred. Thus it is suggested that the qualitative immunostick-ELISA represents a potential in vitro alternative to the mouse-bioassay for assessing the BoNt-C1 production potential in environmental samples. In contrast, qualitative BoNt-C1 gene fragment detection via PCR led to an overestimation of the actual toxin production

  20. Commentary: Writing and evaluating qualitative research reports

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An overview of qualitative methods is provided, particularly for reviewers and authors who may be less familiar with qualitative research. A question and answer format is used to address considerations for writing and evaluating qualitative research. When producing qualitative research, individuals ...

  1. Qualitative Research Articles: Guidelines, Suggestions and Needs

    ERIC Educational Resources Information Center

    Crescentini, Alberto; Mainardi, Giuditta

    2009-01-01

    Purpose: The purpose of this paper is to give ideas and suggestions to avoid some typical problems of qualitative articles. The aim is not to debate quality in qualitative research but to indicate some practical solutions. Design/methodology/approach: The paper discusses the design of qualitative research and the structure of a qualitative article…

  2. Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

    PubMed Central

    Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

    2016-01-01

    AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher

  3. Detection of major diarrheagenic bacterial pathogens by multiplex PCR panels.

    PubMed

    Sjöling, Åsa; Sadeghipoorjahromi, Leila; Novak, Daniel; Tobias, Joshua

    2015-03-01

    Diarrheal diseases remain a major threat to the youngest population in low- and middle-income countries. The main bacterial pathogens causing diarrhea are diarrheagenic Escherichia coli (DEC) that consists of enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic EHEC and enteroinvasive E. coli (EIEC), Salmonella, Shigella spp. (S. dysenteria, S. sonnei, S. flexneri) Campylobacter (C. coli, C. jejuni), Vibrio (V. vulnificus, V. parahaemolyticusm, V. cholerae), Yersinia enterocolitica and Aeromonas hydrophila. The aim of this study was to set up rapid multiplex PCR (mPCR) panels to identify these diarrheagenic pathogens based on their specific virulence genes. Primers against specific target genes were combined into three mPCR panels: one for diarrheal E. coli, one for pathogens causing mainly bloody diarrhea, and the third for the remaining pathogens. The panels were tested against a set of stool samples from Swedish children with diarrhea and controls and the analysis identified bacterial pathogens in 14/54 (26%) of the samples. These results show that our three developed mPCR panels can detect main bacterial diarrheagenic pathogens in clinical samples. PMID:25542594

  4. Halal authenticity of gelatin using species-specific PCR.

    PubMed

    Shabani, Hessam; Mehdizadeh, Mehrangiz; Mousavi, Seyed Mohammad; Dezfouli, Ehsan Ansari; Solgi, Tara; Khodaverdi, Mahdi; Rabiei, Maryam; Rastegar, Hossein; Alebouyeh, Mahmoud

    2015-10-01

    Consumption of food products derived from porcine sources is strictly prohibited in Islam. Gelatin, mostly derived from bovine and porcine sources, has many applications in the food and pharmaceutical industries. To ensure that food products comply with halal regulations, development of valid and reliable analytical methods is very much required. In this study, a species-specific polymerase chain reaction (PCR) assay using conserved regions of mitochondrial DNA (cytochrome b gene) was performed to evaluate the halal authenticity of gelatin. After isolation of DNA from gelatin powders with known origin, conventional PCR using species-specific primers was carried out on the extracted DNA. The amplified expected PCR products of 212 and 271 bp were observed for porcine and bovine gelatin, respectively. The sensitivity of the method was tested on binary gelatin mixtures containing 0.1%, 1%, 10%, and 100% (w/w) of porcine gelatin within bovine gelatin and vice versa. Although most of the DNA is degraded due to the severe processing steps of gelatin production, the minimum level of 0.1% w/w of both porcine and bovine gelatin was detected. Moreover, eight food products labeled as containing bovine gelatin and eight capsule shells were subjected to PCR examination. The results showed that all samples contained bovine gelatin, and the absence of porcine gelatin was verified. This method of species authenticity is very useful to verify whether gelatin and gelatin-containing food products are derived from halal ingredients. PMID:25872445

  5. Novel real-time PCR detection assay for Brucella suis

    PubMed Central

    Hänsel, C.; Mertens, K.; Elschner, M. C.; Melzer, F.

    2015-01-01

    Introduction Brucella suis is the causative agent of brucellosis in suidae and is differentiated into five biovars (bv). Biovars 1 and 3 possess zoonotic potential and can infect humans, whereas biovar 2 represents the main source of brucellosis in feral and domestic pigs in Europe. Both aspects, the zoonotic threat and the economic loss, emphasize the necessity to monitor feral and domestic pig populations. Available serological or PCR based methods lack sensitivity and specificity. Results Here a bioinformatics approach was used to identify a B. suis specific 17 bp repeat on chromosome II (BS1330_II0657 locus). This repeat is common for B. suis bv 1 to 4 and was used to develop a TaqMan probe assay. The average PCR efficiency was determined as 95% and the limit of detection as 12,5 fg/µl of DNA, equally to 3.7 bacterial genomes. This assay has the highest sensitivity of all previously described B. suis specific PCR assays, making it possible to detect 3-4 bacterial genomes per 1 µl of sample. The assay was tested 100% specific for B. suis and negative for other Brucella spp. and closely related non-Brucella species. Conclusions This novel qPCR assay could become a rapid, inexpensive and reliable screening method for large sample pools of B. suis 1 to 4. This method will be applicable for field samples after validation. PMID:26392898

  6. Genus identification of toxic plant by real-time PCR.

    PubMed

    Matsuyama, Shuji; Nishi, Katsuji

    2011-03-01

    Some plants have toxicities that are dangerous for humans. In the case of poisoning by toxic plants, a rapid and easy screening test is required for accurate medical treatment or forensic investigation. In this study, we designed specific primer pairs for identification of toxic plants, such as subgenus Aconitum, genus Ricinus, genus Illicium, and genus Scopolia, by internal transcribed spacer sequences of nuclear ribosomal DNA. Allied species of target plants, foods, and human DNA were not detected, but each primer pair provided a specific PCR product from the target plant using real-time PCR. This method can detect the subgenus Aconitum, genus Ricinus, and genus Scopolia with template DNA of 10 pg, respectively, and genus Illicium with 1 pg. Furthermore, each primer pair provided the exact PCR product from digested target plants in artificial gastric fluid. When a trace unknown plant sample in forensic investigation is collected from stomach contents, this PCR assay may be useful for screening toxic plants. PMID:20623131

  7. Screening ancient tuberculosis with qPCR: challenges and opportunities

    PubMed Central

    Harkins, Kelly M.; Buikstra, Jane E.; Campbell, Tessa; Bos, Kirsten I.; Johnson, Eric D.; Krause, Johannes; Stone, Anne C.

    2015-01-01

    The field of ancient DNA (aDNA) has rapidly accelerated in recent years as a result of new methods in next-generation sequencing, library preparation and targeted enrichment. Such research is restricted, however, by the highly variable DNA preservation within different tissues, especially when isolating ancient pathogens from human remains. Identifying positive candidate samples via quantitative PCR (qPCR) for downstream procedures can reduce reagent costs, increase capture efficiency and maximize the number of sequencing reads of the target. This study uses four qPCR assays designed to target regions within the Mycobacterium tuberculosis complex (MTBC) to examine 133 human skeletal samples from a wide geographical and temporal range, identified by the presence of skeletal lesions typical of chronic disseminated tuberculosis. Given the inherent challenges working with ancient mycobacteria, strict criteria must be used and primer/probe design continually re-evaluated as new data from bacteria become available. Seven samples tested positive for multiple MTBC loci, supporting them as strong candidates for downstream analyses. Using strict and conservative criteria, qPCR remains a fast and effective screening tool when compared with screening by more expensive sequencing and enrichment technologies. PMID:25487341

  8. Assessment of the real-time PCR and different digital PCR platforms for DNA quantification.

    PubMed

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca

    2016-01-01

    Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method. PMID:26521179

  9. Hypervirulent Clostridium difficile PCR-Ribotypes Exhibit Resistance to Widely Used Disinfectants

    PubMed Central

    Dawson, Lisa F.; Valiente, Esmeralda; Donahue, Elizabeth H.; Birchenough, George; Wren, Brendan W.

    2011-01-01

    The increased prevalence of Clostridium difficile infection (CDI) has coincided with enhanced transmissibility and severity of disease, which is often linked to two distinct clonal lineages designated PCR-ribotype 027 and 017 responsible for CDI outbreaks in the USA, Europe and Asia. We assessed sporulation and susceptibility of three PCR-ribotypes; 012, 017 and 027 to four classes of disinfectants; chlorine releasing agents (CRAs), peroxygens, quaternary ammonium compounds (QAC) and biguanides. The 017 PCR-ribotype, showed the highest sporulation frequency under these test conditions. The oxidizing biocides and CRAs were the most efficacious in decontamination of C. difficile vegetative cells and spores, the efficacy of the CRAs were concentration dependent irrespective of PCR-ribotype. However, there were differences observed in the susceptibility of the PCR-ribotypes, independent of the concentrations tested for Virkon®, Newgenn®, Proceine 40® and Hibiscrub®. Whereas, for Steri7® and Biocleanse® the difference observed between the disinfectants were dependent on both PCR-ribotype and concentration. The oxidizing agent Perasafe® was consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five Log10 reduction in spore titre. The PCR-ribotype and concentration dependent differences in the efficacy of the disinfectants in this study indicate that disinfectant choice is a factor for llimiting the survival and transmission of C. difficile spores in healthcare settings. PMID:22039420

  10. Detection of Leptospira DNA in Patients with Aseptic Meningitis by PCR

    PubMed Central

    Romero, Eliete C.; Billerbeck, Ana E. C.; Lando, Valéria S.; Camargo, Eide D.; Souza, Candida C.; Yasuda, Paulo H.

    1998-01-01

    Samples of cerebrospinal fluid from 103 patients with aseptic meningitis were tested by PCR for detection of leptospires, and the results were compared with those of the microscopic agglutination test (MAT) and an enzyme-linked immunosorbent assay for detection of immunoglobulin M (ELISA-IgM). Of these samples, 39.80% were positive by PCR and 8.74 and 3.88% were positive by MAT and ELISA-IgM, respectively. PMID:9574730

  11. The myth of induction in qualitative nursing research.

    PubMed

    Bergdahl, Elisabeth; Berterö, Carina M

    2015-04-01

    In nursing today, it remains unclear what constitutes a good foundation for qualitative scientific inquiry. There is a tendency to define qualitative research as a form of inductive inquiry; deductive practice is seldom discussed, and when it is, this usually occurs in the context of data analysis. We will look at how the terms 'induction' and 'deduction' are used in qualitative nursing science and by qualitative research theorists, and relate these uses to the traditional definitions of these terms by Popper and other philosophers of science. We will also question the assertion that qualitative research is or should be inductive. The position we defend here is that qualitative research should use deductive methods. We also see a need to understand the difference between the creative process needed to create theory and the justification of a theory. Our position is that misunderstandings regarding the philosophy of science and the role of inductive and deductive logic and science are still harming the development of nursing theory and science. The purpose of this article is to discuss and reflect upon inductive and deductive views of science as well as inductive and deductive analyses in qualitative research. We start by describing inductive and deductive methods and logic from a philosophy of science perspective, and we examine how the concepts of induction and deduction are often described and used in qualitative methods and nursing research. Finally, we attempt to provide a theoretical perspective that reconciles the misunderstandings regarding induction and deduction. Our conclusion is that openness towards deductive thinking and testing hypotheses is needed in qualitative nursing research. We must also realize that strict induction will not create theory; to generate theory, a creative leap is needed. PMID:25413613

  12. Rapid and sensitive detection of Feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform.

    PubMed

    Wilkes, Rebecca Penrose; Kania, Stephen A; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chang, Hsiu-Hui; Ma, Li-Juan; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2015-07-01

    Feline immunodeficiency virus (FIV) is an important infectious agent of cats. Clinical syndromes resulting from FIV infection include immunodeficiency, opportunistic infections, and neoplasia. In our study, a 5' long terminal repeat/gag region-based reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) was developed to amplify all known FIV strains to facilitate point-of-need FIV diagnosis. The RT-iiPCR method was applied in a point-of-need PCR detection platform--a field-deployable device capable of generating automatically interpreted RT-iiPCR results from nucleic acids within 1 hr. Limit of detection 95% of FIV RT-iiPCR was calculated to be 95 copies standard in vitro transcription RNA per reaction. Endpoint dilution studies with serial dilutions of an ATCC FIV type strain showed that the sensitivity of lyophilized FIV RT-iiPCR reagent was comparable to that of a reference nested PCR. The established reaction did not amplify any nontargeted feline pathogens, including Felid herpesvirus 1, feline coronavirus, Feline calicivirus, Feline leukemia virus, Mycoplasma haemofelis, and Chlamydophila felis. Based on analysis of 76 clinical samples (including blood and bone marrow) with the FIV RT-iiPCR, test sensitivity was 97.78% (44/45), specificity was 100.00% (31/31), and agreement was 98.65% (75/76), determined against a reference nested-PCR assay. A kappa value of 0.97 indicated excellent correlation between these 2 methods. The lyophilized FIV RT-iiPCR reagent, deployed on a user-friendly portable device, has potential utility for rapid and easy point-of-need detection of FIV in cats. PMID:26185125

  13. Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR

    PubMed Central

    Pagliarulo, Vincenzo; George, Ben; Beil, Stephen J; Groshen, Susan; Laird, Peter W; Cai, Jie; Willey, James; Cote, Richard J; Datar, Ram H

    2004-01-01

    Background Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as β-actin (ACTB). Results The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired student's t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns. Conclusion StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further

  14. Quantitative and qualitative analysis of urine component in the toilet set using Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Chung, So Hyun; Park, Kwang Suk; Choi, Jong Min; Lee, Won Jin

    2004-07-01

    As a part of non-invasive and unaware measurement of physiological signal in the house of live-alone person, Raman spectroscopy was applied for urine component analysis in the toilet set. 785nm, 250-300mW output solid state diode laser and 2048 element linear silicon TE cooled CCD array were incorporated for this system. Several tests were performed for setting up Raman spectroscopy in non-constrained situation: toilet set in the house. The effect of dark current, integration time, warming up time of laser, property of probe and interference of water in the toilet were tested and controlled for appropriate measurement in this environment. The spectra were obtained immediately when the subject uses the toilet set, and they can be transmitted to the server though Bluetooth. Those spectra were pre-processed for removing or correcting the effect of undesired light scattering, sample path-length difference and baseline-effect. The preprocessed data were enhanced for more exact result of multivariate analysis. The training data was prepared for predicting unknown component and its concentration by using multivariate methods. Several kinds of multivariate methods: PCA, PCR, PLS were performed to validate what is the fittest method in this environment. Through quantitative and qualitative analysis of Raman spectroscopy"s spectra obtained in the house's toilet set, we could know the component and its concentration of urine which can be index of disease.

  15. Comparison of Simplexa HSV 1 & 2 PCR with culture, immunofluorescence, and laboratory-developed TaqMan PCR for detection of herpes simplex virus in swab specimens.

    PubMed

    Gitman, Melissa R; Ferguson, David; Landry, Marie L

    2013-11-01

    The Simplexa HSV 1 & 2 direct PCR assay was compared with conventional cell culture, cytospin-enhanced direct fluorescent antibody (DFA), and a laboratory-developed real-time TaqMan PCR (LDT HSV PCR) using extracted nucleic acid for the detection of herpes simplex virus (HSV) in dermal, genital, mouth, ocular, and other swab samples. One hundred seventy-one swabs were tested prospectively, and 58 were positive for HSV (34 HSV-1 and 24 HSV-2). Cytospin-DFA detected 50 (86.2%), conventional cell culture 51 (87.9%), Simplexa direct 55 (94.8%), and LDT HSV PCR 57 (98.3%) of 58 true positives. Simplexa direct detected more positives than DFA and culture, but the differences were not significant (P = 0.0736 and P = 0.3711, respectively, by the McNemar test). Samples that were positive by all methods (n = 48) were strong positives (LDT cycle threshold [CT] value, 14.4 to 26.1). One strongly positive sample was falsely negative by LDT HSV PCR due to a failure of TaqMan probe binding. Three samples falsely negative by Simplexa direct had high CT values by LDT HSV PCR (LDT CT, 35.8 to 38.2). Omission of the DNA extraction step by Simplexa direct led to a drop in sensitivity compared to the sensitivity of LDT HSV PCR using extracted samples (94.8% versus 98.3%, respectively), but the difference was not significant (P = 0.6171). Simplexa HSV 1 & 2 direct PCR was the most expensive but required the least training of the assays used, had the lowest hands-on time and fastest assay time (75 min, versus 3 h by LDT HSV PCR), and provided the HSV type. PMID:24006008

  16. Utility of PCR, Culture, and Antigen Detection Methods for Diagnosis of Legionellosis

    PubMed Central

    Chen, Derrick J.; Procop, Gary W.; Vogel, Sherilynn; Yen-Lieberman, Belinda

    2015-01-01

    The goal of this retrospective study was to evaluate the performance of different diagnostic tests for Legionnaires' disease in a clinical setting where Legionella pneumophila PCR had been introduced. Electronic medical records at the Cleveland Clinic were searched for Legionella urinary antigen (UAG), culture, and PCR tests ordered from March 2010 through December 2013. For cases where two or more test methods were performed and at least one was positive, the medical record was reviewed for relevant clinical and epidemiologic factors. Excluding repeat testing on a given patient, 19,912 tests were ordered (12,569 UAG, 3,747 cultures, and 3,596 PCR) with 378 positive results. The positivity rate for each method was 0.4% for culture, 0.8% for PCR, and 2.7% for UAG. For 37 patients, at least two test methods were performed with at least one positive result: 10 (27%) cases were positive by all three methods, 16 (43%) were positive by two methods, and 11 (30%) were positive by one method only. For the 32 patients with medical records available, clinical presentation was consistent with proven or probable Legionella infection in 84% of the cases. For those cases, the sensitivities of culture, PCR, and UAG were 50%, 92%, and 96%, respectively. The specificities were 100% for culture and 99.9% for PCR and UAG. PMID:26292304

  17. Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations.

    PubMed

    Kraytsberg, Yevgenya; Khrapko, Konstantin

    2005-09-01

    A critical review of the clone-by-clone approach to the analysis of complex spectra of somatic mutations is presented. The study of a priori unknown somatic mutations requires painstaking analysis of complex mixtures of multiple mutant and non-mutant DNA molecules. If mutant fractions are sufficiently high, these mixtures can be dissected by the cloning of individual DNA molecules and scanning of the individual clones for mutations (e.g., by sequencing). Currently, the majority of such cloning is performed using PCR fragments. However, post-PCR cloning may result in various PCR artifacts - PCR errors and jumping PCR - and preferential amplification of certain mutations. This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future. PMID:16149882

  18. Discourse Tracing as Qualitative Practice

    ERIC Educational Resources Information Center

    LeGreco, Marianne; Tracy, Sarah J.

    2009-01-01

    This article introduces a qualitative research method called "discourse tracing". Discourse tracing draws from contributions made by ethnographers, discourse critics, case study scholars, and process tracers. The approach offers new insights and an attendant language about how we engage in research designed specifically for the…

  19. Qualitative Research in Rehabilitation Counseling

    ERIC Educational Resources Information Center

    Hanley-Maxwell, Cheryl; Al Hano, Ibrahim; Skivington, Michael

    2007-01-01

    Qualitative research approaches offer rehabilitation scholars and practitioners avenues into understanding the lives and experiences of people with disabilities and those people and systems with whom they interact. The methods used often parallel those used in counseling and appear to be well matched with the field of rehabilitation counseling.…

  20. Reconsidering Constructivism in Qualitative Research

    ERIC Educational Resources Information Center

    Lee, Cheu-Jey George

    2012-01-01

    This article examines constructivism, a paradigm in qualitative research that has been propagated by Egon Guba, Yvonna Lincoln, and Norman Denzin. A distinction is made between whether the basic presuppositions of constructivism are credible compared to those of a competing paradigm and whether constructivism's beliefs are internally consistent.…

  1. Qualitative Research in Educational Gerontology.

    ERIC Educational Resources Information Center

    Applewhite, Steven Lozano

    1997-01-01

    Quantitative methods such as logical positivism often view nondominant groups as deviant and purport to be objective. Qualitative methods such as ethnography help educational gerontologists understand diverse elderly populations and allow elders to participate in the process of defining reality and producing knowledge. (SK)

  2. Historical Perspectives toward Qualitative Research

    ERIC Educational Resources Information Center

    Watras, Joseph

    2009-01-01

    The keynote address on which this article is based considers four stages or types of studies that qualitative researchers undertake in the field of education. The reason that I explored this focus was to illustrate the benefits and the dangers of designing studies to serve policy makers. The research that I selected sought to uncover information…

  3. [Quantitative and qualitative nursing research].

    PubMed

    Nieminen, H; Sansoni, J

    1998-01-01

    The aim of this article is to open a discussion on Nursing research methods. Authors give some thoughts on qualitative nursing research and underlining the difference between positivistic and teleological vision. Relationship between inductive and deductive thinking is discussed. PMID:10474458

  4. Caregiving: A Qualitative Concept Analysis

    ERIC Educational Resources Information Center

    Hermanns, Melinda; Mastel-Smith, Beth

    2012-01-0