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Sample records for qualitative pcr testing

  1. HCG blood test - qualitative

    MedlinePlus

    ... qualitative; Serum HCG - qualitative; HCG in blood serum - qualitative ... Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:chap ...

  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.

  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. Qualitative and quantitative PCR methods for detection of three lines of genetically modified potatoes.

    PubMed

    Rho, Jae Kyun; Lee, Theresa; Jung, Soon-Il; Kim, Tae-San; Park, Yong-Hwan; Kim, Young-Mi

    2004-06-02

    Qualitative and quantitative polymerase chain reaction (PCR) methods have been developed for the detection of genetically modified (GM) potatoes. The combination of specific primers for amplification of the promoter region of Cry3A gene, potato leafroll virus replicase gene, and potato virus Y coat protein gene allows to identify each line of NewLeaf, NewLeaf Y, and NewLeaf Plus GM potatoes. Multiplex PCR method was also established for the simple and rapid detection of the three lines of GM potato in a mixture sample. For further quantitative detection, the realtime PCR method has been developed. This method features the use of a standard plasmid as a reference molecule. Standard plasmid contains both a specific region of the transgene Cry3A and an endogenous UDP-glucose pyrophosphorylase gene of the potato. The test samples containing 0.5, 1, 3, and 5% GM potatoes were quantified by this method. At the 3.0% level of each line of GM potato, the relative standard deviations ranged from 6.0 to 19.6%. This result shows that the above PCR methods are applicable to detect GM potatoes quantitatively as well as qualitatively.

  5. 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.

  6. Rapid Qualitative Urinary Tract Infection Pathogen Identification by SeptiFast® Real-Time PCR

    PubMed Central

    Malinka, Thomas; Klaschik, Sven; Weber, Stefan U.; Schewe, Jens-Christian; Stüber, Frank; Book, Malte

    2011-01-01

    Background Urinary tract infections (UTI) are frequent in outpatients. Fast pathogen identification is mandatory for shortening the time of discomfort and preventing serious complications. Urine culture needs up to 48 hours until pathogen identification. Consequently, the initial antibiotic regimen is empirical. Aim To evaluate the feasibility of qualitative urine pathogen identification by a commercially available real-time PCR blood pathogen test (SeptiFast®) and to compare the results with dipslide and microbiological culture. Design of study Pilot study with prospectively collected urine samples. Setting University hospital. Methods 82 prospectively collected urine samples from 81 patients with suspected UTI were included. Dipslide urine culture was followed by microbiological pathogen identification in dipslide positive samples. In parallel, qualitative DNA based pathogen identification (SeptiFast®) was performed in all samples. Results 61 samples were SeptiFast® positive, whereas 67 samples were dipslide culture positive. The inter-methodological concordance of positive and negative findings in the gram+, gram- and fungi sector was 371/410 (90%), 477/492 (97%) and 238/246 (97%), respectively. Sensitivity and specificity of the SeptiFast® test for the detection of an infection was 0.82 and 0.60, respectively. SeptiFast® pathogen identifications were available at least 43 hours prior to culture results. Conclusion The SeptiFast® platform identified bacterial DNA in urine specimens considerably faster compared to conventional culture. For UTI diagnosis sensitivity and specificity is limited by its present qualitative setup which does not allow pathogen quantification. Future quantitative assays may hold promise for PCR based UTI pathogen identification as a supplementation of conventional culture methods. PMID:21359187

  7. Evaluation of Altona Diagnostics RealStar Zika Virus RT-PCR Test Kit for Zika virus PCR testing.

    PubMed

    L'Huillier, Arnaud G; Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike; Gubbay, Jonathan B

    2017-03-15

    Background: With the emerging ZIKA virus (ZIKV) epidemic, accessible real-time reverse-transcription PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR Test Kit has been approved for Emergency Use Authorization by the FDA. Our aim was to verify Altona-PCR, by comparing it to the CDC-designed dual target ZIKV virus rRT-PCR reference assay (Reference-PCR), and describe demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada.Methods: A large set of clinical specimens were tested for ZIKV by Altona-PCR and Reference-PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting ZIKV NS5 gene.Results: 671 serum specimens were tested by Reference-PCR: 58 (8.6%) were positive, 193 (28.8%) equivocal and 420 (62.6%) negative. Ninety percent of Reference-PCR positive patients were tested in the first 5 days after symptom onset. Altona-PCR was performed on 284/671 tested specimens by Reference-PCR. Altona-PCR was positive in 53/58 (91%) Reference-PCR positive and 16/193 (8%) Reference-PCR equivocal specimens; ZIKV NS5 PCR was positive in all 68 Altona-PCR positive specimens, and negative in all 181 Altona-PCR negative specimens that underwent NS5 PCR.Conclusion: Most ZIKV PCR positive cases are detected in the first five days of illness. Altona-PCR has very good sensitivity (91%) and specificity (97%) compared to Reference-PCR. Altona-PCR can be used for ZIKV diagnostic testing, with less extensive verification requirements compared to a laboratory developed test.

  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.

  9. Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

    PubMed

    Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

    2011-01-01

    In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

  10. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    PubMed

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  11. PCR (Polymerase Chain Reaction) Testing for Leishmaniasis

    DTIC Science & Technology

    1993-10-20

    reactions. Prior to temperature cycling, this moderately heat stable enzyme selectively excises uracil residues which have been incorporated into DNA... temperature . Despite these precautions, some non-specific annealing of PCR primers does occur, even with single copy gene detection, and more so with...Mallinckrodt) in slightly hypotonic saline and allowed to remain at ambient temperature for 5 minutes, during which time RBC’s are lysed. Centrifugation

  12. Validation of a carnation-specific gene, ANS, used as an endogenous reference gene in qualitative and real-time quantitative PCR for carnations.

    PubMed

    Zhu, Hong; Jiang, Lingxi; Tao, Shiru; Lin, Heyan; Wang, Jinbin; Tan, Furong; Zhao, Kai; Wu, Xiao; Li, Peng; Pan, Aihu; Jia, Junwei; Tang, Xueming

    2011-01-01

    The validation of the anthocyanin synthase (ANS) gene as a carnation endogenous reference gene applicable both in classical and real-time PCR methods is a prerequisite for the development of PCR assays for genetically modified (GM) carnation detection. This is important due to the fact that GM carnation lines, developed by Florigene Pty Ltd, have been approved for commercialization. In this study, both methods were tested on 14 different carnation cultivars, and identical amplification products were obtained with all of them. No amplification products were observed with samples from 14 other plant species, which demonstrated that the system was specific to carnation. The results of Southern blot analysis confirmed that the ANS gene had a low copy number in the 10 tested carnation varieties. In qualitative and real-time PCR assays, the LOD values of 0.05 and 0.005 ng carnation DNA, respectively, were validated. Moreover, the real-time PCR system was validated with high PCR efficiency and linearity. Thus, the ANS gene had species specificity, low heterogeneity, and low copy number among the tested cultivars. These results provide evidence that the gene can be used as an endogenous reference gene of carnation, as well as in qualitative and quantitative PCR systems.

  13. Qualitative Examination of Children's Naming Skills through Test Adaptations.

    ERIC Educational Resources Information Center

    Fried-Oken, Melanie

    1987-01-01

    The Double Administration Naming Technique assists clinicians in obtaining qualitative information about a client's visual confrontation naming skills through administration of a standard naming test; readministration of the same test; identification of single and double errors; cuing for double naming errors; and qualitative analysis of naming…

  14. 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…

  15. 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

  16. Evaluation of Aspergillus PCR protocols for testing serum specimens.

    PubMed

    White, P Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J G; McCulloch, Elaine; Barnes, Rosemary A; Donnelly, J Peter; Loeffler, Juergen

    2011-11-01

    A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

  17. PCR test for detecting Taenia solium cysticercosis in pig carcasses.

    PubMed

    Sreedevi, Chennuru; Hafeez, Mohammad; Kumar, Putcha Anand; Rayulu, Vukka Chengalva; Subramanyam, Kothapalli Venkata; Sudhakar, Krovvidi

    2012-01-01

    Polymerase chain reaction (PCR) test was employed to detect Taenia solium DNA in muscle lesions for validation of the meat inspection results of slaughtered pigs. Two sets of oligonucleotide primers, one targeted against the large subunit rRNA gene (TBR primers) and the other targeted against cytochrome c oxidase subunit 1 gene (Cox1 primers) of T. solium were used in this study. On reactivity in PCR test, the TBR primers and the Cox1 primers yielded products of 286 and 984 bp, respectively, in cysticercosis positive cases. Both the sets of primers were found to be highly specific, since they did not yield any PCR product in negative controls. A total of 225 pig carcasses were screened for cysticercosis by meat inspection, out of which 25 carcasses with visible cysts (16 viable and 9 degenerated cysts) were also confirmed to be positive for cysticercosis in PCR test. However, out of the 35 carcasses with suspected lesions on meat inspection, only two were found to be positive for cysticercosis in PCR test. The detection limits for both the primer sets were analyzed. The TBR primer set could detect up to 10 pg of cysticercus DNA, whereas the Cox1 primer set could detect only up to 1 ng. It is evident from the study that PCR test is an efficient tool for validation of meat inspection results and also to rule out ambiguity in carcass judgment of suspected cases of porcine cysticercosis.

  18. 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…

  19. 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.

  20. 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.

  1. 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).

  2. Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism.

    PubMed

    Guo, Longhua; Qiu, Bin; Chi, Yuwu; Chen, Guonan

    2008-09-01

    In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.

  3. Identification of Candida spp. by phenotypic tests and PCR

    PubMed Central

    Marinho, Sandra Aparecida; Teixeira, Alice Becker; Santos, Otávio Silveira; Cazanova, Ricardo Flores; Ferreira, Carlos Alexandre Sanchez; Cherubini, Karen; de Oliveira, Sílvia Dias

    2010-01-01

    The correct identification of Candida species is of great importance, as it presents prognostic and therapeutical significance, allowing an early and appropriate antifungical therapy. The purpose of this study was to identify isolates of Candida spp. from oral mucosa of 38 patients with oral candidosis evaluated in 2004 by phenotypic methods and PCR, discriminating C. albicans from the other Candida species. The tests used for phenotypic analysis were germ-tube and chlamydoconidia production, culture in CHROMAgar™ Candida, carbohydrate assimilation test, growth at 45ºC and culture in Tween 80 agar. Genotypic confirmation was performed by PCR. Phenotypic tests showed that 63.2% strains formed germ-tubes, 73.7% produced chlamydoconidia, and 63.2% showed green colonies in chromogenic medium, presumptively indicating C. albicans or C. dubliniensis. The carbohydrate assimilation test confirmed these results. A total of 21% strains were identified as C. krusei and 13.2% were indicative of C. tropicalis. Of these later strains, three produced chlamydoconidia. The association of other phenotypic tests with culture in Tween 80 agar identified 95.8% of strains as C. albicans and 4.2% as C. dubliniensis. All 24 strains indicative of C. albicans and C. dubliniensis were confirmed by PCR as C. albicans. PMID:24031493

  4. Multiplex PCR testing for nine different sexually transmitted infections.

    PubMed

    Kriesel, John D; Bhatia, Amiteshwar S; Barrus, Cammie; Vaughn, Mike; Gardner, Jordan; Crisp, Robert J

    2016-12-01

    Current sexually transmitted infection (STI) testing is not optimal due to delays in reporting or missed diagnoses due to a lack of comprehensive testing. The FilmArray® (BioFire Diagnostics, LLC, Salt Lake City, Utah) is a user-friendly, fully automated, multiplex PCR system that is being developed for rapid point-of-care use. A research-use-only STI panel including multiple PCR primer sets for each organism was designed to detect Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, Ureaplasma urealyticum, Haemophilus ducreyi, and herpes simplex virus (HSV) types 1 and 2. Standard clinical testing included Gram stain, nucleic acid amplification, wet mount examination, herpes simplex virus culture, and syphilis IgG. Standard clinical tests were not available for all the organisms tested by the FilmArray STI panel. Two hundred and ninety-five clinical specimens from 190 subjects were directly compared to standard testing. Urine (n = 146), urethral/cervical swabs (31), oral swabs (60), rectal swabs (43), and ulcer swabs (15) were tested. Among the tested samples, FilmArray detected C. trachomatis in 39 (13%), N. gonorrhoeae in 20 (7%), T. vaginalis in nine (3%), HSV 1 in five (2%), HSV 2 in five (2%), U. urealyticum in 36 (12%), M. genitalium in eight (3%), and T. pallidum in 11 (4%). Concordance between the FilmArray STI panel and standard nucleic acid amplification testing for C. trachomatis was 98% and for N. gonorrhoeae was 97%. Multiplex PCR STI testing has the potential to improve public health by providing rapid, sensitive, and reliable results within the clinic or nearby laboratory.

  5. Triplet repeat primed PCR simplifies testing for Huntington disease.

    PubMed

    Jama, Mohamed; Millson, Alison; Miller, Christine E; Lyon, Elaine

    2013-03-01

    Diagnostic and predictive testing for Huntington disease (HD) requires an accurate determination of the number of CAG repeats in the Huntingtin (HHT) gene. Currently, when a sample appears to be homozygous for a normal allele, additional testing is required to confirm amplification from both alleles. If the sample still appears homozygous, Southern blot analysis is performed to rule out an undetected expanded HTT allele. Southern blot analysis is expensive, time-consuming, and labor intensive and requires high concentrations of DNA. We have developed a chimeric PCR process to help streamline workflow; true homozygous alleles are easily distinguished by this simplified method, and only very large expanded alleles still require Southern blot analysis. Two hundred forty-six HD samples, previously run with a different fragment analysis method, were analyzed with our new method. All samples were correctly genotyped, resulting in 100% concordance between the methods. The chimeric PCR assay was able to identify expanded alleles up to >150 CAG repeats. This method offers a simple strategy to differentiate normal from expanded CAG alleles, thereby reducing the number of samples reflexed to Southern blot analysis. It also provides assurance that expanded alleles are not routinely missed because of allele dropout.

  6. [PCR "real time" to analyze the quantitative and qualitative relations microbiota of periodontal pockets].

    PubMed

    Zorina, O A; Kulakov, A A; Boriskina, O A; Rebrikov, D V

    2011-01-01

    The introduction of a broad medical practice PCR "real time" is just beginning and dentistry is no exception. Modern molecular genetic methods provide numerous opportunities for diagnosis, assessment and prediction in patients with inflammatory periodontal diseases. Early and accurate diagnosis can allow in the future reduce the incidence of periodontitis and the progression of its course.

  7. Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative.

    PubMed

    White, P Lewis; Barnes, Rosemary A; Springer, Jan; Klingspor, Lena; Cuenca-Estrella, Manuel; Morton, C Oliver; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem J G; Mengoli, Carlo; Donnelly, J Peter; Heinz, Werner J; Loeffler, Juergen

    2015-09-01

    Aspergillus PCR testing of serum provides technical simplicity but with potentially reduced sensitivity compared to whole-blood testing. With diseases for which screening to exclude disease represents an optimal strategy, sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma than those in serum. The aim of the current investigation was to confirm analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting. Standardized Aspergillus PCR was performed on plasma and serum samples concurrently obtained from hematology patients in a multicenter retrospective anonymous case-control study, with cases diagnosed according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both kinds of samples. The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, respectively, and for plasma, they were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average, plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average, serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases. These results confirm the analytical finding that the sensitivity of Aspergillus PCR using plasma is superior to that using serum. PCR positivity occurs earlier when testing plasma and provides sufficient sensitivity for the screening of invasive aspergillosis while maintaining methodological simplicity.

  8. Why test for tuberculosis? A qualitative study from South Africa.

    PubMed

    Skinner, D; Claassens, M

    2016-12-21

    Setting: Early testing and treatment initiation are crucial for controlling the tuberculosis (TB) epidemic, especially in high-burden countries such as South Africa. Objective: To explore reasons why patients opted to test for TB and the context in which they were tested. Design: This qualitative study was nested in a larger study evaluating patients who did not initiate anti-tuberculosis treatment after diagnosis. In-depth interviews were conducted with 41 patients across five provinces of South Africa. Results: While most patients presented for testing because of their symptoms, unfortunately many waited until their symptoms were severe and thus remained infectious for longer. Outreach campaigns and TB screening at primary health care facilities were perceived favourably, although some respondents were unclear as to the nature of the tests being performed and had concerns about the implications. Positive health care worker attitudes towards presumptive TB patients contributed towards prompt testing and treatment initiation. Conclusion: As patients often delayed presenting for testing, strategies to engage early with presumptive TB patients so that testing and treatment can commence without delay should be a priority for TB programmes.

  9. Why test for tuberculosis? A qualitative study from South Africa

    PubMed Central

    Skinner, D.

    2016-01-01

    Setting: Early testing and treatment initiation are crucial for controlling the tuberculosis (TB) epidemic, especially in high-burden countries such as South Africa. Objective: To explore reasons why patients opted to test for TB and the context in which they were tested. Design: This qualitative study was nested in a larger study evaluating patients who did not initiate anti-tuberculosis treatment after diagnosis. In-depth interviews were conducted with 41 patients across five provinces of South Africa. Results: While most patients presented for testing because of their symptoms, unfortunately many waited until their symptoms were severe and thus remained infectious for longer. Outreach campaigns and TB screening at primary health care facilities were perceived favourably, although some respondents were unclear as to the nature of the tests being performed and had concerns about the implications. Positive health care worker attitudes towards presumptive TB patients contributed towards prompt testing and treatment initiation. Conclusion: As patients often delayed presenting for testing, strategies to engage early with presumptive TB patients so that testing and treatment can commence without delay should be a priority for TB programmes. PMID:28123955

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

    PubMed

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

    2011-01-01

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

  11. 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...

  12. 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

  13. [Retrospective study of the implementation of the qualitative PCR technique in biological samples for monitoring toxoplasmosis in pediatric patients receiving hematopoietic stem cell transplantation].

    PubMed

    Nigro, Mónica G; Figueroa, Carlos; Ledesma, Bibiana A

    2014-01-01

    Toxoplasmosis is an opportunistic infection caused by the parasite Toxoplasma gondii. The infection is severe and difficult to diagnose in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). Twelve patients receiving HSCT were monitored post-transplant, by qualitative PCR at the Children's Hospital S.A.M.I.C. "Prof. Dr. Juan P. Garrahan". The monitoring of these patients was defined by a history of positive serology for toxoplasmosis in the donor or recipient and because their hematologic condition did not allow the use of trimethoprim-sulfamethoxazole for prophylaxis. During the patients' monitoring, two of them with positive PCR results showed signs of illness by T. gondii and were treated with pyrimethamine-clindamycin. In two other patients, toxoplasmosis was the cause of death and an autopsy finding, showing negative PCR results. Four patients without clinical manifestations received treatment for toxoplasmosis because of positive PCR detection. In four patients there were no signs of toxoplasmosis disease and negative PCR results during follow-up. The qualitative PCR technique proved useful for the detection of toxoplasmosis reactivation in HSCT recipients, but has limitations in monitoring and making clinical decisions due to the persistence of positive PCR over time and manifestations of toxicity caused by the treatment.

  14. Qualitative research on point-of-care testing strategies and programs for HIV.

    PubMed

    Engel, Nora; Pant Pai, Nitika

    2015-01-01

    Point-of-care (POC) testing in communities, home settings and primary healthcare centers plays an important role in cutting delays in HIV diagnosis and in the uptake of voluntary testing and counseling. Qualitative research methods have important potential to overcome the current challenges in expanding HIV POC testing programs and strategies, by examining the diagnostic processes, complex inter-relationships and patterns involved in making POC diagnostics work in real-world settings. This article reviews existing qualitative studies on POC testing strategies and programs for HIV. Qualitative research on POC diagnostics around the uptake of POC tests, the actual diagnostic and testing processes involved, the influence of POC tests on clinical decision-making, communication of decisions and decisions exercised by patients are limited. Equally limited are studies that explore adaptation of POC programs to various socio-cultural contexts. More qualitative research is needed to inform test developers, funders and policymakers.

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

    PubMed

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

    2012-01-01

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

  16. 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.

  17. Comparison of PCR, Wright agglutination test and blood culture for diagnosis of brucellosis in suspected patients.

    PubMed

    Hekmatimoghaddam, Seyedhosssein; Sadeh, Maryam; Khalili, Mohammad Bagher; Mollaabedin, Mansour; Sazmand, Alireza

    2013-11-15

    Brucellosis has long been prevalent in Iran, with considerable medical and economic importance. Timely diagnosis is needed for early management and effective prevention of its consequences in human beings and animals. Current diagnostic methods impose peculiar challenges in terms of analytical method performance. This study compares diagnostic sensitivity, specificity, predictive Value of Positive (PVP) and Predictive Value of Negative (PVN) for Polymerase Chain Reaction (PCR), Wright agglutination test and blood culture used for patients suspected of brucellosis. In 120 patients clinically suspected of brucellosis and referred by physicians to the Yazd central Medical Laboratory, some relevant demographic, occupational, nutritional and clinical data were collected. Also, venous blood samples were drawn for diagnosis of brucellosis using PCR, Wright agglutination test and blood culture techniques. The most frequent symptom of patients was arthralgia (82 cases, 68.3%). PCR was positive in 25 cases (20.8%), wright test in 21 patients (17.5%) and blood culture in 6 cases (5%). In 20 out of 21 wright-positive cases, PCR was positive and all of the culture-positive patients had positive PCR. Sensitivity, specificity, PVP and PVN of blood culture compared to PCR (as the gold standard test) were 24, 100, 100 and 86%, respectively, but the above parameters when PCR is compared with blood culture (as gold standard) were 100, 83, 24 and 95%, respectively. PCR has better analytical performances than blood culture for diagnosis of brucellosis and is suitable for confirmation of Wright-positive cases.

  18. 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.

  19. A proficiency testing method for detecting antibodies against Brucella abortus in quantitative and qualitative serological tests.

    PubMed

    Gall, D; Nielsen, K; Nicola, A; Renteria, T

    2008-12-01

    A proficiency testing panel for detecting antibodies against Brucella abortus was developed and evaluated by both primary binding and conventional serological tests, using the guidelines of the World Organisation for Animal Health and the International Organization for Standardization Guide 43-1. All serological tests were judged satisfactory. Among the primary binding tests, the competitive enzyme-linked immunosorbent assay (ELISA 2) and the indirect enzyme-linked immunosorbent assay (ELISA 1), with standard deviation indices (z-scores) of -0.06 and 0.10, respectively, performed best. Similarly, E(n) numbers (i.e. a way of comparing different measurements of performance) of 0 for both the competitive ELISA 2 and the indirect ELISA 1 indicated that these tests performed best in the initial round of proficiency testing. The conventional serological tests all passed the panel. Comparing data from both the quantitative and qualitative tests demonstrated that this proficiency testing scheme was fit for the purpose for which it was designed.

  20. 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…

  1. 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

    2012-04-27

    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.

  2. Statistical tests for comparison of quantitative and qualitative models developed with near infrared spectral data

    NASA Astrophysics Data System (ADS)

    Roggo, Y.; Duponchel, L.; Ruckebusch, C.; Huvenne, J.-P.

    2003-06-01

    Near-infrared spectroscopy (NIRS) has been applied for both qualitative and quantitative evaluation of sugar beet. However, chemometrics methods are numerous and a choice criterion is sometime difficult to determine. In order to select the most accurate chemometrics method, statistical tests are developed. In the first part, quantitative models, which predict sucrose content of sugar beet, are compared. To realize a systematic study, 54 models are developed with different spectral pre-treatments (Standard Normal Variate (SNV), Detrending (D), first and second Derivative), different spectral ranges and different regression methods (Principal Component Regression (PCR), Partial Least Squares (PLS), Modified PLS (MPLS)). Analyze of variance and Fisher's tests are computed to compare respectively bias and Standard Error of Prediction Corrected for bias (SEP(C)). The model developed with full spectra pre-treated by SNV, second derivative and MPLS methods gives accurate results: bias is 0.008 and SEP(C) is 0.097 g of sucrose per 100 g of sample on a concentration range between 14 and 21 g/100 g. In the second part, McNemar's test is applied to compare the classification methods. The classifications are used with two data sets: the first data set concerns the disease resistance of sugar beet and the second deals with spectral differences between four spectrometers. The performances of four well-known classification methods are compared on the NIRS data: Linear Discriminant Analysis (LDA), K Nearest Neighbors method (KNN), Simple Modeling of Class Analogy (SIMCA) and Learning Vector Quantization neural network (LVQ) are computed. In this study, the most accurate method (SIMCA) has a prediction rate of 81.9% of good classification on the disease resistance determination and has 99.4% of good classification on the instrument data set.

  3. TQAP for Verification of Qualitative Lead Test Kits

    EPA Science Inventory

    There are lead-based paint test kits available to help home owners and contractors identify lead-based paint hazards before any Renovation, Repair, and Painting (RRP) activities take place so that proper health and safety meaures can be enacted. However, many of these test kits ...

  4. An Improved Method for Students' Flame Tests in Qualitative Analysis.

    ERIC Educational Resources Information Center

    Bare, William D.; Bradley, Tom; Pulliam, Elizabeth

    1998-01-01

    Describes a revised laboratory set-up for performing flame tests that enables students to pay more attention to the results of the tests and less attention to the manipulation of equipment. Employs a watch glass to add the analytical solutions to the flame. (DDR)

  5. 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.

  6. PCR artifact in testing for homologous recombination in genomic editing in zebrafish

    PubMed Central

    Won, Minho

    2017-01-01

    We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. The donor construct was flanked with homology arms of about 1 kb at the 5’ and 3’ ends. Injected embryos (G0) were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have undergone the desired homologous recombination, as tested by PCR using primer pairs extending from genomic DNA outside the homology region to a site within the donor cassette. However, Southern blots revealed that no recombination had taken place. We conclude that recombination happened during PCR in vitro between the donor integrated elsewhere in the genome and the lnx2a locus. We conclude that PCR alone may be insufficient to verify homologous recombination in genome editing experiments in zebrafish. PMID:28362803

  7. Specific PCR detection of tiger, leopard, and lion ingredients from test samples.

    PubMed

    Cao, Jijuan; Xu, Junyi; Liu, Ran; Yu, Ke; Wang, Changwen

    2011-01-01

    A PCR method was developed for specific detection of tiger, leopard, and lion DNA from test specimens for inspection and quarantine or for law-enforced animal protection. Three pairs of specific primers were designed based on the mitochondrial cytochrome b gene of tiger, leopard, and lion and used in the PCR testing. To mimic the effect of food processing on the sensitivity of the test, the tiger muscle and bovine bonemeal powder samples were treated at 133 degrees C for 30 min. At this processing condition, the method was sensitive enough to detect as low as 0.05% of tiger-derived ingredients from the mixed bonemeal powders. The data demonstrate that our PCR method is convenient and economic, with high sensitivity and repeatability, and can be used to detect and identify tiger, leopard, and lion ingredients from various test samples.

  8. Further Validation of the Qualitative Scoring System for the Modified Bender-Gestalt Test.

    ERIC Educational Resources Information Center

    Brannigan, Gary G.; And Others

    1995-01-01

    Compares the Qualitative Scoring System and the Developmental Scoring Systems, both Bender-Gestalt tests, in predicting achievement on the Metropolitan Achievement Test (MAT). In this study, first through fourth graders (n=409) from regular elementary schools were subjected to both tests; both systems correlated significantly with school…

  9. Comparison of PCR and common clinical tests for the diagnosis of H. pylori in dyspeptic patients.

    PubMed

    Pacheco, N; Mago, V; Gómez, I; Gueneau, P; Guelrud, M; Reyes, N; Pericchi, L R; Domínguez-Bello, M G

    2001-04-01

    Helicobacter pylori has been recognized as a major gastric pathogen. The objective of this study was to assess the diagnostic value of common clinical tests to detect H. pylori infection, by comparison with PCR. Serum and gastric biopsy specimens from 106 dyspeptic patients were examined. Serology was performed with Pyloriset Dry test, and biopsies were examined histologically, for rapid urease activity and PCR amplification of an ureA gene segment of H. pylori. PCR primers were specific for H. pylori and required at least 1.47 pg of H. pylori DNA, corresponding to about 800 bacterial cells. According to serology, histology, rapid urease, and PCR, positive results were respectively found in 56%, 86%, 64%, and 85% of dyspeptic patients, primarily with gastritis. Relative to PCR, the sensitivity (and specificity) was 55% (38%) for serology, 86% (13%) for histology, 70% (69%) for urease. When combining histology and urease, Bayesian analysis of data indicated no advantage of using combined methods over rapid urease test alone. Histology should not any longer be considered a gold standard test for Helicobacter pylori. Urea breath test still seems the first option for non invasive diagnostic. If an invasive diagnostic is justified, highly specific and sensitive molecular methods should be used to examine specimens.

  10. Clinical characteristics of patients who test positive for Clostridium difficile by repeat PCR.

    PubMed

    Green, Daniel A; Stotler, Brie; Jackman, Dana; Whittier, Susan; Della-Latta, Phyllis

    2014-11-01

    The high sensitivity of PCR assays for diagnosing Clostridium difficile infection (CDI) has greatly reduced the need for repeat testing after a negative result. Nevertheless, a small subset of patients do test positive within 7 days of a negative test. The aim of this study was to evaluate the clinical characteristics of these patients to determine when repeat testing may be appropriate. The results of all Xpert C. difficile PCR (Cepheid, Sunnyvale CA) tests performed in the clinical microbiology laboratory at New York-Presbyterian Hospital, Columbia University Medical Center (NYPH/CUMC) from 1 May 2011 through 6 September 2013, were reviewed. A retrospective case-control study was performed, comparing patients who tested positive within 7 days of a negative test result to a random selection of 50 controls who tested negative within 7 days of a negative test result. During the study period, a total of 14,875 tests were performed, of which 1,066 were repeat tests (7.2%). Eleven of these repeat tests results were positive (1.0%). The only risk factor independently associated with repeat testing positive was history of a prior CDI (odds ratio [OR], 19.6 [95% confidence interval {CI}, 4.0 to 19.5], P < 0.001). We found that patients who test positive for C. difficile by PCR within 7 days of a negative test are more likely to have a history of CDI than are patients who test negative with repeat PCR. This finding may be due to the high rate of disease relapse or the increased likelihood of empirical therapy leading to false-negative results in these patients.

  11. 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.

  12. Respirator Qualitative/Quantitative Fit Test Method Analysis

    DTIC Science & Technology

    1980-08-01

    carefully fitted to sample the atmosphere in the oral /nasal or visual cavity. Leakage is expressed as a ratio of the ambient challenge atmosphere...concentra- fifty (LD5O) tested administration (mg/kg) tion (ppm) tion (mq/m3 ) (mg/kg) Human Oral 500 (estimate) Human Inhalation 700 Cat Inhalation 35,000...Rabbit Oral 7,422 Guinea Subcutaneous 5,000 pig (Ref 67, 178, 216) TABLE 5. STANNIC CHLORIDE TOXICITY DATA Lowest Route of Lethal dose lethal Species

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. Event-specific qualitative and quantitative PCR detection methods for transgenic rapeseed hybrids MS1xRF1 and MS1xRF2.

    PubMed

    Wu, Yuhua; Wu, Gang; Xiao, Ling; Lu, Changming

    2007-10-17

    Except for the events RT73, MS8, RF3, and T45, event-specific detection methods for most commercialized genetically modified (GM) rapeseed varieties have not been established, and as a result, the enforcement of genetically modified organism labeling policies has been hindered. The genetically modified rapeseeds, MS1xRF1 and MS1xRF2, are 2 of 11 approved GM-rapeseed varieties for commercialization. In this study, the right border junction fragments between the gene construct and the rapeseed genome of events RF1, RF2, and MS1 were isolated using the commercially available GenomeWalker technology. Homology analysis indicated that the gene construct of RF1 integrated upstream of the nuclease gene, and that of the RF2 and MS1 inserted into the exon region of a gene encoding for an unknown protein. The event-specific primer pairs and corresponding probes were designed on the basis of the revealed right border junction fragments. Then, we successfully developed the identification and quantification methods for the gene-stacked hybrids MS1xRF1 and MS1xRF2 using those primers and probes. The relative limit of detection in the qualitative polymerase chain reaction (PCR) was 0.013% for the RF2 and MS1 assays using 100 ng of rapeseed DNA per reaction and 0.13% for the RF1 assay. The absolute limit of detection in the quantitative PCR was approximately one to two initial copies for each of the three event-specific assays. The evaluation of the real-time PCR assays revealed that the qualitative and quantitative methods developed by focusing on the gene-stacked hybrids MS1xRF1 and MS1xRF2 were highly specific, sensitive, and suitable for samples with a low quantity of DNA.

  18. Adaptive and Qualitative Changes in Encoding Strategy With Experience: Evidence From the Test-Expectancy Paradigm

    PubMed Central

    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 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

  19. 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.

  20. 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.

  1. Comparison of QIAGEN automated nucleic acid extraction methods for CMV quantitative PCR testing.

    PubMed

    Miller, Steve; Seet, Henrietta; Khan, Yasmeen; Wright, Carolyn; Nadarajah, Rohan

    2010-04-01

    We examined the effect of nucleic acid extraction methods on the analytic characteristics of a quantitative polymerase chain reaction (PCR) assay for cytomegalovirus (CMV). Human serum samples were extracted with 2 automated instruments (BioRobot EZ1 and QIAsymphony SP, QIAGEN, Valencia, CA) and CMV PCR results compared with those of pp65 antigenemia testing. Both extraction methods yielded results that were comparably linear and precise, whereas the QIAsymphony SP had a slightly lower limit of detection (1.92 log(10) copies/mL vs 2.26 log(10) copies/mL). In both cases, PCR was more sensitive than CMV antigen detection, detecting CMV viremia in 12% (EZ1) and 21% (QIAsymphony) of antigen-negative specimens. This study demonstrates the feasibility of using 2 different extraction techniques to yield results within 0.5 log(10) copies/mL of the mean value, a level that would allow for clinical comparison between different laboratory assays.

  2. Comparison of quantitative and qualitative tests for glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    LaRue, Nicole; Kahn, Maria; Murray, Marjorie; Leader, Brandon T; Bansil, Pooja; McGray, Sarah; Kalnoky, Michael; Zhang, Hao; Huang, Huiqiang; Jiang, Hui; Domingo, Gonzalo J

    2014-10-01

    A barrier to eliminating Plasmodium vivax malaria is inadequate treatment of infected patients. 8-Aminoquinoline-based drugs clear the parasite; however, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency are at risk for hemolysis from these drugs. Understanding the performance of G6PD deficiency tests is critical for patient safety. Two quantitative assays and two qualitative tests were evaluated. The comparison of quantitative assays gave a Pearson correlation coefficient of 0.7585 with significant difference in mean G6PD activity, highlighting the need to adhere to a single reference assay. Both qualitative tests had high sensitivity and negative predictive value at a cutoff G6PD value of 40% of normal activity if interpreted conservatively and performed under laboratory conditions. The performance of both tests dropped at a cutoff level of 45%. Cytochemical staining of specimens confirmed that heterozygous females with > 50% G6PD-deficient cells can seem normal by phenotypic tests.

  3. Accuracy of malaria diagnosis by microscopy, rapid diagnostic test, and PCR methods and evidence of antimalarial overprescription in non-severe febrile patients in two Tanzanian hospitals.

    PubMed

    Nicastri, Emanuele; Bevilacqua, Nazario; Sañé Schepisi, Monica; Paglia, Maria G; Meschi, Silvia; Ame, Shaali M; Mohamed, Jape A; Mangi, Sabina; Fumakule, Robert; Di Caro, Antonino; Capobianchi, Maria R; Kitua, Andrew; Molteni, Fabrizio; Racalbuto, Vincenzo; Ippolito, Giuseppe

    2009-05-01

    The study was aimed to evaluate the malaria over/underdiagnosis and over/underprescription of antimalarial drugs. Between February and March 2007 blood samples were collected from 336 non-severe febrile outpatients attended in two peripheral Tanzanian hospitals. Microscopy and a rapid diagnostic test (RDT) were done locally and the accuracy evaluated by qualitative polymerase chain reaction (PCR) for Plasmodium spp. The testing was performed at National Institute for Infectious Diseases Lazzaro Spallanzani (INMI), Rome, Italy. As a result of PCR, we identified 26 malaria cases out of 336 (7.7%) patients. Microscopy and RDT accuracies were 93.5% and 97.6%, respectively. Overprescription and underdiagnosis rates were 29.3% and 30.8%, respectively. On-field training, clinical management of febrile illness, and malaria microscopy in remote settings should be considered.

  4. 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…

  5. 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…

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

    PubMed

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

    2015-12-09

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

  7. 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

    2012-08-16

    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%.

  8. Optimisation of forensic genetics procedures used in disputed paternity testing: adjustment of the PCR reaction volume.

    PubMed

    Marjanović, Damir; Bakal, Narcisa; Kovacević, Lejla; Hodzić, Melisa; Haverić, Anja; Haverić, Sanin; Ibrulj, Slavica; Durmić, Adaleta

    2006-05-01

    Standard molecular techniques, with only a slight modification, are very useful in obtaining and interpreting the final results in the field of forensic genetic. Data obtained through such analysis are highly reliable and can be used as a very powerful tool that produces valuable results. However, success and swiftness of DNA typing of biological evidence either that found at a crime scene or used in disputed paternity testing, depends on the optimization of numerous factors. One of the most important and critical phases that ensures reliability of the whole procedure is the choice of the most suitable volume for the amplification protocol. Buccal swabs were collected from volunteers. DNA was extracted by Qiagen Dnaeasy Tissue Kit. PowerPlex 16 kit was used to simultaneously amplify 15 STR loci by PCR. Amplification was carried out as described previously. The tested total working reaction volumes were 5, 10 and 25 microl. The PCR amplification was carried out in PE Gene Amp PCR System Thermal Cycler (ABI, Foster City, CA). Amplification products were analyzed on an ABI PRISM 377 instrument (ABI, Foster City, CA) in 5% bis-acrilamide gel. Amplification was generally successful for all the tested reaction volumes. Lower partial to complete DNA profiles ratio, the quality of obtained STR profiles, significantly reduced amount of reaction's components give advantage to 5 microl reaction volume over other two tested volumes in this case.

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

    PubMed Central

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

    1996-01-01

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

  10. 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.

  11. Trypanosoma evansi: A comparison of PCR and parasitological diagnostic tests in experimentally infected mice.

    PubMed

    Fernández, D; González-Baradat, B; Eleizalde, M; González-Marcano, E; Perrone, T; Mendoza, M

    2009-01-01

    Trypanosoma evansi is the causative agent of equine trypanosomosis, disease that affects horse's productivity and health. Parasitological and molecular methods are mostly used to detect the infection. The aim of this work was evaluate PCR sensitivity to detect T. evansi using the primers 21/22-mer, ITS1, ESAG 6/7 and TBR 1/2 designed from repetitive (multicopies) genomic sequences. The results were compare with two parasitological tests in mice, micro-haematocrite centrifugation technique and direct microscopic examination. The results shows (a) that the minimum amount of DNA from blood of highly parasitaemic mice that was detectable by PCR was 0.001 ng, using the ESAG6/7 and TBR1/2 primer, (b) using TBR1/2 primer for parasites purified could detect 0.000001 ng and (c) in the prepatent period PCR detect the presence of parasites earlier than parasitological techniques. Nevertheless, the percentage of detection for PCR varies depending on primer employed with 60% and 66% for ITS1 and 21/22-mer, and 80% for ESAG6/7 and TBR1/2. Consequently, TBR1/2 and ESAG6/7 were the best primers to monitor T. evansi infections in mice. For epidemiological application, such comparative evaluation should be made for detection of T. evansi in livestock such as horses.

  12. A PCR detection method for testing Mycoplasma contamination of veterinary vaccines and biological products.

    PubMed

    Ingebritson, A L; Gibbs, C P; Tong, C; Srinivas, G B

    2015-02-01

    A rapid test method was developed for detecting mycoplasma contamination in veterinary biological products. The method reduces testing time by 2 weeks and shows comparable sensitivity to the current agar-based detection model. The primary goals for the development of the test were to reduce the testing time, incorporate a method that was easily adaptable across the veterinary biologics industry and reduce the subjective interpretation of results. We found that biological enrichment is necessary to maintain sensitivity of the detection method when compared to the standard culture-based test and that periodic sampling of enrichment cultures is essential to detect a wide variety of mycoplasma species that may be present as contaminants. The PCR detection method is comparable to the agar-based model and can reduce the overall testing time by up to 14 days.

  13. Comparison of N95 disposable filtering facepiece fits using bitrex qualitative and TSI Portacount quantitative fit testing.

    PubMed

    Clapham, S J

    2000-01-01

    As a means of evaluating the use of denatonium benzoate (bitrex) as a qualitative fit test agent with filtering facepiece respirators, the bitrex qualitative and TSI Protacount(R) quantitative fit-test methods were compared using N95 filtering facepieces. Seventy-nine paired tests (trial) were performed. Detection of bitrex during a qualitative fit test or measurement of a fit factor of <100 during a quantitative fit test constituted a failure. Qualitative and quantitative methods were performed using identical test protocols. Data were analyzed using pass/fail criteria, and matched-pair analysis methods were applied. The results of this study indicate that the use of bitrex during qualitative fit testing of N95 disposable filtering facepieces results in an increase in failure and/or rejection in cases where a TSI Portacount (plus N95 companion accessory) quantitatively establishes an acceptable fit.

  14. 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.

  15. ‘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

  16. 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.

  17. Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests.

    PubMed

    Hokynar, Kati; Norja, Päivi; Laitinen, Harri; Palomäki, Pekka; Garbarg-Chenon, Antoine; Ranki, Annamari; Hedman, Klaus; Söderlund-Venermo, Maria

    2004-05-01

    Parvovirus B19 causes a variety of diseases in humans, with outcomes ranging from asymptomatic to severe, such as chronic anemia in immunocompromised patients or fetal hydrops and death after maternal infection during pregnancy. The virus may be transmitted via plasma-derived products. According to the results of solvent-detergent safety studies, an upper limit of B19 DNA in plasma pools was recently defined. To restrict the input of B19 virus into production pools, a quantitative nucleic acid test is a prerequisite. We examined the suitability of the two commercial quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus) for detection, quantification, and differentiation of the three known B19 genotypes, including the newly described erythrovirus variants (genotypes 2 and 3). The former kit was highly sensitive for genotype 1 but was not suitable for detection of genotype 2 or one of two genotype 3 strains. The latter kit detected and differentiated all three genotypes, albeit with lower sensitivity for one of the genotype-3 strains. We furthermore assessed the prevalence of the three B19 virus genotypes in blood donors, by screening pooled plasma samples derived from 140,160 Finnish blood-donor units. None of the pools contained detectable levels of B19 virus genotypes 2 or 3. The origin, mode of transmission, and clinical significance of these genotypes are unknown and deserve further study. The RealArt Parvo B19 LC PCR is suitable for detection, quantification, and differentiation of all three B19 virus genotypes in molecular and clinical research.

  18. Prophylactic strategies for acute hemolysis secondary to plasma-incompatible platelet transfusions: correlation between qualitative hemolysin test and isohemagglutinin titration

    PubMed Central

    Landim, Cinthia Silvestre; Gomes, Francisco Carlos Almeida; Zeza, Bernardete Martin; Mendrone-Júnior, Alfredo; Dinardo, Carla Luana

    2015-01-01

    Objective Brazilian legislation has recently suggested the use of the qualitative hemolysin test instead of isohemagglutinin titers as prophylaxis for acute hemolysis related to plasma-incompatible platelet transfusions. The efficacy of this test in preventing hemolytic reactions has never been evaluated while isohemagglutinin titers have been extensively studied. The main objective of this study was to evaluate the correlation between the results of these two tests. The impact of each type of prophylaxis on the platelet inventory management and the ability of the qualitative hemolysin test to prevent red cell sensitization after the transfusion of incompatible units were also studied. Methods A total of 246 donor blood samples were evaluated using both isohemagglutinin titers and the qualitative hemolysin test, and the results were statistically compared. Subsequently, 600 platelet units were tested using the hemolysin assay and the percentage of units unsuitable for transfusion was compared to historical data using isohemagglutinin titers (cut-off: 100). Moreover, ten patients who received units with minor ABO incompatibilities that were negative for hemolysis according to the qualitative hemolysin test were evaluated regarding the development of hemolysis and red cell sensitization (anti-A or anti-B). Results Isohemagglutinin titration and the results of qualitative hemolysin test did not correlate. The routine implementation of the qualitative hemolysin test significantly increased the percentage of platelet units found unsuitable for transfusions (15–65%; p-value <0.001). Furthermore the qualitative hemolysin test did not prevent red blood cell sensitization in a small exploratory analysis. Conclusion Qualitative hemolysin test results do not correlate to those of isohemagglutinin titers and its implementation as the prophylaxis of choice for hemolysis associated with plasma-incompatible platelet transfusions lacks clinical support of safety and

  19. Will droplet digital PCR become the test of choice for detecting and quantifying ocular Chlamydia trachomatis infection? Maybe not.

    PubMed

    Schachter, Julius

    2013-11-01

    Evaluation of: Roberts CH, Last A, Molina-Gonzalez S et al. Development and evaluation of a next-generation digital PCR diagnostic assay for ocular chlamydia trachomatis infections. J. Clin. Microbiol. 51(7), 2195-2203 (2013). Trachoma is the leading infectious cause of blindness in developing countries. Currently, there is no program to eliminate blinding trachoma as a public health problem. We need better diagnostic tests for research and to assess progress in control programs. Roberts et al. adapted droplet digital PCR (ddPCR), an emulsion PCR process that performs absolute quantitation of nucleic acids, to detect and quantify Chlamydia trachomatis infections. They compared the results with ddPCR on conjunctival swab specimens collected in trachoma-endemic area to results using Roche's Amplicor® C. trachomatis/Neisseria gonorrhoeae (CT/NG) PCR and found that ddPCR sensitivity was 73.3%. The authors concluded that 'ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatis infections'. This reviewer disagrees, feeling that if the stated sensitivity is accurate, it is too low, and suggests there may be good reasons to adapt commercially available tests for this purpose.

  20. Prevalence of PCR detectable malaria infection among febrile patients with a negative Plasmodium falciparum specific rapid diagnostic test in Zanzibar.

    PubMed

    Baltzell, Kimberly A; Shakely, Deler; Hsiang, Michelle; Kemere, Jordan; Ali, Abdullah Suleiman; Björkman, Anders; Mårtensson, Andreas; Omar, Rahila; Elfving, Kristina; Msellem, Mwinyi; Aydin-Schmidt, Berit; Rosenthal, Philip J; Greenhouse, Bryan

    2013-02-01

    We screened for malaria in 594 blood samples from febrile patients who tested negative by a Plasmodium falciparum-specific histidine-rich protein-2-based rapid diagnostic test at 12 health facilities in Zanzibar districts North A and Micheweni, from May to August 2010. Screening was with microscopy, polymerase chain reaction (PCR) targeting the cytochrome b gene (cytbPCR) of the four major human malaria species, and quantitative PCR (qPCR). The prevalence of cytbPCR-detectable malaria infection was 2% (12 of 594), including 8 P. falciparum, 3 Plasmodium malariae, and 1 Plasmodium vivax infections. Microscopy identified 4 of 8 P. falciparum infections. Parasite density as estimated by microscopy or qPCR was > 4,000 parasites/μL in 5 of 8 cytbPCR-detectable P. falciparum infections. The infections that were missed by the rapid diagnostic test represent a particular challenge in malaria elimination settings and highlight the need for more sensitive point-of-care diagnostic tools to improve case detection of all human malaria species in febrile patients.

  1. Comparative Evaluation of Norovirus Infection in Children with Acute Gastroenteritis by Rapid Immunochromatographic Test, RT-PCR and Real-time RT-PCR.

    PubMed

    Kumthip, Kattareeya; Khamrin, Pattara; Saikruang, Wilaiporn; Supadej, Kanittapon; Ushijima, Hiroshi; Maneekarn, Niwat

    2017-03-02

    Immunochromatographic (IC) test for norovirus detection is a rapid and simple detection method. This study evaluated the sensitivity and specificity of a recent version of R-Biopharm RIDA®QUICK Norovirus IC assay for norovirus detection in fecal specimens from children hospitalized with acute gastroenteritis. Fecal specimens were tested by IC kit in comparison with gold standard reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. The IC kit showed high sensitivity and specificity comparable with PCR-based methods. None of false positive and false negative was found and the assay did not cross-react with other gastroenteritis viruses. The IC assay could detect genogroup I.5 (GI.5) and a wide range of genotypes in the GII noroviruses including GII.3, GII.4, GII.6, GII.7, GII.14, GII.15, GII.21, and also newly emerging GII.17 norovirus. In conclusion, this norovirus IC kit could be an alternative choice for rapid screening or a quick diagnostic tool for norovirus detection in fecal specimens of acute gastroenteritis patients.

  2. 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

  3. Screening biological stains with qPCR versus lateral flow immunochromatographic test strips: a quantitative comparison using analytical figures of merit.

    PubMed

    Oechsle, Crystal Simson; Haddad, Sandra; Sgueglia, Joanne B; Grgicak, Catherine M

    2014-01-01

    Biological fluid identification is an important facet of evidence examination in forensic laboratories worldwide. While identifying bodily fluids may provide insight into which downstream DNA methods to employ, these screening techniques consume a vital portion of the available evidence, are usually qualitative, and rely on visual interpretation. In contrast, qPCR yields information regarding the amount and proportion of amplifiable genetic material. In this study, dilution series of either semen or male saliva were prepared in either buffer or female blood. The samples were subjected to both lateral flow immunochromatographic test strips and qPCR analysis. Analytical figures of merit-including sensitivity, minimum distinguishable signal (MDS) and limit of detection (LOD)-were calculated and compared between methods. By applying the theory of the propagation of random errors, LODs were determined to be 0.05 μL of saliva for the RSID™ Saliva cards, 0.03 μL of saliva for Quantifiler(®) Duo, and 0.001 μL of semen for Quantifiler(®) Duo. In conclusion, quantitative PCR was deemed a viable and effective screening method for subsequent DNA profiling due to its stability in different matrices, sensitivity, and low limits of detection.

  4. [Laboratory diagnosis of Treponema pallidum infection in patients with early syphilis and neurosyphilis through a PCR-based test].

    PubMed

    García, Patricia; Grassi, Bruno; Fich, Félix; Salvo, Aurelio; Araya, Luis; Abarzúa, Fernando; Soto, Julia; Poggi, Helena; Lagos, Marcela; Vásquez, Patricia; León, Eugenia P; Pérez, Carlos; Wozniak, Aniela

    2011-08-01

    Syphilis is a sexually transmitted disease caused by Treponema pallidum. The diagnosis is based mainly in clinical presentation and non-specific assays. PCR-based diagnosis has been suggested as an attractive alternative method. The aim of this study was the validation of a PCR-based test for the diagnosis of early syphilis (ES) and neurosyphilis (NS). Clinical samples of mucocutaneous lesions and cerebrospinal fluid (CSF) specimens from patients previously diagnosed for ES and NS respectively using an enlarged gold standard, were tested by PCR. The reaction was done using primers targeting the tpN47 gene. Twenty out of 21 mucocutaneous samples from patients diagnosed with ES were positive by PCR, with a clinical sensitivity of 95%. Four out of 8 CSF samples from patients previously diagnosed with NS were positive by PCR, with a clinical sensitivity of 50%. The clinical specificity for both ES and NS was 100%. The PCR sensitivity and specificity for mucocutaneous samples allowed us to implement this assay in our laboratory for routine diagnosis. Although the sensitivity of the PCR in CSF was low, it may be useful to support clinical diagnosis.

  5. 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.

  6. 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.

  7. 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

  8. Comparison of the Qualitative and Developmental Scoring Systems for the Modified Version of the Bender-Gestalt Test.

    ERIC Educational Resources Information Center

    Brannigan, Gary G.; Brunner, Nancy A.

    1993-01-01

    Examined two scoring systems for Modified Version of the Bender-Gestalt Test. Administered Bender-Gestalt and Otis-Lennon School Ability Test to 75 first-grade and 84 second-grade students. Both systems were significantly correlated with school ability. Results of tests for differences between correlations indicated that Qualitative Scoring System…

  9. Novel methylation specific real-time PCR test for the diagnosis of Klinefelter syndrome

    PubMed Central

    Mehta, Akanksha; Mielnik, Anna; Schlegel, Peter N; Paduch, Darius A

    2014-01-01

    The aim of this study was to design a molecular assay for the diagnosis of Klinefelter syndrome (KS), based on the detection of supernumerary X-chromosomes (X-chs). DNA was extracted from peripheral blood samples of twenty-six 47,XXY males; two 46,XY/47,XXY males; twenty-two 46,XY males; and 15 females; and deaminated. Methylation-specific quantitative polymerase chain reaction (MS-qPCR) was performed using primers for unmethylated and methylated copies of the X-ch inactive-specific transcript (XIST-U and XIST-M) gene. X-ch disomy was determined on the basis of XIST methylation status. Degree of mosaicism in the 46,XY/47,XXY males was compared with karyotype and fluorescent in situ hybridization (FISH) results. Data analysis was performed using the Roche® LightCycler software V. 3.5.3., including determination of crossing points (CPs) by fit-point analysis and melting curve analysis. X-ch disomy was detected in all female controls and KS patients; male controls expressed XIST-M only. CPs ranged from 29.5 to 32.5 (standard deviation (s.d.) 0.8) for XIST-U and from 29 to 31 (s.d. 0.6) for XIST-M. Limit of detection of mosaicism was 1%. Based on XIST-U/XIST-M ratios for the two 47,XXY/46,XY patients, the calculated degree of mosaicism (1.8% and 17.8%) was comparable to FISH results (2.3% and 15%, respectively). Turnaround time from DNA deamination to final data analysis was under 9 h. We conclude that MS-qPCR is a sensitive, specific and rapid test for the detection of X-ch disomy, with applicability for the screening and diagnosis of KS, even in the setting of low grade 47,XXY/46,XY mosaicism. PMID:24923458

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

    PubMed

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

    2008-10-01

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

  11. Patients' understanding of genetic susceptibility testing in mainstream medicine: qualitative study on thrombophilia

    PubMed Central

    Saukko, Paula M; Ellard, Sian; Richards, Suzanne H; Shepherd, Maggie H; Campbell, John L

    2007-01-01

    Background UK and US policy initiatives have suggested that, in the future, patients and clinicians in mainstream medicine could use genetic information to prevent common illnesses. There are no studies on patients' experience and understanding of the process of testing for common genetic susceptibilities in mainstream medicine. Methods Qualitative interviews with 42 individuals who had undergone testing for a genetic susceptibility for deep vein thrombosis in primary and secondary care in the UK. Results Some participants, often from higher social classes, had a good understanding of the test and its implications. They had often sought additional information on thrombophilia from relatives and from the Internet. Others, often from less privileged backgrounds, had a poorer understanding of the test – seven individuals were unaware of having had the genetic test. Features of genetic information led to misunderstandings: (i) at referral, (ii) when communicating results, and (iii) when making sense of the implications of testing. Participants' accounts indicated that non-specialist doctors may feel obliged to refer a patient for a genetic test they know little about, because a patient requests it after a relative had tested positive. Sometimes a referral for a genetic test was lost under information overload when multiple tests and issues were considered. The inconsistent and informal ways of communicating test results – for example by phone – in mainstream medicine also led to confusion. Participants did not generally overestimate their risk, but some were uncertain about whether they were taking the right preventive actions and/or whether their children were at risk. Information about genetic susceptibilities was difficult to make sense of, as it related to ambiguous risks for participants and family members, complicated and unfamiliar terminology and multiple genes and preventive strategies. Conclusion Policy visions of clinicians and patients in mainstream

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

    PubMed Central

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

    1995-01-01

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

  13. Agreement between quantitative and qualitative sensory testing of changes in oro-facial somatosensory sensitivity.

    PubMed

    Agbaje, J; De Laat, A; Constantinus, P; Svensson, P; Baad-Hansen, L

    2017-01-01

    Qualitative somatosensory testing (QualST) is a simple chairside test. It can be used to roughly assess the presence or absence of altered somatosensory function. To use QualST clinically, it is important to assess its agreement with quantitative sensory testing (QST). The aims of this study were to assess the agreement between QST and QualST when testing the modulation of facial sensitivity by capsaicin in healthy participants and to explore the agreement between QST and QualST in assessing the intraoral sensory function in clinical atypical odontalgia (AO) patients. Eighteen healthy pain-free adults and data from 27 AO patients were included in the study. Thirteen QST and three QualST parameters were evaluated at each site. Z-scores were computed for healthy participants, and Loss-Gain scores were created. The agreement observed between QST and QualST in participants with no alterations in facial sensation (placebo) was good, that is ranging from 89% to 94%. A poorer agreement was seen after capsaicin application in all test modalities with agreement ranging from 50% to 72%. The commonest misclassification observed was participants classified as normal according to QST, but hyper- or hyposensitive according to QualST after capsaicin application, especially for cold and pinprick. A similar trend was observed in AO patients where patients classified as normal using QST were misclassified as hypersensitive and in few patients as hyposensitive by QualST. In conclusion, the study showed that QualST may be used as a screening tool in the clinical setting, especially to show that subjects have normal sensory function.

  14. Development of novel noninvasive prenatal testing protocol for whole autosomal recessive disease using picodroplet digital PCR

    PubMed Central

    Chang, Mun Young; Kim, Ah Reum; Kim, Min Young; Kim, Soyoung; Yoon, Jinsun; Han, Jae Joon; Ahn, Soyeon; Kang, Changsoo; Choi, Byung Yoon

    2016-01-01

    We developed a protocol of noninvasive prenatal testing (NIPT), employing a higher-resolution picodroplet digital PCR, to detect genetic imbalance in maternal plasma DNA (mpDNA) caused by cell-free fetal DNA (cffDNA). In the present study, this approach was applied to four families with autosomal recessive (AR) congenital sensorineural hearing loss. First, a fraction of the fetal DNA in mpDNA was calculated. Then, we made artificial DNA mixtures (positive and negative controls) to simulate mpDNA containing the fraction of cffDNA with or without mutations. Next, a fraction of mutant cluster signals over the total signals was measured from mpDNA, positive controls, and negative controls. We determined whether fetal DNA carried any paternal or maternal mutations by calculating and comparing the sum of the log-likelihood of the study samples. Of the four families, we made a successful prediction of the complete fetal genotype in two cases where a distinct cluster was identified for each genotype and the fraction of cffDNA in mpDNA was at least 6.4%. Genotyping of only paternal mutation was possible in one of the other two families. This is the first NIPT protocol potentially applicable to any AR monogenic disease with various genotypes, including point mutations. PMID:27924908

  15. 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

  16. [THE COMPARATIVE ANALYSIS OF RESULTS OF DETECTION OF CARCINOGENIC TYPES OF HUMAN PAPILLOMA VIRUS BY QUALITATIVE AND QUANTITATIVE TESTS].

    PubMed

    Kuzmenko, E T; Labigina, A V; Leshenko, O Ya; Rusanov, D N; Kuzmenko, V V; Fedko, L P; Pak, I P

    2015-05-01

    The analysis of results of screening (n = 3208; sexually active citizen aged from 18 to 59 years) was carried out to detect oncogene types of human papilloma virus in using qualitative (1150 females and 720 males) and quantitative (polymerase chain reaction in real-time (843 females and 115 males) techniques. The human papilloma virus of high oncogene type was detected in 65% and 68.4% of females and in 48.6% and 53% of males correspondingly. Among 12 types of human papilloma virus the most frequently diagnosed was human papilloma virus 16 independently of gender of examined and technique of analysis. In females, under application of qualitative tests rate of human papilloma virus 16 made up to 18.3% (n = 280) and under application of quantitative tests Rte of human papilloma virus made up to 14.9% (n = 126; p ≤ 0.05). Under examination of males using qualitative tests rate of human papilloma virus 16 made up to 8.3% (n = 60) and under application of qualitative tests made up to 12.2% (n = 14; p ≥ 0.05). Under application of qualitative tests rate of detection on the rest ofoncogene types of human papilloma virus varied in females from 3.4% to 8.4% and in males from 1.8% to 5.9%. Under application of qualitative tests to females rate of human papilloma virus with high viral load made up to 68.4%, with medium viral load - 2.85% (n = 24) and with low viral load -0.24% (n = 2). Under application of quantitative tests in males rate of detection of types of human papilloma virus made up to 53% and at that in all high viral load was established. In females, the most of oncogene types of human papilloma virus (except for 31, 39, 59) are detected significantly more often than in males.

  17. 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.

  18. Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR

    PubMed Central

    Gonçalves, Maria Gisele; Fukasawa, Lucila Okuyama; Oliveira, Rosangela Siqueira; Salgado, Maristela Marques; Harrison, Lee H; Shutt, Kathleen A; Sacchi, Claudio Tavares

    2015-01-01

    Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC™ MGIT™ 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity. PMID:23147147

  19. Evaluation of Quantitative Real-Time PCR as a Hepatitis C Virus Supplementary Test After RIBA Discontinuation.

    PubMed

    Gong, Shunyou; Schmotzer, Christine L; Zhou, Lan

    2016-09-01

    Laboratory testing plays a major role in hepatitis C virus (HCV) diagnosis and patient follow-up. The high false positive rates of HCV screening tests require confirmation through a supplementary test. According to the 2003 CDC guidelines, recombinant immunoblot assay (RIBA) is indispensible to confirm positive screening results and differentiate biologic false positivity from true HCV exposure. However, RIBA has been permanently discontinued since 2011. In the 2013 update of its guidelines, CDC called for further studies on HCV laboratory testing without RIBA. In this study, we analyzed the applicability of quantitative real-time PCR (qPCR) as a supplementary HCV diagnostic test. By comparing our HCV testing performances before and after RIBA discontinuation, we found that omitting RIBA has no significant effect on the accurate and efficient identification of HCV infection, provided that HCV antibody signal-to-cutoff ratio is considered. Furthermore, we proposed a new HCV testing algorithm that incorporates semiquantitative assessment of HCV antibody positivity and HCV viral load measurement by qPCR. By following the algorithm, we were able to address confirmation of positive HCV screening results and to provide useful information generally required by clinicians, including the needs of further laboratory testing or clinical follow-up, as well as HCV viral titers.

  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. [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

  2. 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. PMID:27630979

  3. 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

  4. 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.

  5. A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.

    PubMed

    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.

  6. 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

  7. The Quantitative-Qualitative Distinction and the Null Hypothesis Significance Testing Procedure

    ERIC Educational Resources Information Center

    Ratnesar, Nimal; Mackenzie, Jim

    2006-01-01

    Conventional discussion of research methodology contrast two approaches, the quantitative and the qualitative, presented as collectively exhaustive. But if qualitative is taken as the understanding of lifeworlds, the two approaches between them cover only a tiny fraction of research methodologies; and the quantitative, taken as the routine…

  8. Detection of feline upper respiratory tract disease pathogens using a commercially available real-time PCR test.

    PubMed

    Litster, A; Wu, C C; Leutenegger, C M

    2015-11-01

    Feline herpesvirus (FHV-1), feline calicivirus (FCV), Bordetella bronchiseptica (Bb), Chlamydia felis (Cf) and Mycoplasma felis (Mf) are common infectious agents identified in cats with upper respiratory tract disease (URTD). Each of these agents can either act as primary pathogens or cause subclinical infections, and pathogen identification can be used to prevent disease transmission in shelters, or to manage individual cats with recurrent URTD. The aim of this study was to compare pathogen detection rates using real-time PCR testing and virus isolation (VI) or bacterial culture in conjunctival, nasal and oropharyngeal swabs from 18 shelter-housed cats with clinical URTD. Co-infections were common; FHV-1 was most prevalent and Cf and FCV were least prevalent. Agents detected by PCR were FCV 2/18 (11%), FHV-1 17/18 (94%), Bb 8/18 (44%) and Mf 15/18 (83%). Agents detected by VI and bacterial culture were FCV 1/18 (6%), FHV-1 12/18 (67%), Bb 8/18 (44%) and Mf 12/18 (67%). Agreement between PCR results and the other two methods was: FHV-1, 57.4%; FCV, 98.1%; Bb, 75.0%; Mf, 60.0%. Discordancies included PCR-positive, VI-negative (FCV, n = 1/54, 1.9%; FHV-1, n = 23/54, 42.6%), PCR-positive, culture-negative (Bb, n = 6/36, 16.7%; Mf, n = 13/36, 36.1%) or PCR-negative, culture-positive (Bb, n = 3/36, 8.3%; Mf, n = 2/36, 5.6%) results. A combination of an oropharyngeal swab and either a conjunctival or a nasal swab submitted for PCR testing was able to detect all infectious agents tested for in each cat. PCR testing was a sensitive and convenient method of detection of infectious agents in cats with clinical signs of URTD.

  9. Test Takers' Experiences with Computer-Administered Listening Comprehension Tests: Interviewing for Qualitative Explorations of Test Validity

    ERIC Educational Resources Information Center

    Gorsuch, Greta

    2004-01-01

    In this study, retrospective interviews were used to investigate reliability (and thus validity) threats to a computerized ESL listening comprehension test administered at a university in the US. The participants in the investigation, six international graduate students, were asked to respond to semi- and open-ended questions during individual…

  10. Direct comparison of flow-FISH and qPCR as diagnostic tests for telomere length measurement in humans.

    PubMed

    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 (R(2) = 0.60; p<0.0001) and patients (R(2) = 0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R(2) = 0.35; p<0.0001) and low correlation for patients (R(2) = 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 (R(2) = 0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R(2) = 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 measurement of human

  11. 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

  12. Evaluation of the hyplex® TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples

    PubMed Central

    2010-01-01

    Background Tuberculosis (TB) is one of the major public health concerns worldwide. The detection of the pathogen Mycobacterium tuberculosis complex (MTBC) as early as possible has a great impact on the effective control of the spread of the disease. In our study, we evaluated the hyplex® TBC PCR test (BAG Health Care GmbH), a novel assay using a nucleic acid amplification technique (NAAT) with reverse hybridisation and ELISA read out for the rapid detection of M. tuberculosis directly in clinical samples. Results A total of 581 respiratory and non-respiratory specimens from our pneumological hospital and the National TB Institute of Uzbekistan were used for the evaluation of the PCR assay. Of these, 292 were classified as TB samples and 289 as non-TB samples based on the results of the TB cultures as reference method. The PCR results were initially used to optimise the cut-off value of the hyplex® TBC test system by means of a ROC analysis. The overall sensitivity of the assay was determined to be 83.1%. In smear-positive TB samples, the sensitivity of the hyplex® TBC PCR test was estimated to 93.4% versus 45.1% in smear-negative samples. The specificity of the test was 99.25%. Of the two specimens (0.75%) with false-positive PCR results, one yielded a culture positive for non-tuberculous mycobacteria. Based on the assumption of a prevalence of 8% TB positives among the samples in our diagnostic TB laboratory, the positive and negative predictive values were estimated to 90.4% and 98.5%, respectively. Conclusions The hyplex® TBC PCR test is an accurate NAAT assay for a rapid and reliable detection of M. tuberculosis in various respiratory and non-respiratory specimens. Compared to many other conventional NAAT assays, the hyplex® TBC PCR test is in a low price segment which makes it an attractive option for developing and emerging countries with high TB burdens. PMID:20356361

  13. Human cytomegalovirus detection by real-time PCR and pp65-antigen test in hematopoietic stem cell transplant recipients: a challenge in low and middle-income countries

    PubMed Central

    Breda, Giovanni; Almeida, Bernado; Carstensen, Suzana; Bonfim, Carmem M; Nogueira, Meri B; Vidal, Luine R; Almeida, Sergio M; Raboni, Sonia M

    2013-01-01

    Aim: Cytomegalovirus (CMV) infection is one of the most common complications in patients submitted to hematopoietic stem cell transplant (HSCT). Pre-emptive therapy has been indicated in patients with laboratory evidence of CMV replication. The aims of this study were to compare real-time PCR or pp65 antigen assay methodologies to detect CMV replication in HSCT patients, define a viral load threshold for initiation of pre-emptive therapy, and assess the feasibility of its implementation in hospitals of countries with low and middle income. Material and methods: Human CMV detection by real-time PCR and pp65 antigen assay was carried out in blood and plasma samples of HSCT patients collected weekly during 3 months. Pre-emptive therapy was based on CMV antigenemia results. Results: Twenty-one patients were monitored with a total of 227 samples collected; 13 (62%) patients were children. A poor correlation was observed between qualitative results, though quantitative results showed statistically significant difference, with higher viral loads detected in patients with positive antigenemia. Compared to a positive antigenemia, a cutoff value of 1067.5 copies/ml, 3.03 log10/ml, for viral load was obtained with 100% sensitivity and 71% specificity. Conclusion: CMV real-time PCR in whole blood was suitable for monitoring HSCT patients. However, its high cost is a limiting factor, and it could be used to monitor selected patients, those with prolonged leukopenia and underweight children, and subsequently switched to pp65 antigen test. Further studies involving larger numbers of patients should be performed to confirm this statement. PMID:24188241

  14. 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

  15. 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…

  16. Correlations Between the Porteus Maze Test Qualitative Score and Age and Recidivism Rates of Female Correctional Inmates.

    ERIC Educational Resources Information Center

    Pearson, Virginia L.

    This study investigated correlations between the Qualitative score of the Porteus Maze Test and age and rates of recidivism of correctional institution inmates. In addition, the study was structured to provide answers to the following questions: (1) Is there a relationship between age and rates of recidivism and the Conformity-Variability score of…

  17. Comparison of a qualitative canine C-reactive protein test to a quantitative test and traditional markers of inflammation - Short communication.

    PubMed

    Smuts, Celia M; Mills, Jennifer N; Gaál, Tibor

    2015-09-01

    Qualitative tests for C-reactive protein (CRP) are available for use in dogs, and provide a rapid in-house method of detecting acute inflammation. The aim of this study was to compare results from a qualitative CRP lateral flow test (Teco CRP FASTest) to those obtained from a quantitative CRP ELISA and to traditional methods of detecting inflammation, including total leukocyte and neutrophil numbers, presence of immature neutrophils and a left shift, presence or absence of toxic changes in neutrophils and plasma fibrinogen concentration in whole blood and serum samples collected from 113 client-owned dogs. More dogs had CRP FASTest positive results than had quantitatively increased CRP (ELISA) or increases in traditional methods used for measuring inflammation. Few dogs had increases in markers of inflammation but no elevated CRP. The qualitative CRP FASTest was found to be a sensitive test for detecting increased CRP concentration and was positive more frequently than were traditional markers of inflammation.

  18. Predictive power of quantitative and qualitative fecal immunochemical tests for hemoglobin in population screening for colorectal neoplasm.

    PubMed

    Huang, Yanqin; Li, Qilong; Ge, Weiting; Cai, Shanrong; Zhang, Suzhan; Zheng, Shu

    2014-01-01

    The aim of this study was to evaluate the performance of qualitative and quantitative fecal immunochemical tests (FITs) in population screening for colorectal neoplasm. A total of 9000 participants aged between 40 and 74 years were enrolled in this study. Each participant received two stool sampling tubes and was asked to simultaneously submit two stool samples from the same bowel movement. The stool samples of each participant were tested using an immunogold labeling FIT dipstick (qualitative FIT) and an automated fecal blood analyzer (quantitative FIT). Colonoscopy was performed for those who test positive in either FIT. The positive predictive values and population detection rates of the FITs for predicting colorectal neoplasm were compared. A total of 6494 (72.16%) participants simultaneously submitted two stool samples. The diagnostic consistency for a positive result between quantitative and qualitative FITs was poor (κ=0.278, 95% confidence interval=0.223-0.333). The positive predictive values of the quantitative FIT were significantly higher than those of the qualitative FIT for predicting large (≥1 cm) adenomas (23 cases, 14.29% and 16 cases, 6.72%, P=0.013) and colorectal cancer (10 cases, 6.21% and 5 cases, 2.10%, P=0.034); however, the population detection rate for advanced neoplasm of the quantitative FIT was not significantly different from that of the qualitative FIT. Quantitative FIT is superior to qualitative FIT in predicting advanced colorectal neoplasm during colorectal cancer screening. Further studies are needed to elucidate the causes of the predictive superiority.

  19. Accurate, fast and cost-effective diagnostic test for monosomy 1p36 using real-time quantitative PCR.

    PubMed

    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.

  20. 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

  1. An alternative and sensitive method based on LCM and Q-PCR for HER2 testing in breast cancer.

    PubMed

    Fetica, Bogdan; Balacescu, Ovidiu; Balacescu, Loredana; Rus, Meda; Berindan-Neagoe, Ioana

    2014-01-01

    Nowadays, HER2 testing in breast cancer represents a necessity for both prognostic and therapy. Despite widespread use of immunohistochemistry (IHC) for assessing HER2 status, there are some limitations to identify truly negative or positive HER2 cases. Fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) could solve the equivocal HER2 IHC cases but there is no consensus on which is the best method. Consequently, finding a sensitive method for HER2 testing is critical for the management of the disease. In addition, tumor heterogeneity is an important factor which could affect accuracy of molecular diagnostics. Laser capture micro-dissection (LCM) is used to isolate pure cell populations from heterogeneous tumor tissue. The combination between LCM and quantitative polymerase chain reaction (Q-PCR), the gold standard in molecular biology for quantifying gene amplification levels, could define an important tool to improve the molecular diagnostics of HER2 status.In our pilot study we used LCM and Q-PCR to evaluate HER2 gene amplification for invasive breast carcinoma samples. The samples were selected based on HER2 status assessed by IHC and CISH. Our results demonstrated high sensitivity of Q-PCR for assessing HER2 DNA amplification as well as a good concordance between Q-PCR and IHC/ CISH assay.

  2. PCR and Mammalian Cell Selection Assays for Short-Term Genotoxicity Testing

    DTIC Science & Technology

    1993-11-05

    quantitative PCR, the specific DNA fragments (-330- and, when observed, 830-bp products) were excised from the gel and their Cerenkov radiation (i.e., light ...amplification bands so that the proced..re could be used to detect newly arising t(14;18) amplification producto wnose exact size could not be predicted. We

  3. Using a field quantitative real-time PCR test to rapidly identify highly viremic rift valley fever cases.

    PubMed

    Njenga, M Kariuki; Paweska, Janusz; Wanjala, Rose; Rao, Carol Y; Weiner, Matthew; Omballa, Victor; Luman, Elizabeth T; Mutonga, David; Sharif, Shanaaz; Panning, Marcus; Drosten, Christian; Feikin, Daniel R; Breiman, Robert F

    2009-04-01

    Approximately 8% of Rift Valley fever (RVF) cases develop severe disease, leading to hemorrhage, hepatitis, and/or encephalitis and resulting in up to 50% of deaths. A major obstacle in the management of RVF and other viral hemorrhagic fever cases in outbreaks that occur in rural settings is the inability to rapidly identify such cases, with poor prognosis early enough to allow for more-aggressive therapies. During an RVF outbreak in Kenya in 2006 to 2007, we evaluated whether quantitative real-time reverse transcription-PCR (qRT-PCR) could be used in the field to rapidly identify viremic RVF cases with risk of death. In 52 of 430 RVF cases analyzed by qRT-PCR and virus culture, 18 died (case fatality rate [CFR] = 34.6%). Levels of viremia in fatal cases were significantly higher than those in nonfatal cases (mean of 10(5.2) versus 10(2.9) per ml; P < 0.005). A negative correlation between the levels of infectious virus particles and the qRT-PCR crossover threshold (C(T)) values allowed the use of qRT-PCR to assess prognosis. The CFR was 50.0% among cases with C(T) values of <27.0 (corresponding to 2.1 x 10(4) viral RNA particles/ml of serum) and 4.5% among cases with C(T) values of >or=27.0. This cutoff yielded 93.8% sensitivity and a 95.5% negative predictive value; the specificity and positive predictive value were 58% and 50%, respectively. This study shows a correlation between high viremia and fatality and indicates that qRT-PCR testing can identify nearly all fatal RVF cases.

  4. Usefulness of PCR and Antigen Latex Agglutination Test with Samples Obtained by Transthoracic Needle Aspiration for Diagnosis of Pneumococcal Pneumonia

    PubMed Central

    García, Amparo; Rosón, Beatriz; Pérez, José Luis; Verdaguer, Ricard; Dorca, Jordi; Carratalà, Jordi; Casanova, Aurora; Manresa, Frederic; Gudiol, Francesc

    1999-01-01

    In a large number of cases, the etiology of community-acquired pneumonia (CAP) is not established. Some cases are probably caused by Streptococcus pneumoniae. Transthoracic needle aspiration (TNA) culture has a limited sensitivity which might be improved by antigen detection or gene amplification techniques. We evaluated the capacity of a PCR assay and a latex agglutination test to detect S. pneumoniae in samples obtained by TNA from 95 patients with moderate-to-severe CAP. Latex agglutination and PCR had sensitivities of 52.2 and 91.3%, specificities of 88.7 and 83.3%, positive predictive values of 62.3 and 65.6%, and negative predictive values of 83.3 and 96.5%, respectively, when culture techniques were used as the “gold standard.” When we considered expanded criteria for the diagnosis of pneumococcal pneumonia as a standard for our calculations, latex agglutination and PCR had sensitivities of 53.6 and 89.7%, specificities of 93.0 and 90.0%, positive predictive values of 78.9 and 81.3%, and negative predictive values of 80.3 and 94.7%, respectively. The additional diagnosis provided by the PCR assay compared to latex agglutination was 12.2% (95% confidence interval of the difference from 0.4 to 20.1%). PCR was more sensitive than TNA culture, particularly in patients who had received prior antibiotic therapy (83.3 versus 33.3%). Although PCR is a very sensitive and specific technique, it has not proved to be cost-effective in clinical practice. Conversely, latex agglutination is a fast and simple method whose results might have significant implications for initial antibiotic therapy. PMID:9986837

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

    PubMed

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

    2014-11-01

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

  6. 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

  7. Simple PCR Assays Improve the Sensitivity of HIV-1 Subtype B Drug Resistance Testing and Allow Linking of Resistance Mutations

    PubMed Central

    Johnson, Jeffrey A.; Li, Jin-Fen; Wei, Xierong; Lipscomb, Jonathan; Bennett, Diane; Brant, Ashley; Cong, Mian-er; Spira, Thomas; Shafer, Robert W.; Heneine, Walid

    2007-01-01

    Background The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing. Methodology We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing. Significance Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations. PMID:17653265

  8. 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…

  9. Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations.

    PubMed

    Springer, Jan; White, P Lewis; Hamilton, Shanna; Michel, Denise; Barnes, Rosemary A; Einsele, Hermann; Löffler, Juergen

    2016-03-01

    Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time

  10. 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.

  11. [Staphylococcus aureus enterotoxin A detection using the polymerase chain reaction (PCR) and its correlation with coagulase and thermonuclease tests].

    PubMed

    Suarez, María José; Arias, María Laura; del Mar Gamboa, María

    2008-03-01

    Staphylococcus aureus is a pathogenic bacterium, widely distributed on nature and associated to general infection and food borne outbreaks. The relationship between this bacterium and food borne outbreaks has been done, historically, using several tests, including coagulase, thermonuclease and actually, PCR for the genes codifying for the enterotoxin responsible of clinical symptoms. The objective of this work is to detect enterotoxin A codifying gene through PCR in a group of S. aureus strains isolated from food samples, and also to correlate the presence of this gene with the production of coagulase and thermonuclease enzymes. A total of 69 staphylococcal strains were analyzed, 58 obtained from non pasteurized milk samples from the Estación Experimental Alfredo Volio Mata and 11 from the Food and Water Microbiology Laboratory collection, Universidad de Costa Rica. Coagulase, thermonuclease and enterotoxin A were analyzed in all the strains, and a statistical correlation was performed in order to verify possible associations. Results show that there is no correlation between the three variables, nevertheless, all coagulase positive strains were thermonuclease positive, and all enterotoxin positive strains were coagulase and thermonuclease positive, but not inversely. These results show that the use of presumptive or indirect tests for establishing entorotoxigenity of S. aureus strains is not truthful, more sensible and specific analysis, as PCR, shall be performed.

  12. A PCR blood test outperforms chromogranin A in carcinoid detection and is unaffected by proton pump inhibitors.

    PubMed

    Modlin, Irvin M; Aslanian, Harry; Bodei, Lisa; Drozdov, Ignat; Kidd, Mark

    2014-12-01

    A critical requirement in neuroendocrine tumor (NET) management is a blood biomarker test that is sensitive, specific and reproducible. We evaluated a PCR-based 51-transcript signature to detect tumors, compared it with chromogranin A (CgA) and examined the confounding effect of proton pump inhibitors (PPIs), which cause falsely elevated CgA levels. The multigene signature was evaluated in two groups. Group 1: 125 prospectively collected NETs: gastroenteropancreatic NETs (n=91, including 42 pancreatic and 40 small intestinal), carcinoids of unknown primary (n=18) and other sites (n=16). Group 2: prospectively collected non-NET patients receiving PPIs (>1 month; dyspepsia, n=19; GERD, n=6; and pancreatitis, n=4) and 50 controls. All samples were analyzed by PCR (marker genes) and ELISA (DAKO-CgA). Sensitivity comparisons included χ(2), non-parametric measurements, and receiver operating characteristic (ROC) curves. Group 1: 123 NETs were PCR-positive (98.4%) compared with 50 (40%) CgA-positive (χ(2)=97.3, P<10(-26)). Significant differences (P<0.001) were noted between pancreas: PCR 95% vs CgA 29.2% (P<10(-9)) and small intestine: 100 vs 58% (P<10(-4)). The multigene test was elevated in all grades (G1-G3), in both local and disseminated disease, and was not normalized by somatostatin analog therapy. It was also elevated in 97% of CgA normal NETs. Group 2: PPI administration increased CgA in 83% and CgA was elevated in 26% of controls. PCR values were not elevated in either group. PCR performance metrics were as follows: sensitivity 98.4%, specificity 100%, positive predictive value 100%, negative predictive value 97.8%, and the ROC-derived area under the curve (AUC) was 0.997. These were significantly better than CgA (all metrics <60%; AUC, 0.54; Z-statistic, 10.44, P<0.0001). A 51-panel multigene blood transcript analysis is significantly more sensitive than plasma CgA for NET detection and is unaffected by acid suppression therapy.

  13. HPV is detectable in virtually all abnormal cervical cytology samples after reinvestigation of HPV negatives with multiple alternative PCR tests.

    PubMed

    Evans, Mark Francis; Adamson, Christine Stewart-Crawford; Schned, Laura Meredith; St John, Timothy Louis; Leiman, Gladwyn; Ashikaga, Takamaru; Cooper, Kumarasen

    2010-09-01

    The demonstration of human papillomavirus (HPV) in 99.7% of cervical carcinoma surgical specimens from around the world required investigations by multiple alternative polymerase chain reaction (PCR) assays. A similar approach may therefore be necessary to best characterize HPV prevalence and genotype distribution among cervical cytology samples. In an earlier study, 752 of 799 (94.1%) abnormal and 82 of 300 (27.3%) normal cytology specimens tested HPV positive after PCR using GP5+/6+primers. This study has reinvestigated the "HPV negative" abnormal samples (20 atypical squamous cells of undetermined significance, 5 low-grade squamous intraepithelial lesion, 14 atypical squamous cells, cannot exclude HSIL, 6 high-grade squamous intraepithelial lesion) and an age-matched cohort of "HPV negative" normal (negative for an intraepithelial lesion or malignancy) samples by PCR using PGMY09/11, FAP59/64, and LCR-E7 primers. PGMY09/11-GP5+/6+ nested PCR was performed on samples that were HPV negative by PGMY09/11 PCR. After the first 3 assays, HPV was detected in 41 of 45 (91.1%) abnormal and in 10 of 47 (21.3%) normal samples (P<0.0001). Eighteen HPV genotypes were detected and in some samples the genotype that was identified differed between the tests. The nondetection of common HPV genotypes (eg, HPVs 6, 11, 16, and 18) was notable. High-grade histopathology was found for 2 patients with HPV52-positive cytopathology. Combined with our earlier study, HPV (40 different genotypes) is shown in 99.5% of abnormal samples (99.8% inclusive of the nested PCR data). These findings show that HPV genotype and prevalence estimates are dependent on the method(s) of detection and indicate that suboptimal analytical sensitivity for one or more of the less common high-risk HPV genotypes could lead to impaired clinical sensitivity. HPV may be causal in almost every instance of abnormal cervical cytology; however, passenger HPV that is incidental to an abnormality may also have been

  14. 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

  15. Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR.

    PubMed

    Ali, Shahzad; Ali, Qurban; Melzer, Falk; Khan, Iahtasham; Akhter, Shamim; Neubauer, Heinrich; Jamal, Syed M

    2014-01-01

    Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell's serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n = 5), aborted fetuses (n = 13), and vaginal swabs (n = 12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.

  16. 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

  17. Narrative-based educational nursing intervention for managing hospitalized older adults at risk for delirium: field testing and qualitative evaluation.

    PubMed

    Bélanger, Louise; Ducharme, Francine

    2015-01-01

    Though delirium is a common complication among hospitalized older adults and the nursing care required in these situations is complex, the subject has received little attention in the literature on continuing nursing education. A study was undertaken to field test and qualitatively evaluate a narrative-based educational intervention for nurses in hospital units with a high incidence of delirium. Triangulated data collection allowed carrying out a qualitative evaluation of the intervention process and outcomes. Process evaluation showed that the intervention was facilitated by the participants' attitudes and diversity of experience, as well as by the use of real care situations, which allowed integrating theory and practice. Outcome evaluation brought to light numerous elements of empirical, ethical and esthetic knowledge expressed by the participants. Study results evidence the applicability of such interventions as part of continuing nursing education and their contribution to knowledge development.

  18. Comparison of the clinical performances of the AdvanSure HPV Screening Real-Time PCR, the Abbott Real-Time High-Risk HPV Test, and the Hybrid Capture High-Risk HPV DNA Test for Cervical Cancer Screening.

    PubMed

    Chung, Hae-Sun; Hahm, Chorong; Lee, Miae

    2014-09-01

    The clinical performance of three human papillomavirus (HPV) DNA commercial assays for cervical cancer screening was evaluated; the AdvanSure HPV Screening Real-Time PCR (AdvanSure PCR; LG Life Sciences) that was developed recently for the detection of both high-risk and low-risk genotypes, the Abbott RealTime High-Risk HPV Test (Abbott PCR; Abbott Molecular) and the Hybrid Capture High-Risk HPV DNA test (HC2; Qiagen). The three different HPV DNA tests were compared using cytology samples obtained from 619 women who underwent routine cervical cancer screening. The gold-standard assay was histopathological confirmation of cervical intraepithelial neoplasia of grade 2 or worse. The clinical sensitivities of the AdvanSure PCR, the Abbott PCR and the HC2 for the detection of cervical intraepithelial neoplasia of grade 2 or worse were 95.5%, 95.5% and 100%, respectively, while the clinical specificities were 61.6%, 86.4% and 83.3%, respectively. There were no significant differences in the clinical sensitivities of the Abbott PCR and the AdvanSure PCR compared to the HC2. The clinical specificities of the Abbott PCR and the AdvanSure PCR for the detection of HPV types 16/18 were 97.8% and 98.5%, respectively. For cervical cancer screening, all three tests showed relatively good clinical sensitivities, but the AdvanSure PCR had lower clinical specificity than the Abbott PCR and the HC2. The AdvanSure PCR and the Abbott PCR assays have the advantage of being automated and the ability to distinguish between HPV types 16/18 and other HPV types. The two real-time PCR assays could be useful tools in HPV testing for cervical cancer screening.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola)

    PubMed Central

    de Boer, Muriel E; de Boer, Tjalf E; Mariën, Janine; Timmermans, Martijn JTN; Nota, Benjamin; van Straalen, Nico M; Ellers, Jacintha; Roelofs, Dick

    2009-01-01

    Background Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation. Results Eight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase (SDHA) and eukaryotic transcription initiation factor 1A (ETIF) genes were found the most stable over the different treatments, while for O. cincta, the beta actin (ACTb) and tyrosine 3-monooxygenase (YWHAZ) genes were the most stable. Conclusion We present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements. PMID:19486513

  4. 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.

  5. 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

  6. A qualitative study of secondary distribution of HIV self-test kits by female sex workers in Kenya

    PubMed Central

    Maman, Suzanne; Murray, Katherine R.; Napierala Mavedzenge, Sue; Oluoch, Lennah; Sijenje, Florence; Agot, Kawango; Thirumurthy, Harsha

    2017-01-01

    Promoting awareness of serostatus and frequent HIV testing is especially important among high risk populations such as female sex workers (FSW) and their sexual partners. HIV self-testing is an approach that is gaining ground in sub-Saharan Africa as a strategy to increase knowledge of HIV status and promote safer sexual decisions. However, little is known about self-test distribution strategies that are optimal for increasing testing access among hard-to-reach and high risk individuals. We conducted a qualitative study with 18 FSW who participated in a larger study that provided them with five oral fluid-based self-tests, training on how to use the tests, and encouragement to offer the self-tests to their sexual partners using their discretion. Women demonstrated agency in the strategies they used to introduce self-tests to their partners and to avoid conflict with partners. They carefully considered with whom to share self-tests, often assessing the possibility for negative reactions from partners as part of their decision making process. When women faced negative reactions from partners, they drew on strategies they had used before to avoid conflict and physical harm from partners, such as not responding to angry partners and forgoing payment to leave angry partners quickly. Some women also used self-tests to make more informed sexual decisions with their partners. PMID:28346527

  7. A qualitative study of secondary distribution of HIV self-test kits by female sex workers in Kenya.

    PubMed

    Maman, Suzanne; Murray, Katherine R; Napierala Mavedzenge, Sue; Oluoch, Lennah; Sijenje, Florence; Agot, Kawango; Thirumurthy, Harsha

    2017-01-01

    Promoting awareness of serostatus and frequent HIV testing is especially important among high risk populations such as female sex workers (FSW) and their sexual partners. HIV self-testing is an approach that is gaining ground in sub-Saharan Africa as a strategy to increase knowledge of HIV status and promote safer sexual decisions. However, little is known about self-test distribution strategies that are optimal for increasing testing access among hard-to-reach and high risk individuals. We conducted a qualitative study with 18 FSW who participated in a larger study that provided them with five oral fluid-based self-tests, training on how to use the tests, and encouragement to offer the self-tests to their sexual partners using their discretion. Women demonstrated agency in the strategies they used to introduce self-tests to their partners and to avoid conflict with partners. They carefully considered with whom to share self-tests, often assessing the possibility for negative reactions from partners as part of their decision making process. When women faced negative reactions from partners, they drew on strategies they had used before to avoid conflict and physical harm from partners, such as not responding to angry partners and forgoing payment to leave angry partners quickly. Some women also used self-tests to make more informed sexual decisions with their partners.

  8. 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

  9. 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.

  10. Usefulness of two Aspergillus PCR assays and Aspergillus galactomannan and β-D-glucan testing of bronchoalveolar lavage fluid for diagnosis of chronic pulmonary aspergillosis.

    PubMed

    Urabe, Naohisa; Sakamoto, Susumu; Sano, Go; Suzuki, Junko; Hebisawa, Akira; Nakamura, Yasuhiko; Koyama, Kazuya; Ishii, Yoshikazu; Tateda, Kazuhiro; Homma, Sakae

    2017-03-22

    We evaluated the usefulness of an Aspergillus galactomannan (GM) test, a β-D-glucan (βDG) test, and two different Aspergillus polymerase chain reaction (PCR) assays of bronchoalveolar lavage fluid (BALF) samples for diagnosis of chronic pulmonary aspergillosis (CPA). BALF samples from 28 patients with and 120 patients without CPA were collected. We calculated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio for each test individually and in combination with other tests. The optical density index values, as determined by receiver operating characteristic analysis, for diagnosis of CPA were 0.5 and 105 for GM and βDG testing of BALF, respectively. The sensitivity and specificity of the GM test, βDG test, and PCR assays 1 and 2 were 77.8% and 90.0%, 77.8% and 72.5%, 86.7% and 84.2%, and 66.7% and 94.2%, respectively. Comparison of PCR assays showed that PCR assay 1 had better sensitivity, negative predictive value, and negative likelihood ratio and PCR assay 2 had better specificity, positive predictive value, and positive likelihood ratio. The combination of the GM and βDG tests had the highest diagnostic odds ratio. The combination of the GM and βDG tests of BALF was more useful than any single test for diagnosing CPA.

  11. Acceptability of Using Electronic Vending Machines to Deliver Oral Rapid HIV Self-Testing Kits: A Qualitative Study

    PubMed Central

    Young, Sean D.; Daniels, Joseph; Chiu, ChingChe J.; Bolan, Robert K.; Flynn, Risa P.; Kwok, Justin; Klausner, Jeffrey D.

    2014-01-01

    Introduction Rates of unrecognized HIV infection are significantly higher among Latino and Black men who have sex with men (MSM). Policy makers have proposed that HIV self-testing kits and new methods for delivering self-testing could improve testing uptake among minority MSM. This study sought to conduct qualitative assessments with MSM of color to determine the acceptability of using electronic vending machines to dispense HIV self-testing kits. Materials and Methods African American and Latino MSM were recruited using a participant pool from an existing HIV prevention trial on Facebook. If participants expressed interest in using a vending machine to receive an HIV self-testing kit, they were emailed a 4-digit personal identification number (PIN) code to retrieve the test from the machine. We followed up with those who had tested to assess their willingness to participate in an interview about their experience. Results Twelve kits were dispensed and 8 interviews were conducted. In general, participants expressed that the vending machine was an acceptable HIV test delivery method due to its novelty and convenience. Discussion Acceptability of this delivery model for HIV testing kits was closely associated with three main factors: credibility, confidentiality, and convenience. Future research is needed to address issues, such as user-induced errors and costs, before scaling up the dispensing method. PMID:25076208

  12. 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.

  13. Standard Setting in Specific-Purpose Language Testing: What Can a Qualitative Study Add?

    ERIC Educational Resources Information Center

    Manias, Elizabeth; McNamara, Tim

    2016-01-01

    This paper explores the views of nursing and medical domain experts in considering the standards for a specific-purpose English language screening test, the Occupational English Test (OET), for professional registration for immigrant health professionals. Since individuals who score performances in the test setting are often language experts…

  14. Prospective clinical evaluation of Amplicor Mycobacterium tuberculosis PCR test as a screening method in a low-prevalence population.

    PubMed Central

    Cartuyvels, R; De Ridder, C; Jonckheere, S; Verbist, L; Van Eldere, J

    1996-01-01

    Of 656 respiratory samples analyzed for Mycobacterium tuberculosis by microscopy, culture, and the Amplicor PCR method, 25 were positive by culture, 12 were positive by microscopy, and 17 were positive by the Amplicor PCR method; 16 samples were Amplicor PCR positive and culture negative. No patient except one with culture-negative, Amplicor PCR-positive samples had clinical indications of tuberculosis. The sensitivity and specificity of the Amplicor PCR compared with those of culture were 68 and 97.4%, respectively. For culture-positive, smear-negative samples, the sensitivity of the Amplicor PCR was 46%. PMID:8818898

  15. Attitudes toward genetic testing for cancer risk after genetic counseling and decision support: a qualitative comparison between hereditary cancer types.

    PubMed

    Wakefield, Claire E; Kasparian, Nadine A; Meiser, Bettina; Homewood, Judi; Kirk, Judy; Tucker, Kathy

    2007-01-01

    This study aimed to qualitatively assess individuals' attitudes toward genetic testing for cancer risk after genetic counseling and decision support. As part of a larger study, 78 women considering genetic testing for hereditary breast/ovarian cancer (HBOC) risk and 22 individuals considering genetic testing for hereditary nonpolyposis colorectal cancer (HNPCC) completed an open-ended table of their perceived pros and cons of genetic testing. The most frequently reported pros were "to help manage my risk of developing cancer," "to help my family," and "to know my cancer risk." With regards to risk management, the HBOC group perceived genetic testing as most helpful in informing their general risk management practices, while the HN-PCC group focused on the potential to clarify their need for bowel cancer screening, suggesting that patients' perceptions of the benefits of genetic testing may differ across cancer syndromes. Individuals in both groups expressed concern about the potential psychological impact of genetic testing. We also found that some affected individuals may not fully comprehend the meaning of their potential test results. Eliciting patients' perceived pros and cons during genetic counseling is likely to be a valuable tool for improving patient care. This data also provides an improved evidence base for the development of patient education tools.

  16. 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

  17. Understanding laboratory testing in diagnostic uncertainty: a qualitative study in general practice.

    PubMed Central

    van der Weijden, Trudy; van Bokhoven, Marloes A; Dinant, Geert-Jan; van Hasselt, Cathelijne M; Grol, Richard P T M

    2002-01-01

    BACKGROUND: Better knowledge of the professional's motives for ordering laboratory tests in the case of diagnostic uncertainty may lead to interventions directed at reducing unnecessary testing. AIM: To gain insight into the general practitioner's (GP's) motives for ordering laboratory tests for patients presenting with unexplained complaints. DESIGN OF STUDY: Semi-structured interviews based on surgery observations. SETTING: Twenty-one general practices in rural and urban areas of The Netherlands. METHOD: Investigation of the GP's perception of determinants of test-ordering behaviour in the situation of diagnostic uncertainty. The interviews were structured by evaluating the consultations and test-ordering performance of that day. RESULTS: Dutch GPs vary considerably in their motives for ordering tests. Numerous motives emerged from the data. Some examples of important themes include: personal routines; tolerance of diagnostic uncertainty; time pressure; and tactical motives for test ordering. Complying with the perceived needs of the patient for reassurance through testing is seen as an easy, cost- and time-effective strategy. A clear hierarchy in the determinants was not found. CONCLUSION: The decision to request laboratory testing is the result of a complex interaction of considerations that are often conflicting. Designers of interventions meant to improve the ordering of tests should be aware of the numerous determinants, and take contextual variables into account. PMID:12528582

  18. [Evaluation of immunochromatography test for rapid detection of influenza A and B viruses using real-time PCR].

    PubMed

    Hara, Michimaru; Sadamasu, Kenji; Takao, Shinichi; Shinkai, Takayuki; Kai, Akemi; Fukuda, Shinji; Shimazu, Yukie; Kuwayama, Masaru; Miyazaki, Kazuo

    2006-09-01

    The sensitivity of rapid diagnostic kits to influenza B is lower than to influenza A. The cause-poor performance of the kit or the scarcity of viruses in type B specimens-has yet to be clarified. Using real-time PCR, we measured the amount of influenza viruses with nasopharyngeal aspirate fluid previously identified by virus isolation culture and passing the rapid diagnosis test by four types of kits, including the ESPLINE Influenza A&B-N (Fujirebio Corp., Japan). We classified the results of virus isolation and rapid diagnosis tests into three groups and examined them: group 1 (12 specimens, influenza B, all negative in tests using four types of kits); group 2 (57 specimens, influenza B, all positive in tests); and group 3 (36 specimens, AH3, all positive in tests). The average amount of viruses in group 1 (6.60 +/- 0.81 log10copies/mL) was significantly lower (p<0.0001) than that in group 2 (8.51 +/- 0.57 log10copies/mL) or group 3 (8.72 +/- 0.63 log10copies/mL). No significant difference was seen in the amount of viruses between groups 2 and 3. We concluded that the cause of low sensitivity in rapid diagnostic kits to influenza B are attributable to the scarcity of viruses in the specimen.

  19. Pre-Clinical Testing of a Real-Time PCR Assay for Diahhreal Disease Agent Cryptosporidium

    DTIC Science & Technology

    2014-05-16

    tests on freeze-dried Shigella ipaH, enterotoxigenic E . coli (ETEC) ST1b, ETEC LT, ETEC ST1a, Cryptosporidium, and Leptospira (Idaho Technologies, Salt...Citrobacter freundii 1 Negative EHEC 1 Negative Enterobacter aerogenes 1 Negative Enterobacter cloacae 1 Negative Enterotoxigenic E . coli (ETEC...2 Negative Enteroinvasive E . coli (EIEC) 1 Negative Escherichia coli 1 Negative K. pneumoniae 1 Negative P. aeruginosa 1 Negative Proteus

  20. General practitioners' barriers and facilitators towards new provider-initiated HIV testing strategies: a qualitative study.

    PubMed

    Joore, Ivo K; van Roosmalen, Sanne Lc; van Bergen, Jan Eam; van Dijk, Nynke

    2017-04-01

    European guidelines recommend offering an HIV test to individuals who display HIV indicator conditions (ICs). UK guidelines recommend performing a 'routine offer of HIV testing' in primary care where HIV prevalence exceeds 2 in 1000. Implementation of new provider-initiated HIV testing strategies in general practice is limited, while the numbers of undiagnosed and late for care HIV patients remain high. We have explored Dutch general practitioners' barriers to and facilitators of both strategies. We combined semi-structured in-depth interviews with focus groups. Nine general practitioners - key informants of sexually transmitted infection/HIV prevention and control - were selected for the interviews. Additionally, we organised focus groups with a broad sample of general practitioners (n = 81). Framework analysis was used to analyse the data. Various barriers were found, related to (1) the content of the guidelines (testing the right group and competing priorities in general practice), (2) their organisational implementation (lack of time, unclear when to repeat the HIV test and overlong list of ICs) and (3) the patient population (creating fear among patients, stigmatising them and fear regarding financial costs). Multiple general practitioners stated that performing a sexual risk assessment of patients is important before applying either strategy. Also, they recommended implementing the IC-guided approach only in high-prevalence areas and combining HIV tests with other laboratory blood tests. General practitioners tend to cling to old patterns of risk-based testing. Promoting awareness of HIV testing and educating general practitioners about the benefits of new provider-initiated HIV testing strategies is important for the actual uptake of HIV testing.

  1. Rapid molecular haemagglutinin subtyping of avian influenza isolates by specific real-time RT-PCR tests.

    PubMed

    Elizalde, Maia; Agüero, Montserrat; Buitrago, Dolores; Yuste, María; Arias, María Luisa; Muñoz, María Jesús; Lelli, Davide; Pérez-Ramírez, Elisa; Moreno-Martin, Ana María; Fernández-Pinero, Jovita

    2014-02-01

    Sixteen haemagglutinin (HA) subtypes of avian influenza viruses (AIV) have been described to date. Rapid subtype identification of any AIV is of major interest because of the possible serious consequences for the poultry industry and even public health. Molecular techniques currently allow immediate accurate subtype characterisation prior to virus isolation. In this study, a set of fourteen specific real-time RT-PCR methods were developed and evaluated for AIV HA subtyping (H1-H4, H6-H8, H10-H16), H5 and H9 being excluded on the basis of the current validity of the European Union (EU) recommended specific assays. Specific primers and probes sets for each HA-subtype were designed to hybridise the largest isolates range within each single subtype, considering the Eurasian lineage as a major target. The robustness and general application of the 14 HA-subtype methods were verified by the analysis of 110 AIV isolates belonging to all 16 HA-subtypes, performed in different laboratories. The developed real-time RT-PCR assays proved to be highly specific and revealed suitable sensitivity, allowing direct HA-subtyping of clinical material. In summary, this study provides for the first time a panel of molecular tests using specific hydrolysis probes for rapid and complete AIV HA-subtype identification.

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

    PubMed

    Smart, Andrew

    2010-12-01

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

  3. 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

  4. General practitioners’ barriers and facilitators towards new provider-initiated HIV testing strategies: a qualitative study

    PubMed Central

    van Roosmalen, Sanne LC; van Bergen, Jan EAM; van Dijk, Nynke

    2016-01-01

    European guidelines recommend offering an HIV test to individuals who display HIV indicator conditions (ICs). UK guidelines recommend performing a ‘routine offer of HIV testing’ in primary care where HIV prevalence exceeds 2 in 1000. Implementation of new provider-initiated HIV testing strategies in general practice is limited, while the numbers of undiagnosed and late for care HIV patients remain high. We have explored Dutch general practitioners’ barriers to and facilitators of both strategies. We combined semi-structured in-depth interviews with focus groups. Nine general practitioners – key informants of sexually transmitted infection/HIV prevention and control – were selected for the interviews. Additionally, we organised focus groups with a broad sample of general practitioners (n = 81). Framework analysis was used to analyse the data. Various barriers were found, related to (1) the content of the guidelines (testing the right group and competing priorities in general practice), (2) their organisational implementation (lack of time, unclear when to repeat the HIV test and overlong list of ICs) and (3) the patient population (creating fear among patients, stigmatising them and fear regarding financial costs). Multiple general practitioners stated that performing a sexual risk assessment of patients is important before applying either strategy. Also, they recommended implementing the IC-guided approach only in high-prevalence areas and combining HIV tests with other laboratory blood tests. General practitioners tend to cling to old patterns of risk-based testing. Promoting awareness of HIV testing and educating general practitioners about the benefits of new provider-initiated HIV testing strategies is important for the actual uptake of HIV testing. PMID:27207253

  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. Qualitative study of how women define and use information about breast symptoms and diagnostic tests.

    PubMed

    Davey, Heather M; Butow, Phyllis N

    2006-10-01

    Unstructured interviews were conducted with 14 women with recent experience of diagnostic testing to explore the definition and role of information in women's experiences of undergoing diagnostic breast tests. Analysis showed that women see information as more than what they are told by healthcare professionals. Information also comes from significant others, administration and reception staff, media, comparison with previous breast symptoms, silences, gestures and behavior of healthcare professionals and significant others, and the number of tests they undergo. Information is about more than understanding. It can provide support and control, reduce anxiety, facilitate question asking and promote women's trust and confidence in themselves and their healthcare professionals. Healthcare professionals need to be aware that women define information more broadly than what they are told, and of the importance women attach to this other information. They also need to be aware that information serves more purposes than helping women understand the diagnostic testing process and their result.

  7. Analysis of Helicobacter pylori Prevalence in Chittagong, Bangladesh, Based on PCR and CLO Test

    PubMed Central

    Habib, Abdul Musaweer; Alam, Md. Jibran; Rudra, Bashudev; Quader, Md. Abdul; Al-Forkan, Mohammad

    2016-01-01

    The pathogenic bacterium Helicobacter pylori is a causative agent of gastric diseases in Bangladesh as well as throughout the world. This study aimed at analyzing the prevalence of H. pylori infection among dyspeptic patients in Chittagong, the second most populous city of Bangladesh, using 16S rRNA-based H. pylori-specific Polymerase Chain Reaction and Campylobacter-like organism test. We found that 67% of the population under study was positive for H. pylori infection. Gastric ulcer and duodenal ulcer disease showed statistically significant association with H. pylori infection; however, no association of H. pylori infection was observed in terms of age and gender. This study would play a crucial role in managing H. pylori-induced gastric diseases by understanding the current trend of H. pylori infection in the Chittagong region of Bangladesh. PMID:27891051

  8. Chlamydia infection in patients with and without cervical intra-epithelial lesions tested by real-time PCR vs. direct immunofluorescence.

    PubMed

    Oliveira, Micheline de Lucena; Amorim, Melania Maria Ramos de; Souza, Paulo Roberto Eleutério de; Albuquerque, Lúcia Cristina Bezerra de; Brandão, Lucas André Cavalcanti; Guimarães, Rafael Lima

    2008-08-01

    This study compares the detection rates of Chlamydia trachomatis by two techniques, direct immunofluorescence (IMF) and real time polymerase chain reaction (PCR), in patients with and without intra-epithelial cervical lesions (SIL) in Recife. We conducted a transversal study involving 35 women with SIL and 35 without SIL attended at Ambulatório Especializado da Mulher, Recife, Brazil. They were tested for Chlamydia trachomatis using two techniques, direct IMF or real time PCR. The rates of Chlamydia trachomatis detection were compared and the association with intra-epithelial cervical lesions was determined using the chi-square test at a 5% level of significance. Concordance between the tests was evaluated using kappa. The global prevalence of Chlamydia infection was 47.1% by direct IMF and 58.6% by real time PCR. A significant association was observed between Chlamydia diagnosis and presence of intra-epithelial cervical lesions, with about 80% positive results by direct IMF and 77.1% by real time PCR. However, the detected rate of infection with Chlamydia trachomatis was significantly greater in patients without intra-epithelial cervical lesions tested by real time PCR (40%) when compared to direct IMF (14.3%). The concordance between the tests was weak, with a kappa coefficient of 0.4. Both real time PCR and direct IMF detected elevated rates of Chlamydia infection in patients with intra-epithelial cervical lesions (80%) but the tests were discordant when patients without cervical lesions were tested, possibly because sensitivity of real time PCR is greater.

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

    PubMed

    Honda, K; Tun, Z; Matoba, R

    2001-09-01

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

  10. 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.

  11. Accessing HIV Testing and Treatment among Men Who Have Sex with Men in China: A Qualitative Study

    PubMed Central

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

    2013-01-01

    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, 7 focus group 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, non-disclosure to families, misconceptions of domestically produced antiretroviral drugs 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

  12. Nasal Methicillin-Resistant Staphylococcus aureus (MRSA) PCR Testing Reduces the Duration of MRSA-Targeted Therapy in Patients with Suspected MRSA Pneumonia.

    PubMed

    Baby, Nidhu; Faust, Andrew C; Smith, Terri; Sheperd, Lyndsay A; Knoll, Laura; Goodman, Edward L

    2017-04-01

    The objective of this study was to evaluate the impact of pharmacist-ordered methicillin-resistant Staphylococcus aureus (MRSA) PCR testing on the duration of empirical MRSA-targeted antibiotic therapy in patients with suspected pneumonia. This is a retrospective analysis of patients who received vancomycin or linezolid for suspected pneumonia before and after the implementation of a pharmacist-driven protocol for nasal MRSA PCR testing. Patients were included if they were adults of >18 years of age and initiated on vancomycin or linezolid for suspected MRSA pneumonia. The primary endpoint was the duration of vancomycin or linezolid therapy. After screening 368 patients, 57 patients met inclusion criteria (27 pre-PCR and 30 post-PCR). Baseline characteristics were similar between the two groups, with the majority of patients classified as having health care-associated pneumonia (68.4%). The use of the nasal MRSA PCR test reduced the mean duration of MRSA-targeted therapy by 46.6 h (74.0 ± 48.9 h versus 27.4 ± 18.7 h; 95% confidence interval [CI], 27.3 to 65.8 h; P < 0.0001). Fewer patients in the post-PCR group required vancomycin serum levels and dose adjustment (48.1% versus 16.7%; P = 0.02). There were no significant differences between the pre- and post-PCR groups regarding days to clinical improvement (1.78 ± 2.52 versus 2.27 ± 3.34; P = 0.54), length of hospital stay (11.04 ± 9.5 versus 8.2 ± 7.8; P = 0.22), or hospital mortality (14.8% versus 6.7%; P = 0.41). The use of nasal MRSA PCR testing in patients with suspected MRSA pneumonia reduced the duration of empirical MRSA-targeted therapy by approximately 2 days without increasing adverse clinical outcomes.

  13. Toward a Model for Field-Testing Patient Decision-Support Technologies: A Qualitative Field-Testing Study

    PubMed Central

    Elwyn, Glyn; Edwards, Adrian; Watson, Eila; Austoker, Joan; Grol, Richard

    2007-01-01

    Background Field-testing is a quality assurance criterion in the development of patient decision-support technologies (PDSTs), as identified in the consensus statement of the International Patient Decision Aids Standards Collaboration. We incorporated field-testing into the development of a Web-based, prostate-specific antigen PDST called Prosdex, which was commissioned as part of the UK Prostate Cancer Risk Management Programme. Objectives The aim of this study was to develop a model for the future field-testing of PDSTs, based on the field-testing of Prosdex. Our objectives were (1) to explore the reactions of men to evolving prototypes of Prosdex, (2) to assess the effect of these responses on the development process, and (3) to develop a model for field-testing PDSTs based on the responses and their effect on the development process. Methods Semistructured interviews were conducted with the men after they had viewed evolving prototypes of Prosdex in their homes. The men were grouped according to the prototype viewed. Men between 40 and 75 years of age were recruited from two family practices in different parts of Wales, United Kingdom. In the interviews, the men were asked for their views on Prosdex, both as a whole and in relation to specific sections such as the introduction and video clips. Comments and technical issues that arose during the viewings were noted and fed back to the developers in order to produce subsequent prototypes. Results A total of 27 men were interviewed, in five groups, according to the five prototypes of Prosdex that were developed. The two main themes from the interviews were the responses to the information provided in Prosdex and the responses to specific features of Prosdex. Within these themes, two of the most frequently encountered categories were detail of the information provided and balance between contrasting viewpoints. Criticisms were encountered, particularly with respect to navigation of the site. In addition, we found

  14. A qualitative study looking at informed choice in the context of non‐invasive prenatal testing for aneuploidy

    PubMed Central

    Hill, Melissa; Chitty, Lyn S.

    2016-01-01

    Abstract Objective To explore women's attitudes towards non‐invasive prenatal testing (NIPT) and determine factors influencing their decisions around uptake of NIPT. Method We conducted qualitative interviews to assess knowledge, attitude and deliberation amongst women offered NIPT in a public health service. In total, 45 women took part in telephone interviews (79% participation rate). Results Most women could recount the key aspects of NIPT discussed during pre‐test counselling but had variable knowledge about Down syndrome. Analysis of women's attitudes towards undergoing NIPT revealed three dominant factors they considered when reflecting on the test: (1) how NIPT compared with alternative testing options, (2) reflections on coping and (3) moral or religious values. Exploring the deliberative process revealed the different paths women take when making decisions. For some, it was an extension of the decision to have Down syndrome screening; some considered it early on following the booking‐in appointment; others made step‐wise decisions about NIPT when it became relevant to them. Conclusion Our findings support the importance of personalised counselling, whereby women and their partners have the opportunity to reflect on the implications of the test results in the context of their own lives and values. Our data highlight the influence of personal circumstances on decision‐making. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd. PMID:27477537

  15. A human papilloma virus testing algorithm comprising a combination of the L1 broad-spectrum SPF10 PCR assay and a novel E6 high-risk multiplex type-specific genotyping PCR assay.

    PubMed

    van Alewijk, Dirk; Kleter, Bernhard; Vent, Maarten; Delroisse, Jean-Marc; de Koning, Maurits; van Doorn, Leen-Jan; Quint, Wim; Colau, Brigitte

    2013-04-01

    Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection and genotyping systems. To improve HPV detection by PCR, the broad-spectrum L1-based SPF10 PCR DNA enzyme immunoassay (DEIA) LiPA system and a novel E6-based multiplex type-specific system (MPTS123) that uses Luminex xMAP technology were combined into a new testing algorithm. To evaluate this algorithm, cervical swabs (n = 860) and cervical biopsy specimens (n = 355) were tested, with a focus on HPV types detected by the MPTS123 assay (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 6, and 11). Among the HPV-positive samples, identifications of individual HPV genotypes were compared. When all MPTS123 targeted genotypes were considered together, good overall agreement was found (κ = 0.801, 95% confidence interval [CI], 0.784 to 0.818) with identification by SPF10 LiPA, but significantly more genotypes (P < 0.0001) were identified by the MPTS123 PCR Luminex assay, especially for HPV types 16, 35, 39, 45, 58, and 59. An alternative type-specific assay was evaluated that is based on detection of a limited number of HPV genotypes by type-specific PCR and a reverse hybridization assay (MPTS12 RHA). This assay showed results similar to those of the expanded MPTS123 Luminex assay. These results confirm the fact that broad-spectrum PCRs are hampered by type competition when multiple HPV genotypes are present in the same sample. Therefore, a testing algorithm combining the broad-spectrum PCR and a range of type-specific PCRs can offer a highly accurate method for the analysis of HPV infections and diminish the rate of false-negative results and may be particularly useful for epidemiological and vaccine studies.

  16. 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

  17. A qualitative analysis of inaccurate responses on the Hooper Visual Organization Test.

    PubMed

    Lopez, Michael N; Lazar, Michael D; Imperio, Sherwin M

    2005-06-01

    The present study investigated the types of inaccurate responses, i.e., Don't Know, Semantic, Visual (nonlinguistic), Phonological, Circumlocutory, and Perseverative, made on the Hooper Visual Organization Test by a heterogeneous sample of 68 brain-damaged and 63 substance abuse patients. The mean age of the brain-damaged and substance abuse groups were 46.0 (SD=13.5) and 43.7 (SD=12.9) yr., respectively. Analysis showed that the brain-damaged group made significantly more visual and perseverative responses than the substance abuse group. There was significantly more variance in the Visual responses than the Semantic responses for the brain-damaged group. The authors conclude that visuospatial ability is the primary factor for successful performance on this test.

  18. 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

  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. Cross-institute evaluations of inhibitor-resistant PCR reagents for direct testing of aerosol and blood samples containing biological warfare agent DNA.

    PubMed

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

    2014-02-01

    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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. Rapid colorimetric testing for pyrazinamide susceptibility of M. tuberculosis by a PCR-based in-vitro synthesized pyrazinamidase method.

    PubMed

    Zhou, Man; Geng, Xuelei; Chen, Jun; Wang, Xude; Wang, Dianbing; Deng, Jiaoyu; Zhang, Zhiping; Wang, Weihua; Zhang, Xian-En; Wei, Hongping

    2011-01-01

    Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug. But PZA susceptibility test is challenging because PZA activity is optimal only in an acid environment that inhibits the growth of M. tuberculosis. For current phenotypic methods, inconsistent results between different labs have been reported. Direct sequencing of pncA gene is being considered as an accurate predictor for PZA susceptibility, but this approach needs expensive sequencers and a mutation database to report the results. An in-vitro synthesized Pyrazinamidase (PZase) assay was developed based on PCR amplification of pncA gene and an in vitro wheat germ system to express the pncA gene into PZase. The activity of the synthesized PZase was used as an indicator for PZA susceptibility. Fifty-one clinical isolates were tested along with pncA sequencing and the BACTEC MGIT 960 methods. The in-vitro synthesized PZase assay was able to detect PZA susceptibility of M. tuberculosis within 24 h through observing the color difference either by a spectrometer or naked eyes. This method showed agreements of 100% (33/33) and 88% (14/16) with the pncA sequencing method, and agreements of 96% (27/28) and 65% (15/23) with the BACTEC MGIT 960 method, for susceptible and resistant strains, respectively. The novel in-vitro synthesized PZase assay has significant advantages over current methods, such as its fast speed, simplicity, no need for expensive equipment, and the potentials of being a direct test, predicting resistance level and easy reading results by naked eyes. After confirmation by more clinical tests, this method may provide a radical change to the current PZA susceptibility assays.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual test in Kenya.

    PubMed

    Chang, Joy; Omuomo, Kenneth; Anyango, Emily; Kingwara, Leonard; Basiye, Frank; Morwabe, Alex; Shanmugam, Vedapuri; Nguyen, Shon; Sabatier, Jennifer; Zeh, Clement; Ellenberger, Dennis

    2014-08-01

    Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0-100.0) and 100.0% (95% CI: 96.0-100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0-100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9copies/mL at 95% CIs (p=0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants.

  11. Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer.

    PubMed

    El Hadi, Hicham; Abdellaoui-Maane, Imane; Kottwitz, Denise; El Amrani, Manal; Bouchoutrouch, Nadia; Qmichou, Zineb; Karkouri, Mehdi; ElAttar, Hicham; Errihani, Hassan; Fernandez, Pedro L; Bakri, Youssef; Sefrioui, Hassan; Moumen, Abdeladim

    2017-03-20

    Accurate measurement of Human epidermal growth factor receptor (HER2) gene expression is central for breast or stomach cancer therapy orientation and prognosis. The current standards testing methods for HER2 expression are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the current study, we explored the use of quantitative real time reverse transcription-PCR (RT-qPCR) as a potential method for the accurate relative quantification of the HER2 gene using formalin fixed paraffin embedded (FFPE) breast cancer biopsy samples. The main aim of the current study is to measure the level of concordance of RT-qPCR based quantification of HER2 overexpression with both IHC and FISH. Accordingly, an endogenous control gene (ECG) is required for this relative quantification and should ideally be expressed equivalently across tested samples. Stably expressed ECGs have been selected from a panel of seven genes using GenEx V6 software which is based on geNorm and NormFinder and statistical methods. Quantification of HER2 gene expression was performed by our RT-qPCR-based test and compared to the results obtained by both IHC and FISH methods. HER2 gene quantification using RT-qPCR test was normalized using the two ECGs (RPL30 and RPL37A) that were successfully identified and selected from a panel of seven genes as the most stable and reliable ECGs. We evaluated a total of 216 FFPE tissue samples from breast cancer patients. The results obtained with RT-qPCR in the current study were compared to both IHC and FISH data collected for the same patients. In addition to an internal evaluation, an external evaluation of this assay was also performed in a recognized pathology center in Europe (Clinic Barcelona Hospital Universitari, Spain) using 116 FFPE breast cancer tissue samples. The results demonstrated a high concordance between RT-qPCR and either IHC (98%) or FISH (72%) methods. Accordantly, the overall concordance was 85%. To our knowledge, this is the

  12. Wet-lab tested microRNA assays for qPCR studies with SYBR® Green and DNA primers in pig tissues.

    PubMed

    Mentzel, Caroline M J; Skovgaard, Kerstin; Córdoba, Sarai; Herrera Uribe, Juber; Busk, Peter K; Cirera, Susanna

    2014-01-01

    MicroRNAs are key post-transcriptional regulators of gene expression that are involved in several biological processes including those that mediate disease pathophysiology. Hence, quantifying microRNA expression levels can provide important and novel insights into disease biology. In recent years, the pig has emerged as an excellent large animal model for studying human diseases and conditions (e.g. obesity) due to similarities in organ size, gastro-intestinal tract, metabolism, immune response, genetics and the availability of relevant tissues that are not normally easily available in humans. We have previously developed two useful tools in the field of microRNA quantitative real time PCR (qPCR): 1) a very specific, sensitive and simple qPCR method based on DNA primers, MiR-specific qPCR; and 2) the free primer-design software miRprimer. The present study integrates in a publicly accessible database all available information on validated porcine microRNA qPCR assays that have utilized these tools. Due to the high phylogenetic conservation in microRNA sequence between pig, humans and other domestic species this database is a very valuable resource for the broader scientist community who are working on microRNAs and want to use readily tested qPCR assays in a simple and cost-effective manner.

  13. 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

  14. How to Make Feedback More Effective? Qualitative Findings from Pilot Testing of an Audit and Feedback Report for Endoscopists

    PubMed Central

    Webster, Fiona; Patel, Jigisha; Rice, Kathleen; Baxter, Nancy; Paszat, Lawrence; Rabeneck, Linda

    2016-01-01

    Background. Audit and feedback (A/F) reports are one of the few knowledge translation activities that can effect change in physician behavior. In this study, we pilot-tested an endoscopist A/F report to elicit opinions about the proposed report's usability, acceptability and usefulness, and implications for knowledge translation. Methods. Semi-structured qualitative interviews were conducted with eleven endoscopists in Ontario, Canada. We tested an A/F report template comprising 9 validated, accepted colonoscopy quality indicators populated with simulated data. Interview transcripts were coded using techniques such as constant comparison and themes were identified inductively over several team meetings. Results. Four interrelated themes were identified: (1) overall perceptions of the A/F report; (2) accountability and consequences for poor performance; (3) motivation to change/improve skills; and (4) training for performance enhancement and available resources. The A/F report was well received; however, participants cited some possible threats to the report's effectiveness including the perceived threat of loss of privileges or licensing and the potential for the data to be dismissed. Conclusions. Participants agreed that A/F has the potential to improve colonoscopy performance. However, in order to be effective in changing physician behavior, A/F must be thoughtfully implemented with attention to the potential concerns of its recipients. PMID:27722149

  15. 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

  16. 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

    2015-11-26

    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.

  17. 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

  18. 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

  19. 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...

  20. Evaluation of commercial universal rRNA gene PCR plus sequencing tests for identification of bacteria and fungi associated with infectious endocarditis.

    PubMed

    Kühn, Christian; Disqué, Claudia; Mühl, Helge; Orszag, Peter; Stiesch, Meike; Haverich, Axel

    2011-08-01

    Two new commercially available universal rRNA gene PCR plus sequencing tests, SepsiTest and universal microbe detection (UMD; Molzym, Bremen, Germany), were evaluated using blood specimens and heart valves from 30 patients with suspected infectious endocarditis (IE). The sensitivity of PCR (85%) was nearly twice as high as that of culture (45%), which in 10/20 IE cases presumably stayed negative as a consequence of growth inhibition of the pathogens by antibiotics. Further, PCR provided the basis for reclassification of 5/10 non-IE cases into IE cases. Culture-negative infections were identified by PCR, including single infections due to streptococci and Gram-negative bacteria (Escherichia coli, Haemophilus parainfluenzae) and mixed infections involving two Gram-positive bacteria or Candida spp. with Gram-positive bacteria. The new commercial tests proved to be of value for the rapid diagnosis of IE, particularly in cases of culture-negative infections. Issues regarding the feasibility of these tests for routine use are discussed.

  1. 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...

  2. [PCR-based analysis of the D1S80 locus and its application in paternity test].

    PubMed

    Que, T; Lin, Y; Li, L

    1998-01-01

    Allelic data for the D1S80 locus was obtained by using the PCR and subsequent analysis with high-resolution horizontal PAGE technique. In a sample of 184 unrelated Chinese, the D1S80 locus had 75 phenotypes and 20 allelic. The distribution of genotype was in agreement with expected values according to the Hardy-Weinberg equilibrium. Analysis of the D1S80 locus by the PCR has been successfully applied in 150 cases of questioned paternity.

  3. Performance of the COBAS AMPLICOR HCV MONITOR Test, Version 2.0, an Automated Reverse Transcription-PCR Quantitative System for Hepatitis C Virus Load Determination

    PubMed Central

    Gerken, G.; Rothaar, T.; Rumi, M. G.; Soffredini, R.; Trippler, M.; Blunk, M. J.; Butcher, A.; Soviero, S.; Colucci, G.

    2000-01-01

    A clinical evaluation of an automated quantitative PCR assay, the COBAS AMPLICOR HCV MONITOR test, version 2.0 (v2.0), was carried out to assess the performance of this test in comparison with that of the previous, manual version, the AMPLICOR HCV MONITOR test, and with that of nested PCR. Serial dilutions of serum samples infected with genotype 1b, 2a, or 3, as well as synthetic RNA transcripts and serum samples derived from 87 patients with chronic hepatitis C and infected with genotype 1a, 1b, 2a, 2b, 3a, 3b, 4, or 5, were analyzed to determine the ability of the system to efficiently quantify various hepatitis C virus (HCV) genotypes. These experiments showed that the COBAS AMPLICOR HCV MONITOR test, v2.0, has mean intra-assay, interassay, and interoperator coefficients of variation that range from 22 to 34.5% and a 3-logarithm dynamic range, which spans from 103 to 106 copies/ml. Compared to the previous, manual version of the test, the COBAS AMPLICOR HCV MONITOR test, v2.0, showed an improved efficacy for all genotypes, especially genotypes 2, 3, and 4, whose estimated concentrations were on average 1 logarithm higher. When used to monitor patients under treatment, however, both versions showed the same patterns of viremia, indicating that the COBAS AMPLICOR HCV MONITOR test, v2.0, and the AMPLICOR HCV MONITOR test were equally effective at detecting relative viremia changes in serial samples. As expected, the automated test was less sensitive than nested PCR; among specimens from a cohort of patients treated with interferon, nested PCR identified three more viremic specimens, which probably contained very low concentrations of HCV RNA. PMID:10834978

  4. 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

  5. Detection of multiple Mycoplasma species in bulk tank milk samples using real-time PCR and conventional culture and comparison of test sensitivities.

    PubMed

    Justice-Allen, A; Trujillo, J; Goodell, G; Wilson, D

    2011-07-01

    The objective of this study was to further validate a SYBR PCR protocol for Mycoplasma spp. by comparing it with standard microbial culture in the detection of Mycoplasma spp. in bulk tank milk samples. Additionally, we identified Mycoplasma spp. present by analysis of PCR-generated amplicons [dissociation (melt) temperature (T(m)), length, and DNA sequence]. The research presented herein tests the hypothesis that the SYBR PCR protocol is as sensitive as conventional culture for the detection of Mycoplasma spp. in bulk tank milk samples. Mycoplasmas cause several important disease syndromes in cattle, including mastitis in dairy cows. The standard diagnostic method at the herd level has been microbial isolation of mycoplasmas on 1 of several specialized media and speciation through biochemical or immunological techniques; repeated sampling schemes are recommended. The development of a real-time SYBR PCR protocol offers advantages in decrease of time to detection, cost, and complexity. The T(m) of the double-stranded DNA generated from the PCR reaction was used to detect the presence of and tentatively identify the species of mycoplasmas other than Mycoplasma bovis. In the SYBR PCR protocol, the presence of multiple species of mycoplasmas is indicated by an atypical dissociation curve. Gel electrophoresis and sequencing of the amplicons was used to confirm the mycoplasma species present when a non-M. bovis organism was detected (T(m) not equal to M. bovis) and used to identify all the mycoplasma species present for the samples with atypical dissociation curves. Mycoplasma bovis was identified in 83% of SYBR PCR mycoplasma-positive bulk tank samples. Another mycoplasma was identified either alone or in addition to M. bovis in 25% of SYBR PCR mycoplasma-positive bulk tank milk samples. Four species of mycoplasma other than M. bovis (Mycoplasma alkalescens, Mycoplasma arginini, Mycoplasma bovigenitalium, and Mycoplasma gateae) were identified in bulk tank milk samples

  6. A novel poisson distribution-based approach for testing boundaries of real-time PCR assays for food pathogen quantification.

    PubMed

    Rossmanith, Peter; Wagner, Martin

    2011-09-01

    The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in food pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.

  7. Fetal Tissues Tested for Microbial Sterility by Culture- and PCR-Based Methods Can be Safely Used in Clinics.

    PubMed

    Vitrenko, Yakov; Kostenko, Iryna; Kulebyakina, Kateryna; Duda, Alla; Klunnyk, Mariya; Sorochynska, Khrystyna

    2017-02-16

    Cell preparations to be used in clinical practice must be free of infectious agents. Safety concerns are especially elevated upon the use of human fetal tissues, which are otherwise highly advantageous in cell therapy. We demonstrate that treating fetal samples with antibiotic, extensive washing, and homogenization prior to cryoconservation efficiently removes microbes in general. Screening a large collection by an automatic culture system showed that 89.2% fetal tissue samples were sterile, while contamination was detected in 10.8% samples. Liver and chorion were contaminated more than the brain, kidney, lung, and soft tissues. Broad-range PCR from the bacterial 16s rRNA gene was adopted as a confirmatory assay; however, the concordance between the culture-based and PCR assays was weak. Taxonomic identification was done for contaminated samples by bacteriological methods and sequencing 16s rRNA PCR products. The two approaches revealed different spectra of taxonomic groups sharing only Lactobacillus, the most frequently found genus. In addition, other representatives of vaginal microbiota were detected by culture-based identification, while PCR product sequencing has also revealed a subset of nosocomial microorganisms. Importantly, species known to cause sepsis were identified by both techniques, arguing for their indispensability and mutual complementarity. We suggest that most contaminations are taken up during collection of fetal material rather than originating from an in utero infection. In conclusion, a rigorous microbiological control by culture and PCR is a prerequisite for safe clinical use of fetal tissue suspensions.

  8. Restriction-Site-Specific PCR as a Rapid Test To Detect Enterohemorrhagic Escherichia coli O157:H7 Strains in Environmental Samples

    PubMed Central

    Kimura, Richard; Mandrell, Robert E.; Galland, John C.; Hyatt, Doreene; Riley, Lee W.

    2000-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield “fingerprint” patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples. PMID:10831431

  9. 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.

  10. Assessment of the Usefulness of Multiplex Real-Time PCR Tests in the Diagnostic and Therapeutic Process of Pneumonia in Hospitalized Children: A Single-Center Experience

    PubMed Central

    Bartkowska-Śniatkowska, Alicja; Jończyk-Potoczna, Katarzyna; Wysocka-Leszczyńska, Joanna; Bobkowski, Waldemar; Fichna, Piotr; Sobkowiak, Paulina; Mazur-Melewska, Katarzyna; Bręborowicz, Anna; Wysocki, Jacek; Januszkiewicz-Lewandowska, Danuta

    2017-01-01

    The aim of the study was assessment of the usefulness of multiplex real-time PCR tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. Methods. The study group included 97 children hospitalized due to pneumonia at the Karol Jonscher Teaching Hospital in Poznań, in whom multiplex real-time PCR tests (FTD respiratory pathogens 33; fast-track diagnostics) were used. Results. Positive test results of the test were achieved in 74 patients (76.3%). The average age in the group was 56 months. Viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). The presence of comorbidities was established in 90 children (92.78%). On the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. Conclusions. Our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. Multiplex real-time PCR tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations. PMID:28182108

  11. Assessment of the Usefulness of Multiplex Real-Time PCR Tests in the Diagnostic and Therapeutic Process of Pneumonia in Hospitalized Children: A Single-Center Experience.

    PubMed

    Gowin, Ewelina; Bartkowska-Śniatkowska, Alicja; Jończyk-Potoczna, Katarzyna; Wysocka-Leszczyńska, Joanna; Bobkowski, Waldemar; Fichna, Piotr; Sobkowiak, Paulina; Mazur-Melewska, Katarzyna; Bręborowicz, Anna; Wysocki, Jacek; Januszkiewicz-Lewandowska, Danuta

    2017-01-01

    The aim of the study was assessment of the usefulness of multiplex real-time PCR tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. Methods. The study group included 97 children hospitalized due to pneumonia at the Karol Jonscher Teaching Hospital in Poznań, in whom multiplex real-time PCR tests (FTD respiratory pathogens 33; fast-track diagnostics) were used. Results. Positive test results of the test were achieved in 74 patients (76.3%). The average age in the group was 56 months. Viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). The presence of comorbidities was established in 90 children (92.78%). On the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. Conclusions. Our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. Multiplex real-time PCR tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations.

  12. Qualitative research.

    PubMed

    Gelling, Leslie

    2015-03-25

    Qualitative research has an important role in helping nurses and other healthcare professionals understand patient experiences of health and illness. Qualitative researchers have a large number of methodological options and therefore should take care in planning and conducting their research. This article offers a brief overview of some of the key issues qualitative researchers should consider.

  13. Comparison of the Eragen Multi-Code Respiratory Virus Panel with Conventional Viral Testing and Real-Time Multiplex PCR Assays for Detection of Respiratory Viruses▿ †

    PubMed Central

    Arens, Max Q.; Buller, Richard S.; Rankin, Anne; Mason, Sheila; Whetsell, Amy; Agapov, Eugene; Lee, Wai-Ming; Storch, Gregory A.

    2010-01-01

    High-throughput multiplex assays for respiratory viruses are an important step forward in diagnostic virology. We compared one such assay, the PLx Multi-Code Respiratory Virus Panel (PLx-RVP), manufactured by Eragen Biosciences, Inc. (Madison, WI), with conventional virologic testing, consisting of fluorescent-antibody staining plus testing with the R-mix system and fibroblast tube cultures. The test set consisted of 410 archived respiratory specimens, mostly nasopharyngeal swabs, including 210 that had been positive by conventional testing for a balanced selection of common respiratory viruses. Specimens yielding discrepant results were evaluated using a panel of respiratory virus PCR assays developed, characterized, and validated with clinical specimens. PLx-RVP increased the total rate of detection of viruses by 35.8%, and there was a 25.7% increase in the rate of detection of positive specimens. Reference PCR assay results corroborated the PLx-RVP result for 54 (82%) of 66 discrepancies with conventional testing. Of the 12 specimens with discrepancies between PLx-RVp and the reference PCRs, 6 were positive for rhinovirus by PLx-RVP and the presence of rhinovirus was confirmed by nucleotide sequencing. The remaining six specimens included five in which the PLx-RVP failed to detect parainfluenza virus and one in which the detection of influenza A virus by PLx-RVP could not be confirmed by the reference PCR. Taking the results of the reference PCR assay results into account, the sensitivities of the PLx-RVP for individual viruses ranged from 94 to 100% and the specificities ranged from 99 to 100%. We conclude that PLx-RVP is a highly accurate system for the detection of respiratory viruses and significantly improves the rate of detection of these viruses compared to that by conventional virologic testing. PMID:20484608

  14. 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.

  15. 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

    ...) Electrophoresis. Mix PCR products (5 to 10 μl) with 2 μl loading buffer (Sigma) and electrophorese on a 2 percent agarose gel containing 0.5 μg/mL ethidium bromide in TAE buffer (40 mM tris; 2 mM EDTA; pH 8.0...

  16. 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

    ...) Electrophoresis. Mix PCR products (5 to 10 μl) with 2 μl loading buffer (Sigma) and electrophorese on a 2 percent agarose gel containing 0.5 μg/mL ethidium bromide in TAE buffer (40 mM tris; 2 mM EDTA; pH 8.0...

  17. 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

    ...) Electrophoresis. Mix PCR products (5 to 10 μl) with 2 μl loading buffer (Sigma) and electrophorese on a 2 percent agarose gel containing 0.5 μg/mL ethidium bromide in TAE buffer (40 mM tris; 2 mM EDTA; pH 8.0...

  18. 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

    ...) Electrophoresis. Mix PCR products (5 to 10 μl) with 2 μl loading buffer (Sigma) and electrophorese on a 2 percent agarose gel containing 0.5 μg/mL ethidium bromide in TAE buffer (40 mM tris; 2 mM EDTA; pH 8.0...

  19. 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.

  20. Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR.

    PubMed

    Wei, Libin; Miao, Hongmei; Zhao, Ruihong; Han, Xiuhua; Zhang, Tide; Zhang, Haiyang

    2013-03-01

    Sesame (Sesamum indicum L.) is an ancient and important oilseed crop. However, few sesame reference genes have been selected for quantitative real-time PCR until now. Screening and validating reference genes is a requisite for gene expression normalization in sesame functional genomics research. In this study, ten candidate reference genes, i.e., SiACT, SiUBQ6, SiTUB, Si18S rRNA, SiEF1α, SiCYP, SiHistone, SiDNAJ, SiAPT and SiGAPDH, were chosen and examined systematically in 32 sesame samples. Three qRT-PCR analysis methods, i.e., geNorm, NormFinder and BestKeeper, were evaluated systematically. Results indicated that all ten candidate reference genes could be used as reference genes in sesame. SiUBQ6 and SiAPT were the optimal reference genes for sesame plant development; SiTUB was suitable for sesame vegetative tissue development, SiDNAJ for pathogen treatment, SiHistone for abiotic stress, SiUBQ6 for bud development and SiACT for seed germination. As for hormone treatment and seed development, SiHistone, SiCYP, SiDNAJ or SiUBQ6, as well as SiACT, SiDNAJ, SiTUB or SiAPT, could be used as reference gene, respectively. To illustrate the suitability of these reference genes, we analyzed the expression variation of three functional sesame genes of SiSS, SiLEA and SiGH in different organs using the optimal qRT-PCR system for the first time. The stability levels of optimal and worst reference genes screened for seed development, anther sterility and plant development were validated in the qRT-PCR normalization. Our results provided a reference gene application guideline for sesame gene expression characterization using qRT-PCR system.

  1. Qualitative modeling.

    PubMed

    Forbus, Kenneth D

    2011-07-01

    Qualitative modeling concerns the representations and reasoning that people use to understand continuous aspects of the world. Qualitative models formalize everyday notions of causality and provide accounts of how to ground symbolic, relational representations in perceptual processes. This article surveys the basic ideas of qualitative modeling and their applications from a cognitive science perspective. It describes the basic principles of qualitative modeling, and a variety of qualitative representations that have been developed for quantities and for relationships between them, providing a kind of qualitative mathematics. Three ontological frameworks for organizing modeling knowledge (processes, components, and field) are summarized, along with research on automatically assembling models for particular tasks from such knowledge. Qualitative simulation and how it carves up time into meaningful units is discussed. We discuss several accounts of causal reasoning about dynamical systems, based on different choices of qualitative mathematics and ontology. Qualitative spatial reasoning is explored, both in terms of relational systems and visual reasoning. Applications of qualitative models of particular interest to cognitive scientists are described, including how they have been used to capture the expertise of scientists and engineers and how they have been used in education. Open questions and frontiers are also discussed, focusing on relationships between ideas developed in the qualitative modeling community and other areas of cognitive science. WIREs Cogni Sci 2011 2 374-391 DOI: 10.1002/wcs.115 For further resources related to this article, please visit the WIREs website.

  2. Detection of malaria infection in blood transfusion: a comparative study among real-time PCR, rapid diagnostic test and microscopy: sensitivity of Malaria detection methods in blood transfusion.

    PubMed

    Hassanpour, Gholamreza; Mohebali, Mehdi; Raeisi, Ahmad; Abolghasemi, Hassan; Zeraati, Hojjat; Alipour, Mohsen; Azizi, Ebrahim; Keshavarz, Hossein

    2011-06-01

    The transmission of malaria by blood transfusion was one of the first transfusion-transmitted infections recorded in the world. Transfusion-transmitted malaria may lead to serious problems because infection with Plasmodium falciparum may cause rapidly fatal death. This study aimed to compare real-time polymerase chain reaction (real-time PCR) with rapid diagnostic test (RDT) and light microscopy for the detection of Plasmodium spp. in blood transfusion, both in endemic and non-endemic areas of malaria disease in Iran. Two sets of 50 blood samples were randomly collected. One set was taken from blood samples donated in blood bank of Bandar Abbas, a city located in a malarious-endemic area, and the other set from Tehran, a non-endemic one. Light microscopic examination on both thin and thick smears, RDTs, and real-time PCR were performed on the blood samples and the results were compared. Thin and thick light microscopic examinations of all samples as well as RDT results were negative for Plasmodium spp. Two blood samples from endemic area were positive only with real-time PCR. It seems that real-time PCR as a highly sensitive method can be helpful for the confirmation of malaria infection in different units of blood transfusion organization especially in malaria-endemic areas where the majority of donors may be potentially infected with malaria parasites.

  3. Detection of Mixed-Species Infections of Plasmodium falciparum and Plasmodium vivax by Nested PCR and Rapid Diagnostic Tests in Southeastern Iran

    PubMed Central

    Ehtesham, Reyhaneh; Fazaeli, Asghar; Raeisi, Ahmad; Keshavarz, Hossein; Heidari, Aliehsan

    2015-01-01

    Coexistence of two species of Plasmodium in a single host has disrupted the diagnosis and treatment of malaria. This study was designed to evaluate the ability of rapid diagnostic test (RDT) kits for the diagnosis of mixed-species malaria infections in southeastern Iran. A total of 100 malaria patients were included in the study out of 164 randomly suspected symptomatic malaria patients from May to November 2012. Nested polymerase chain reaction (PCR) was also used to judge the ability of microscopy versus RDT kits for detecting mixed species. The sensitivity of light microscopy for the detection of mixed-species malaria infections was 16.6% (95% confidence interval [CI] = 3–49.1). Nested PCR revealed 12 patients with mixed-species infection. The CareStart Pv/Pf Combo kit detected 58% of the mixed-species infections, which were determined by nested PCR (sensitivity = 58.3%; 95% CI = 28.5–83.5). For identifying P. falciparum, P. vivax, and mixed-species infections, the concordance rates (kappa statistics) of microscopy and CareStart Pv/Pf Combo kit with nested PCR were 0.76 and 0.79, respectively (P = 0.001). This study underlines the effectiveness of RDT kits to improve the differentiation of mixed-species malaria infections in endemic areas where the prevalence of chloroquine resistance is high. PMID:25962771

  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

    ... and re-suspend the pellet in 25-50 μl of 0.1 percent DEP (Diethyl Pyrocarbonate; Sigma) water. Boil at... μl taq-polymerase (5 U), 32 μl DEP water. (ii) 18 μl water, 25 μl PCR mix (Promega), 1 μl Reverse... Agriculture or an endorsement over other products not mentioned. Temperature ( °C) Duration Cycles 94...

  5. Development and Field Testing of a Real-Time PCR Assay for Caprine Arthritis-Encephalitis-Virus (CAEV)

    PubMed Central

    Brajon, Giovanni; Mandas, Daniela; Liciardi, Manuele; Taccori, Flavia; Meloni, Mauro; Corrias, Franco; Montaldo, Caterina; Coghe, Ferdinando; Casciari, Cristina; Giammarioli, Monica; Orrù, Germano

    2011-01-01

    Caprine arthritis/encephalitis (CAE) of goats and occasionally sheep are persistent virus infections caused by a lentivirus (CAEV). This viral infection results in arthritis in adult animals and encephalitis in kids. Prognosis for the encephalitic form is normally poor, with substantial economic loss for the farm. In this context an early/fast laboratory diagnosis for CAEV infection could be useful for effective prophylactic action. In this work we performed a quantitative real time PCR designed on the CAEV env gene to detect/quantify in goat/sheep samples, viral RNA or proviral DNA forms of CAEV. This procedure was validated in 15 sheep, experimentally infected with CAEV or with a highly correlated lentivirus (visna maedi, MVV); in addition, a total of 37 clinical goat specimens recruited in CAEV positive herds were analyzed and compared using serological analysis (Elisa and AGID). All samples infected with MVV resulted negative. In sheep experimentally infected with CAEV, proviral DNA was detectable 15 days post infection, whereas the serological methods revealed an indicative positivity after 40-60 days.This method showed a sensitivity of 102 env fragments/PCR) with a linear dynamic range of quantitation from 103 to 107 env fragments/PCR; the R2 correlation coefficient was 0.98. All subjects with a clinical diagnosis for Caprine Arthritis-Encephalitis (CAE) resulted CAEV DNA positive. PMID:22888382

  6. Impact of Routine Real-Time PCR Testing of Imported Malaria over 4 Years of Implementation in a Clinical Laboratory

    PubMed Central

    Shokoples, Sandra; Mukhi, Shamir N.; Scott, Allison N.

    2013-01-01

    In clinical laboratories, diagnosis of imported malaria is commonly performed by microscopy. However, the volume of specimens is generally low and maintaining proficiency in reading blood smears, particularly at the species level, is challenging in this setting. To address this problem, the Provincial Laboratory for Public Health (ProvLab) in Alberta, Canada, implemented real-time PCR for routine confirmation of all smear-positive samples in the province. Here we report our experience over a 4-year period (2008 to 2012) with this new diagnostic algorithm. While detection of Plasmodium falciparum by microscopy alone was accurate, real-time PCR served as an important adjunct to microscopy for the identification of non-falciparum species. In 18% of cases, the result was reported as non-falciparum or the species could not be identified by microscopy alone, and in all cases, the species was resolved by real-time PCR. In another 4% of cases, the species was misidentified by microscopy. To enhance surveillance for malaria, we integrated our demographic, clinical, and laboratory data into a new system developed by the Canadian Network for Public Health Intelligence, called the Malaria System for Online Surveillance (SOS). Using this application, we characterized our patient populations and travel history to identify risk factors associated with malaria infection abroad. PMID:23554200

  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. Testing the Viability of the READ Scale (Reference Effort Assessment Data)©: Qualitative Statistics for Academic Reference Services

    ERIC Educational Resources Information Center

    Gerlich, Bella Karr; Berard, G. Lynn

    2010-01-01

    The READ Scale (Reference Effort Assessment Data) is a six-point scale tool for recording qualitative statistics by placing an emphasis on recording effort, knowledge, skills, and teaching used by staff during a reference transaction. Institutional research grants enabled the authors to conduct a national study of the READ Scale at 14 diverse…

  9. Fully automated sample preparation microsystem for genetic testing of hereditary hearing loss using two-color multiplex allele-specific PCR.

    PubMed

    Zhuang, Bin; Gan, Wupeng; Wang, Shuaiqin; Han, Junping; Xiang, Guangxin; Li, Cai-Xia; Sun, Jing; Liu, Peng

    2015-01-20

    A fully automated microsystem consisting of a disposable DNA extraction and PCR microchip, as well as a compact control instrument, has been successfully developed for genetic testing of hereditary hearing loss from human whole blood. DNA extraction and PCR were integrated into a single 15-μL reaction chamber, where a piece of filter paper was embedded for capturing genomic DNA, followed by in-situ PCR amplification without elution. Diaphragm microvalves actuated by external solenoids together with a "one-way" fluidic control strategy operated by a modular valve positioner and a syringe pump were employed to control the fluids and to seal the chamber during thermal cycling. Fully automated DNA extractions from as low as 0.3-μL human whole blood followed by amplifications of 59-bp β-actin fragments can be completed on the microsystem in about 100 min. Negative control tests that were performed between blood sample analyses proved the successful elimination of any contamination or carryover in the system. To more critically test the microsystem, a two-color multiplex allele-specific PCR (ASPCR) assay for detecting c.176_191del16, c.235delC, and c.299_300delAT mutations in GJB2 gene that accounts for hereditary hearing loss was constructed. Two allele-specific primers, one labeled with TAMRA for wild type and the other with FAM for mutation, were designed for each locus. DNA extraction from blood and ASPCR were performed on the microsystem, followed by an electrophoretic analysis on a portable microchip capillary electrophoresis system. Blood samples from a healthy donor and five persons with genetic mutations were all accurately analyzed with only two steps in less than 2 h.

  10. Empirical testing of 16S rRNA gene PCR primer pairs reveals variance in target specificity and efficacy not suggested by in silico analysis.

    PubMed

    Morales, Sergio E; Holben, William E

    2009-05-01

    Phylogenetic and "fingerprinting" analyses of the 16S rRNA genes of prokaryotes have been a mainstay of microbial ecology during the last two decades. However, many methods and results from studies that rely on the 16S rRNA gene for detection and quantification of specific microbial taxa have seemingly received only cursory or even no validation. To directly examine the efficacy and specificity of 16S rRNA gene-based primers for phylum-, class-, and operational taxonomic unit-specific target amplification in quantitative PCR, we created a collection of primers based solely on an extensive soil bacterial 16S rRNA gene clone library containing approximately 5,000 sequences from a single soil sample (i.e., a closed site-specific library was used to create PCR primers for use at this site). These primers were initially tested in silico prior to empirical testing by PCR amplification of known target sequences and of controls based on disparate phylogenetic groups. Although all primers were highly specific according to the in silico analysis, the empirical analyses clearly exhibited a high degree of nonspecificity for many of the phyla or classes, while other primers proved to be highly specific. These findings suggest that significant care must be taken when interpreting studies whose results were obtained with target specific primers that were not adequately validated, especially where population densities or dynamics have been inferred from the data. Further, we suggest that the reliability of quantification of specific target abundance using 16S rRNA-based quantitative PCR is case specific and must be determined through rigorous empirical testing rather than solely in silico.

  11. How well does physician selection of microbiologic tests identify Clostridium difficile and other pathogens in paediatric diarrhoea? Insights using multiplex PCR-based detection.

    PubMed

    Stockmann, C; Rogatcheva, M; Harrel, B; Vaughn, M; Crisp, R; Poritz, M; Thatcher, S; Korgenski, E K; Barney, T; Daly, J; Pavia, A T

    2015-02-01

    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 (GI) 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 three (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 Clostridium 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.

  12. RNA and DNA bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine PCR tests.

    PubMed

    Ninove, Laetitia; Nougairede, Antoine; Gazin, Celine; Thirion, Laurence; Delogu, Ilenia; Zandotti, Christine; Charrel, Remi N; De Lamballerie, Xavier

    2011-02-09

    Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.

  13. Comparison of visual motor development in Hong Kong and the USA assessed on the Qualitative Scoring System for the Modified Bender-Gestalt test.

    PubMed

    Chan, P W

    2001-02-01

    This study compared the visuomotor development of young children in Hong Kong and the USA assessed on the Qualitative Scoring System for the Modified Bender-Gestalt test. 744 children aged 4:6 to 8:5 years from 6 kindergartens and 6 primary schools in Hong Kong were administered the Modified Bender-Gestalt test. The Qualitative Scoring System was used to measure the children's visuomotor development. Their visuomotor scores were then compared with norms for children in the USA. Analysis indicated significant differences across all age groups of 4:6 to 8:5 years in 6-mo. units. Consistent with previous research, children in Hong Kong outperformed their western peers. Percentile scores and T scores for children in Hong Kong in each age group were reported.

  14. 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.

  15. Sequence-modified primers for the differential RT-PCR detection of Andean potato latent and Andean potato mild mosaic viruses in quarantine tests.

    PubMed

    Koenig, Renate; Ziebell, Heiko

    2014-05-01

    To enable the differential PCR detection of Andean potato latent virus (APLV) and Andean potato mild mosaic virus (APMMV) strains, sense primers were designed that correspond to regions directly upstream of the coat protein genes. Their differentiating power was increased by A->C or T->C replacements in their 3'-terminal parts. Together with the broad-specificity antisense primer EM3, primer AL-a-mod3C detected all APLV strains tested, but none of the APMMV strains. Primer AM-a-mod4C yielded PCR products with all APMMV preparations, but also with some APLV preparations. Sequence analysis revealed that this was not due to a lack of primer specificity, but to the sensitive detection of contaminating APMMV in some of our APLV preparations.

  16. 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.

  17. Evaluation of PCR testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification.

    PubMed

    Krafft, A E; Russell, K L; Hawksworth, A W; McCall, S; Irvine, M; Daum, L T; Connoly, J L; Reid, A H; Gaydos, J C; Taubenberger, J K

    2005-04-01

    Viral culture isolation has been widely accepted as the "gold standard" for laboratory confirmation of viral infection; however, it requires ultralow temperature specimen storage. Storage of specimens in ethanol at room temperature could expand our ability to conduct active surveillance and retrospective screenings of viruses with rapid and inexpensive real-time PCR tests, including isolates from remote regions where freezing specimens for culture is not feasible. Molecular methods allow for rapid identification of viral pathogens without the need to maintain viability. We hypothesized that ethanol, while inactivating viruses, can preserve DNA and RNA for PCR-based methods. To evaluate the use of ethanol-stored specimens for augmenting surveillance for detection of influenza viruses A and B and adenoviruses (AdV), paired nasal swab specimens were collected from 384 recruits with febrile respiratory illness at Fort Jackson, S.C., in a 2-year study. One swab was stored at ambient temperature in 100% ethanol for up to 6 months, and the other swab was stored at -70 degrees C in viral medium. For viral detection, frozen specimens were cultured for a variety of respiratory viruses, and ethanol-fixed specimens were tested with TaqMan (TM) probe and LightCycler SYBR green (SG) melting curve assays with at least two different PCR targets for each virus. The sensitivities of the TM and SG assays on specimens stored in ethanol for 1 month were 75% and 58% for influenza A, 89% and 67% for influenza B, and 93 to 98% and 57% for AdV, respectively. Lower specificities of the real-time assays corresponded to the increased detection of PCR-positive but culture-negative specimens. Influenza virus RNA was detected as well or better after 6 months of storage in ethanol.

  18. Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201.

    PubMed

    Peng, Linda X; Wallace, Morgan; Andaloro, Bridget; Fallon, Dawn; Fleck, Lois; Delduco, Dan; Tice, George

    2011-01-01

    The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.

  19. Development and evaluation of a test for tuberculosis in live European badgers (Meles meles) based on measurement of gamma interferon mRNA by real-time PCR.

    PubMed

    Sawyer, J; Mealing, D; Dalley, D; Davé, D; Lesellier, S; Palmer, S; Bowen-Davies, J; Crawshaw, T R; Chambers, M A

    2007-08-01

    A real-time PCR assay for the measurement of gamma interferon (IFN-gamma) mRNA in European badger (Meles meles) blood cultures was developed. The levels of IFN-gamma mRNA in blood cultures stimulated with either bovine or avian tuberculin or specific mycobacterial antigens were compared with those in a nonstimulated control blood culture as the basis for determining the tuberculosis (TB) status of live badgers. The assay was validated by testing 247 animals for which there were matching data from postmortem examination and culture of tissues. Relative changes in the levels of IFN-gamma mRNA in response to bovine tuberculin and specific antigens were found to be greater among badgers with tissues positive for TB on culture. The test was at its most accurate (87% of test results were correct) by using blood cultures containing bovine tuberculin as the antigen and when the response to avian tuberculin was taken into account by subtracting the avian tuberculin response from the bovine tuberculin response. At a specificity of 90.7%, the test was 70.6% sensitive. At the same specificity, the current serological enzyme-linked immunosorbent assay for TB in badgers was only 53% sensitive. This work demonstrates that measurement of IFN-gamma mRNA by real-time PCR is a valid method for the detection of TB in live badgers and may provide an alternative to the current serological methods of diagnosis, the Brock test. The testing procedure can be completed within 5 h of receipt of the blood culture samples. In addition, the use of a molecular biology-based test offers the potential to fully automate the testing procedure through the use of robotics.

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

    PubMed

    Paes, Eliana Ferreira; de Assis, Angela Maria; Teixeira, Cirbia S Campos; Aoki, Francisco Hideo; Teixeira, Julio Cesar

    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, negative 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.

  1. Assessment accommodations on tests of academic achievement for students who are deaf or hard of hearing: a qualitative meta-analysis of the research literature.

    PubMed

    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 accommodations. The meta-analysis found that the results of analyses of group effects of accommodated versus unaccommodated test formats are often not significant, test-level factors exist that can affect how students perceive the assessments, and differences exist in how test items function across different conditions. Student-level factors, including educational context and academic proficiency, influence accommodations' role in assessment processes. The results of this analysis highlight the complexity of and intersections between student-level factors, test-level factors, and larger policy contexts. Findings are discussed within the context of larger changes in academic assessment, including computer-based administration and high-stakes testing.

  2. Variation in MRSA identification results from different generations of Xpert MRSA real-time PCR testing kits from nasal swabs.

    PubMed

    Rabaan, Ali A; Bazzi, Ali M

    2017-02-06

    GeneXpert MRSA kits (Cepheid) are based on a multiplex, real-time PCR method for methicillin-resistant Staphylococcus aureus (MRSA) detection, with primers to detect each SCCmec type and the chromosomal orfX-SCCmec junction. Modifications in recent kit versions were proposed to help overcome false-positive issues in earlier kit versions. The main objective of this study was to determine whether use of any version of the GeneXpert MRSA multiplex, real-time PCR kits yielded higher than expected MRSA+ results. We also estimated the level of MRSA in our healthcare facility as a proportion of total S. aureus between 2010 and 2015. We examined results from five generations of the kits used between 2008 and 2015. Results were from nasal swab samples from 16,431 patients in the Johns Hopkins Aramco Healthcare facility in Saudi Arabia. The percentage of isolates scored as MRSA+ for the original Xpert MRSA kit was 18.57%, compared to 6.93±1.12% (mean±SD) for the other four kits. The Xpert MRSA-SA Nasal kit yielded 6.48% Invalid results, compared to 0.73±0.28% for the other four kits. The succeeding Xpert MRSA-SA Nasal G3 and Xpert MRSA-SA Nasal Complete G3 kits yielded Invalid results rates of 0.29% and 1.04% respectively. Levels of MRSA-positive isolates as a percentage of total S. aureus-containing samples ranged between 19.81% and 26.74%. In conclusion, the original Xpert MRSA kit yielded higher than expected rates of MRSA+. Issues with over-estimation of MRSA+ and/or numerous Invalid results have been overcome in the most recent modified kits.

  3. 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.

  4. A qualitative study of the complete set of solutions of the differential equation of motion of a test particle in the equatorial plane of the Kerr gravitational field

    NASA Technical Reports Server (NTRS)

    Montgomery, H. E.; Chan, F. K.

    1973-01-01

    A study is made of the mathematical solution of the differential equation of motion of a test particle in the equatorial plane of the Kerr gravitational field, using S (Schwarzschild-like) coordinates. A qualitative solution of this equation leads to the conclusion that there can only be 25 different types of orbits. For each value of a, the results are presented in a master diagram for which h and e are the parameters. A master diagram divides the h, e parameter space into regions such that at each point within one of these regions the types of admissible orbits are qualitatively the same. A pictorial representation of the physical orbits in the r, phi plane is also given.

  5. 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.

  6. 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

  7. Zika Virus Testing Considerations: Lessons Learned from the First 80 Real-Time Reverse Transcription-PCR-Positive Cases Diagnosed in New York State.

    PubMed

    St George, Kirsten; Sohi, Inderbir S; Dufort, Elizabeth M; Dean, Amy B; White, Jennifer L; Limberger, Ronald; Sommer, Jamie N; Ostrowski, Stephanie; Wong, Susan J; Backenson, P Bryon; Kuhles, Daniel; Blog, Debra; Taylor, Jill; Hutton, Brad; Zucker, Howard A

    2017-02-01

    The performance and interpretation of laboratory tests for Zika virus (ZKV) continue to be evaluated. Serology is cross-reactive, laborious, and frequently difficult to interpret, and serum was initially solely recommended for molecular diagnosis. ZKV testing was initiated in January 2016 in New York State for symptomatic patients, pregnant women, their infants, and patients with Guillain-Barré syndrome who had traveled to areas with ZKV transmission. Subsequently, eligibility was expanded to pregnant women with sexual partners with similar travel histories. Serum and urine collected within 4 weeks of symptom onset or within 6 weeks of travel were tested with real-time reverse transcription-PCR (RT-PCR) assays targeting the ZKV envelope and NS2B genes. In this review of lessons learned from the first 80 positive cases in NYS, ZKV RNA was detected in urine only in 50 patients, in serum only in 19 patients, and in both samples concurrently in 11 patients, with average viral loads in urine a log higher than those in serum. Among 93 positive samples from the 80 patients, 41 were positive on both gene assays, 52 were positive on the envelope only, and none were positive on the NS2B only. Of the 80 infected patients, test results for 74 (93%) would have defined their infection status as not detected or equivocal if the requirement for positive results from two assay targets (two-target-positive requirement) in the initial federal guidance to public health laboratories was enforced, if urine was not tested, or if the extended eligibility time for molecular testing was not implemented. These changes facilitated more extensive molecular diagnosis of ZKV, reducing reliance on time-consuming and potentially inconclusive serology.

  8. 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

  9. Optimizing Infant HIV Diagnosis in Resource-Limited Settings: Modeling the Impact of HIV DNA PCR Testing at Birth

    PubMed Central

    Chiu, Alexander; Modi, Surbhi; Rivadeneira, Emilia D.; Koumans, Emilia H.

    2017-01-01

    Background Early antiretroviral therapy (ART) initiation in HIV-infected infants significantly improves survival but is often delayed in resource-limited settings. Adding HIV testing of infants at birth to the current recommendation of testing at age 4–6 weeks may improve testing rates and decrease time to ART initiation. We modeled the benefit of adding HIV testing at birth to the current 6-week testing algorithm. Methods Microsoft Excel was used to create a decision-tree model of the care continuum for the estimated 1,400,000 HIV-infected women and their infants in sub-Saharan Africa in 2012. The model assumed average published rates for facility births (42.9%), prevention of mother-to-child HIV transmission utilization (63%), mother-to-child-transmission rates based on prevention of mother-to-child HIV transmission regimen (5%–40%), return of test results (41%), enrollment in HIV care (52%), and ART initiation (54%). We conducted sensitivity analyses to model the impact of key variables and applied the model to specific country examples. Results Adding HIV testing at birth would increase the number of infants on ART by 204% by age 18 months. The greatest increase is seen in early ART initiations (543% by age 3 months). The increase would lead to a corresponding increase in survival at 12 months of age, with 5108 fewer infant deaths (44,550, versus 49,658). Conclusion Adding HIV testing at birth has the potential to improve the number and timing of ART initiation of HIV-infected infants, leading to a decrease in infant mortality. Using this model, countries should investigate a combination of HIV testing at birth and during the early infant period. PMID:27792684

  10. The Qualitative Scoring MMSE Pentagon Test (QSPT): A New Method for Differentiating Dementia with Lewy Body from Alzheimer’s Disease

    PubMed Central

    Caffarra, Paolo; Gardini, Simona; Dieci, Francesca; Copelli, Sandra; Maset, Laura; Concari, Letizia; Farina, Elisabetta; Grossi, Enzo

    2013-01-01

    The differential diagnosis across different variants of degenerative diseases is sometimes controversial. This study aimed to validate a qualitative scoring method for the pentagons copy test (QSPT) of Mini-Mental State Examination (MMSE) based on the assessment of different parameters of the pentagons drawing, such as number of angles, distance/intersection, closure/opening, rotation, closing-in, and to verify its efficacy to differentiate dementia with Lewy Body (DLB) from Alzheimer's disease (AD). We established the reliability of the qualitative scoring method through the inter-raters and intra-subjects analysis. QSPT was then applied to forty-six AD and forty-six DLB patients, using two phases statistical approach, standard and artificial neural network respectively. DLB patients had significant lower total score in the copy of pentagons and number of angles, distance/intersection, closure/opening, rotation compared to AD. However the logistic regression did not allow to establish any suitable modeling, whereas using Auto-Contractive Map (Auto-CM) the DLB was more strongly associated with low scores in some qualitative parameters of pentagon copying, i.e. number of angles and opening/closure and, for the remaining subitems of the MMSE, in naming, repetition and written comprehension, and for demographic variables of gender (male) and education (6–13 years). Twist system modeling showed that the QSPT had a good sensitivity (70.29%) and specificity (78.67%) (ROC-AUC 0.74). The proposed qualitative method of assessment of pentagons copying used in combination with non-linear analysis, showed to be consistent and effective in the differential diagnosis between Lewy Body and Alzheimer’s dementia. PMID:23396218

  11. 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

  12. 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

  13. Performance of the Cepheid CT/NG Xpert Rapid PCR Test for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

    PubMed

    Gaydos, Charlotte A; Van Der Pol, Barbara; Jett-Goheen, Mary; Barnes, Mathilda; Quinn, Nicole; Clark, Carey; Daniel, Grace E; Dixon, Paula B; Hook, Edward W

    2013-06-01

    Tests for Chlamydia trachomatis and Neisseria gonorrhoeae, which can provide results rapidly to guide therapeutic decision-making, offer patient care advantages over laboratory-based tests that require several days to provide results. We compared results from the Cepheid GeneXpert CT/NG (Xpert) assay to results from two currently approved nucleic acid amplification assays in 1,722 female and 1,387 male volunteers. Results for chlamydia in females demonstrated sensitivities for endocervical, vaginal, and urine samples of 97.4%, 98.7%, and 97.6%, respectively, and for urine samples from males, a sensitivity of 97.5%, with all specificity estimates being ≥ 99.4%. Results for gonorrhea in females demonstrated sensitivities for endocervical, vaginal, and urine samples of 100.0%, 100.0%, and 95.6%, respectively, and for urine samples from males, a sensitivity of 98.0%, with all estimates of specificity being ≥ 99.8%. These results indicate that this short-turnaround-time test can be used to accurately test patients and to possibly do so at the site of care, thus potentially improving chlamydia and gonorrhea control efforts.

  14. The role of social support on HIV testing and treatment adherence: A qualitative study of HIV-infected refugees in southwestern Uganda.

    PubMed

    Rouhani, Shada A; O'Laughlin, Kelli N; Faustin, Zikama M; Tsai, Alexander C; Kasozi, Julius; Ware, Norma C

    2016-01-19

    Little is known about the factors that encourage or discourage refugees to test for HIV, or to access and adhere to HIV care. In non-refugee populations, social support has been shown to influence HIV testing and utilisation of services. The present study enrolled HIV-infected refugees on anti-retroviral therapy (ART) in Uganda, who participated in qualitative interviews on HIV testing, treatment, and adherence. Interviews were analysed for themes about four types of social support: emotional, informational, instrumental, and appraisal support. A total of 61 interviews were analysed. Four roles for these types of social support were identified: (1) informational support encouraged refugees to test for HIV; (2) emotional support helped refugees cope with a diagnosis of HIV; (3) instrumental support facilitated adherence to ART and (4) after diagnosis, HIV-infected refugees provided informational and emotional support to encourage other refugees to test for HIV. These results suggest that social support influences HIV testing and treatment among refugees. Future interventions should capitalise on social support within a refugee settlement to facilitate testing and treatment.

  15. Feasibility of implementing rapid oral fluid HIV testing in an urban University Dental Clinic: a qualitative study

    PubMed Central

    2012-01-01

    Background More than 1 million individuals in the U.S. are infected with HIV; approximately 20% of whom do not know they are infected. Early diagnosis of HIV infection results in earlier access to treatment and reductions in HIV transmission. In 2006, the CDC recommended that health care providers offer routine HIV screening to all adolescent and adult patients, regardless of community seroprevalence or patient lifestyle. Dental providers are uniquely positioned to implement these recommendations using rapid oral fluid HIV screening technology. However, thus far, uptake into dental practice has been very limited. Methods The study utilized a qualitative descriptive approach with convenience samples of dental faculty and students. Six in-depth one-on-one interviews were conducted with dental faculty and three focus groups were conducted with fifteen dental students. Results Results were fairly consistent and indicated relatively high levels of acceptability. Barriers and facilitators of oral fluid HIV screening were identified in four primary areas: scope of practice/practice enhancement, skills/knowledge/training, patient service/patient reactions and logistical issues. Conclusions Oral fluid HIV screening was described as having benefits for patients, dental practitioners and the public good. Many of the barriers to implementation that were identified in the study could be addressed through training and interdisciplinary collaborations. PMID:22571324

  16. A qualitative analysis of Emergency Department physicians' practices and perceptions in relation to test result follow-up.

    PubMed

    Callen, Joanne; Georgiou, Andrew; Prgomet, Mirela; Paoloni, Richard; Westbrook, Johanna

    2010-01-01

    Follow-up of abnormal test results for discharged Emergency Department (ED) patients is a critical safety issue. This study aimed to explore ED physicians' perceptions, practices, and suggestions for improvements of test result follow-up when using an electronic provider order entry system to order all laboratory and radiology tests and view results. Interviews were conducted with seven ED physicians and one clinical information system support person. Interviews were analyzed to elicit key concepts relating to physicians' perceptions of test result follow-up and how the process could be improved. Results described the current electronic test result follow-up system with two paper-based manual back-up systems for microbiology and radiology results. The key issues for physicians were: responsibility for test follow-up; the unique ED environment and time pressures, and the role of the family physician in test result follow-up. The key suggestion for improvement was a complete integrated electronic information system with on-line result endorsement. The study highlighted the complexity of the test result follow-up process and the importance of engaging clinicians in devising solutions for improvements.

  17. 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.

  18. 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.

  19. Pre-Clinical Testing of Real-Time PCR Assays for Diarrheal Disease Agents of Genera Escherichia and Shigella

    DTIC Science & Technology

    2014-05-16

    met or exceeded to qualify as a candidate for FDA clearance. In addition to test activities, Enterotoxigenic Escherichia coli Detection Kit and...Enterotoxigenic Escherichia coli Detection Kit and Shigella Detection Kit pre-IDE documents were prepared to serve as a point of departure for discussion...assays for enterotoxigenic Escherichia coli (ETEC) toxin genes, Heat Stable Ia (STIa), Heat Stable Ib (STIb), and Heat Labile (LT) and Shigella

  20. 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…

  1. Equal Performance of Self-Collected and Health Care Worker-Collected Pharyngeal Swabs for Group A Streptococcus Testing by PCR

    PubMed Central

    Murray, Martha A.; Schulz, Lorie A.; Furst, Joseph W.; Homme, Jason H.; Jenkins, Sarah M.; Uhl, James R.; Patel, Robin; Cockerill, Franklin C.; Myers, Jane F.

    2014-01-01

    A process employing patient- or parent-collected pharyngeal swabs for group A Streptococcus (GAS) testing would expedite diagnosis and treatment, reduce patient exposure to the health care setting, and decrease health care costs. Our aim was to determine the concordance between patient- or parent-collected (self-collected) and health care worker (HCW)-collected pharyngeal swabs for detection of GAS by PCR. From 9 October 2012 to 21 March 2013, patients presenting with a sore throat meeting criteria for GAS testing and not meeting criteria for severe disease were offered the opportunity to collect their own pharyngeal swab. The HCW also collected a swab. Paired swabs were tested by GAS real-time PCR, allowing semiquantitative comparisons between positive results. Of the 402 participants, 206 had a swab collected by the patient and 196 a swab collected by the parent. The percent positivity results were 33.3% for HCW-collected swabs and 34.3% for self-collected swabs (P = 0.41). The overall concordance between the two collection strategies was 94.0% (95% confidence interval [CI], 91.3 to 96.0). Twenty-four of the paired swabs had discordant results, with 10 and 14 positives detected only with the HCW- and self-collected swabs, respectively (P = 0.41). The person collecting the swab in the self-collected arm, the order of collection, and prior swab collection training did not influence results. Among the 124 specimens that were positive by both collection methods, the amount of GAS DNA was higher in the self-collected versus the HCW-collected swabs (P = 0.008). Self-collected pharyngeal swabs provide a reliable alternative to HCW collection for detection of GAS and offer a strategy for improved health care delivery. PMID:25502528

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

    PubMed Central

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

    1995-01-01

    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 frame-shifting deletions in the dystrophin gene that are detectable by a 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 a 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. Images Figure 2 PMID:7668256

  3. Simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. after their exposure to petroleum hydrocarbons.

    PubMed

    Kuyukina, Maria S; Ivshina, Irena B; Serebrennikova, Marina K; Rubtsova, Ekaterina V; Krivoruchko, Anastasiya V

    2013-08-01

    A method of simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. was developed that allowed the estimation of immobilized Rhodococcus opacus and Rhodococcus ruber survival after their exposure to petroleum hydrocarbon mixture. Spectrophotometric INT assay revealed high tolerance of gel-immobilized rhodococci to petroleum hydrocarbons, while among two Rhodococcus strains studied, R. ruber tolerated better to hydrocarbons compared to R. opacus. These findings were confirmed by respirometry results that showed increased respiratory activity of gel-immobilized Rhodococcus strains after 10-day incubation with 3% (v/v) petroleum hydrocarbon mixture. Moreover, jointly incubated rhodococcal strains demonstrated higher oxidative activities toward petroleum hydrocarbons than individual strains. Both Rhodococcus species were recovered successfully in cryogel granules using 16S rDNA-targeted PCR, even though the granules were previously stained with INT and extracted with ethanol. The method developed can be used for rapid detection and monitoring of gel-immobilized bacterial inocula in bioreactors or contaminated soil systems.

  4. The burden of knowing: balancing benefits and barriers in HIV testing decisions. a qualitative study from Zambia

    PubMed Central

    2012-01-01

    Background Client-initiated HIV counselling and testing has been scaled up in many African countries, in the form of voluntary counselling and testing (VCT). Test rates have remained low, with HIV-related stigma being an important barrier to HIV testing. This study explored HIV testing decisions in one rural and one urban district in Zambia with high HIV prevalence and available antiretroviral treatment. Methods Data were collected through 17 in-depth interviews and two focus group discussions with individuals and 10 in-depth interviews with counsellors. Interpretive description methodology was employed to analyse the data. Results 'To know your status' was found to be a highly charged concept yielding strong barriers against HIV testing. VCT was perceived as a diagnostic device and a gateway to treatment for the severely ill. Known benefits of prevention and early treatment were outweighed by a perceived burden of knowing your HIV status related to stigma and fear. The manner in which the VCT services were organised added to this burden. Conclusions This study draws on social stigma theory to enhance the understanding of the continuity of HIV related stigma in the presence of ART, and argues that the burden of knowing an HIV status and the related reluctance to get HIV tested can be understood both as a form of label-avoidance and as strong expressions of the still powerful embodied memories of suffering and death among non-curable AIDS patients over the last decades. Hope lies in the emerging signs of a reduction in HIV related stigma experienced by those who had been tested for HIV. Further research into innovative HIV testing service designs that do not add to the burden of knowing is needed. PMID:22222028

  5. 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.

  6. 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

  7. Distinction between persistent and transient infection in a bovine viral diarrhoea (BVD) control programme: appropriate interpretation of real-time RT-PCR and antigen-ELISA test results.

    PubMed

    Hanon, J-B; Van der Stede, Y; Antonissen, A; Mullender, C; Tignon, M; van den Berg, T; Caij, B

    2014-04-01

    Control of bovine viral diarrhoea (BVD) in Belgium is currently implemented on a voluntary basis at herd level and mainly relies on detection and culling of persistently infected (PI) animals. The present field study was conducted during the winter of 2010/2011 to assess the performances of diagnostic assays used in the testing scheme for BVD as proposed by the two Belgian regional laboratories. Individual blood samples were collected from 4972 animals, and individual samples from the same herd were pooled (maximum of 30 individual samples per pool) and screened for the presence of Bovine Viral Diarrhoea Virus (BVDV)-specific RNA using a commercial real-time RT-PCR test (ADIAGENE). Individual samples from positive pools were then tested in parallel with the same RT-PCR test and with an antigen-capture ELISA test (IDEXX) to detect viremic animals. This study demonstrated that individual results differed according to the type of assay used (P < 0.001): 140 animals (2.8%) were positive by RT-PCR and 72 (1.4%) by antigen-ELISA. A second blood sample was taken 40 days later from 74 PCR positive animals to detect persistent viremia: 17 (23%) of these were still PCR positive and considered to be PI and the 57 that no longer tested positive were assumed to be transiently infected (TI) animals. All PI animals were positive also by antigen-ELISA at both time points. Among TI animals, 10 (16%) were positive by antigen-ELISA at the first but none at the second sampling. A highly significant difference in cycle threshold (Ct ) values obtained by RT-PCR was observed between PI and TI animals. ROC analysis was performed to establish thresholds to confirm with high probability that an animal is PI, based on the result of RT-PCR test performed on a single individual blood sample.

  8. 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

  9. Qualitative study of primary care clinicians’ views on point-of-care testing for C-reactive protein for acute respiratory tract infections in family medicine

    PubMed Central

    Hardy, Victoria; Thompson, Matthew; Keppel, Gina A; Alto, William; Dirac, M Ashworth; Neher, Jon; Sanford, Christopher; Hornecker, Jaime; Cole, Allison

    2017-01-01

    Objective To explore clinicians views of the barriers and facilitators to use of C-reactive protein (CRP) point-of-care tests (POCT) in US family medicine clinics for the management of acute respiratory tract infections (ARTIs) in adults. Setting Five family medicine clinics across two US states. Participants 30 clinicians including 18 physicians, 9 physician residents, 2 physician assistants and 1 nurse practitioner, took part in the study. Design A qualitative study using a grounded theory approach to thematically analyse focus group interviews. Results These clinicians had limited access to diagnostic tests for patients with ARTI, and very little knowledge of CRP POCT. Three major themes were identified and included the potential clinical role of CRP POCT, concerns related to implementing CRP POCT and evidence needed prior to wider adoption in family medicine. Clinicians believed CRP POCT could support decision-making for some presentations of ARTIs and patient populations when used in conjunction with clinical criteria. Clinicians had concerns about possible overuse and inaccuracy of CRP POCT which they believed might increase antibiotic prescribing rates. Other concerns identified included integration of the test with clinic workflows and cost-effectiveness. Conclusions Clinicians stand at the forefront of antibiotic stewardship efforts, but have few diagnostic tests to help them confidently manage ARTIs. CRP POCT may facilitate some aspects of clinical practice. Incorporating CRP POCT with clinical guidelines may strengthen utility of this test, when there is diagnostic uncertainty. PMID:28122829

  10. Responses to FTO genetic test feedback for obesity in a sample of overweight adults: a qualitative analysis.

    PubMed

    Meisel, S F; Wardle, J

    2014-01-01

    Current evidence indicates that genetic testing for obesity risk has limited affective or behavioral impact, but few studies have explored the effects among individuals who self-identify as having weight problems. Here, we report findings from in-depth telephone interviews with seven overweight or obese volunteers who were genotyped for one weight-related gene (FTO), which may offer interesting insights into motivations to seek out genetic testing and immediate reactions to it. All participants had a BMI > 25. The gene test identified one participant as homozygous for the 'higher-risk' variant (AA), three heterozygous (AT), and three homozygous for the 'lower-risk' variant (TT) of FTO. All participants said they took part to find an explanation for their personal struggle with weight control. Those with one or two higher-risk variants experienced relief and saw the result as confirming their private assumption that they were susceptible to weight gain for reasons perceived as 'external' to them. However, at the same time, they described themselves as more motivated to overcome their genetic predisposition. Those with lower-risk variants reported brief disappointment, but then focused on alternative explanations, reinforcing the multifactorial nature of obesity. Despite objectively low 'information value,' all individuals derived some 'personal' benefit from FTO genetic test feedback. However, improving education about the multifactorial nature of complex conditions is important to decrease polarized thinking and associated genetic determinism and stigma to derive the greatest benefits of novel genetic technologies for individuals and their health.

  11. Applications of Digital PCR for Clinical Microbiology.

    PubMed

    Kuypers, Jane; Jerome, Keith R

    2017-03-15

    Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute quantification without a standard curve, improved precision, improved accuracy in the presence of inhibitors, and more accurate quantitation when amplification efficiency is low, make dPCR the assay of choice for several specimen testing applications. This mini-review will discuss the advantages and disadvantages of dPCR compared to qPCR, its applications in clinical microbiology and the considerations for implementation of the method in a clinical laboratory.

  12. Racial/ethnic differences in the criterion-related validity of cognitive ability tests: a qualitative and quantitative review.

    PubMed

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

    2011-09-01

    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 ( = .33, N = 903,779), average correlations were lower for Black samples ( = .24, N = 112,194) and Hispanic samples ( = .30, N = 51,205) and approximately equal for Asian samples ( = .33, N = 80,705). Despite some moderating effects (e.g., type of performance criterion, decade of data collection, job complexity), validity favored White over Black and Hispanic test takers in almost all conditions that included a sizable number of studies. Black-White validity comparisons were possible both across and within the 3 broad domains that use cognitive ability tests for high-stakes selection and placement: civilian employment, educational admissions, and the military. The trend of lower Black validity was repeated in each domain; however, average Black-White validity differences were largest in military studies and smallest in educational and employment studies. Further investigation of the reasons for these validity differences is warranted.

  13. 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.

  14. [The methodological assessment and qualitative evaluation of psychometric performance tests based on the example of modern tests that assess reading and spelling skills].

    PubMed

    Galuschka, Katharina; Rothe, Josefine; Schulte-Körne, Gerd

    2015-09-01

    This article looks at a means of objectively evaluating the quality of psychometric tests. This approach enables users to evaluate psychometric tests based on their methodological characteristics, in order to decide which instrument should be used. Reading and spelling assessment tools serve as examples. The paper also provides a review of German psychometric tests for the assessment of reading and spelling skills. This method facilitates the identification of psychometric tests.of high methodological quality which can be used for the assessment of reading and spelling skills. Reading performance should ideally be assessed with the following instruments: ELFE 1-6, LGVT 6-12, LESEN 6-7, LESEN 8-9, or WLLP-R. The tests to be used for the evaluation of spelling skills are DERET 1-2+, DERET 3-4+, WRT 1+, WRT 2+, WRT 3+, WRT 4+ or HSP 1-10.

  15. RhC Phenotyping, Adsorption/Elution Test, and SSP-PCR: The Combined Test for D-Elute Phenotype Screening in Thai RhD-Negative Blood Donors.

    PubMed

    Srijinda, Songsak; Suwanasophon, Chamaiporn; Visawapoka, Unchalee; Pongsavee, Malinee

    2012-01-01

    The Rhesus (Rh) blood group is the most polymorphic human blood group and it is clinically significant in transfusion medicine. Especially, D antigen is the most important and highly immunogenic antigen. Due to anti-D, it is the cause of the hemolytic disease of the newborn and transfusion reaction. About 0.1%-0.5% of Asian people are RhD-negative, whereas in the Thai population, the RhD-negative blood type only occurs in 0.3%. Approximately 10%-30% of RhD-negative in Eastern Asian people actually were D-elute (DEL) phenotype, the very weak D antigen that cannot be detected by indirect antiglobulin test (IAT). There are many reports about anti-D immunization in RhD-negative recipients through the transfusion of red blood cells from individuals with DEL phenotype. D-elute phenotype screening in Thai RhD-negative blood donors was studied to distinguish true RhD-negative from DEL phenotype. A total of 254 Thai serologically RhD-negative blood donors were tested for RhCE phenotypes and anti-D adsorption/elution test. In addition, RhC(+) samples were tested for RHD 1227A allele by SSP-PCR technique. The RhD-negative phenotype samples consisted of 131 ccee, 4 ccEe, 1 ccEE, 101 Ccee, 16 CCee, and 1 CcEe. The 42 Ccee and 8 CCee phenotype samples were typed as DEL phenotype and 96% of DEL samples were positive for RHD 1227A allele. The incidence of RhC(+) was 46.4%, and 48 of the 118 RhC(+) samples were positive for both anti-D adsorption/elution test and SSP-PCR technique for RHD 1227A allele. The sensitivity and specificity were 96% and 100%, respectively, for RHD 1227A detection as compared with the adsorption/elution test. In conclusion, RhC(+) phenotype can combine with anti-D adsorption/elution test and RHD 1227A allele SSP-PCR technique for distinguishing true RhD-negative from DEL phenotype.

  16. 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

  17. 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

  18. 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

  19. 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

  20. Sequence homologies between Mycoplasma and Chlamydia spp. lead to false-positive results in chlamydial cell cultures tested for mycoplasma contamination with a commercial PCR assay.

    PubMed

    Maass, Viola; Kern, Jan Marco; Poeckl, Matthias; Maass, Matthias

    2011-10-01

    Mycoplasma contamination is a frequent problem in chlamydial cell culture. After obtaining contradictory contamination results, we compared three commercial PCR kits for mycoplasma detection. One kit signaled contamination in mycoplasma-free Chlamydia pneumoniae cultures. Sequencing of cloned PCR products revealed primer homology with the chlamydial genome as the basis of this false-positive result.

  1. 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...

  2. Detection and monitoring of virus infections by real-time PCR.

    PubMed

    Watzinger, F; Ebner, K; Lion, T

    2006-01-01

    The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.

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

    PubMed

    Nsojo, Anthony; Aboud, Said; Lyamuya, Eligius

    2010-10-01

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

  4. Relationship of bovine viral diarrhea virus persistent infection to incidence of diseases on dairy farms based on bulk tank milk test by RT-PCR.

    PubMed

    Kozasa, Takashi; Tajima, Motoshi; Yasutomi, Ichiro; Sano, Kimihiro; Ohashi, Kazuhiko; Onuma, Misao

    2005-03-20

    The prevalence of bovine viral diarrhea virus (BVDV) in dairy herds in Hokkaido, Japan, was estimated by reverse transcription polymerase chain reaction (RT-PCR) using bulk tank milk samples. Sixteen out of 265 dairy herds were identified as BVDV positive, and at least one persistently infected (PI) cattle was recognized in each of the positive herds except for two herds of which, owners did not agree to examine individual cows. The proportion of positive herds with a history of BVDV PI was significantly higher than that with no history of BVDV PI (odds ratio (OR) 4.25, 95% confidence interval (CI) 1.471-12.278, p = 0.004). The herds examined for BVDV were divided into two groups, high and low disease incidence groups based on the occurrence of diseases such as diarrhea, pneumonia or abortion in the past 1 year. The BVDV positive herds in the high disease incidence group were significantly more than that in the low disease incidence group (OR 2.92, CI 1.110-7.683, p = 0.024). It was observed that there were significantly (p = 0.008) more PI calves or heifers in farms of high disease incidence group than in farms of low disease incidence group. These results suggested that bulk tank milk test was available method for the detection of PI animals in dairy herds, and the existence of PI non-lactating cows in herd correlated with the incidence of diseases of the diarrhea or respiratory disorders.

  5. Comparison of the novel Partec rapid malaria test to the conventional Giemsa stain and the gold standard real-time PCR.

    PubMed

    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

    2010-08-01

    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.

  6. Development of a Qualitative Quantitative Polymerase Chain Reaction Test to Identify Patients Failing First-Line Therapy to Non-Nucleotide Reverse Transcriptase Inhibitor.

    PubMed

    L Machado, Sergio; Gonçalves, Gabriel S; Dudley, Dawn; O'Connor, David; Keiko Toma, Helena; Couto Fernandes, José Carlos; Tanuri, Amilcar

    2017-01-11

    Antiretroviral therapy (ART) can be compromised by selection of drug resistance strains, which can be promoted by lack of adherence during therapy and drug tolerance, and some of these drug-resistant strains can persist for years as minority populations. The K103N drug resistance mutation is selected by the use of non-nucleotide reverse transcriptase inhibitors, including nevirapine or efavirenz (EFV), used in low-income countries. Here we describe the use of a less expensive qualitative point mutation polymerase chain reaction (PMqPCRK103N) targeting K103N mutation. To validate the use of this methodology, we tested previously sequenced samples from patients treated with highly active ART with viral loads above 2,000 copies/ml and compared the results of our assay with Illumina deep sequencing. Due to its low cost and high specificity, this test is particularly suitable for low-income countries to screen for pretreatment resistance in patients either initiating ART or failing first-line regimens containing EFV.

  7. 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.

  8. Implementing routine provider-initiated HIV testing in public health care facilities in Kenya: a qualitative descriptive study of nurses' experiences.

    PubMed

    Evans, Catrin; Ndirangu, Eunice

    2011-10-01

    Routine "provider-initiated testing and counselling" (PITC) for HIV has been implemented amidst concern over how consent, confidentiality and counselling (the 3Cs) can be maintained in under-resourced health care settings. In Kenya, PITC has been rolled out since 2005, HIV prevalence is 7.1% and more than 86% of adults have not been tested. Kenyan nurses are the main cadre implementing PITC, but little is known about their experiences of incorporating HIV testing into everyday practice and the challenges faced in maintaining the 3Cs within their work environments. This study aimed to explore these issues and adopted a qualitative multi-method design using a convenience sampling approach. Two focus group discussions (total n=12) and 13 in-depth individual interviews were undertaken with nurses from 11 different public health care facilities in Nairobi and its surrounding areas (including in-patient and outpatient settings). Data were analysed thematically. Nurses identified a range of personal, client and health system challenges in the everyday application of PITC. These included (i) the contradictions of normalising a highly stigmatised disease and the difficulty in providing client-centred care within a routinised and target-oriented work culture; (ii) the challenge of dealing with ethically complex client situations in which the principles of the 3Cs could be difficult to uphold; and (iii) lack of time, resources, space and recognition within workplace environments (especially in-patient settings) that, likewise, led to problems with maintaining the 3Cs. In-patient nurses in particular identified problems associated with testing in a multi-disciplinary context, suggesting that other health professionals appeared to routinely flout the PITC guidelines. In conclusion, this study shows that the process of translating policy into practice is invariably complex and that more research is needed to explore PITC practices, particularly in in-patient settings. Nurses

  9. 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

    2016-07-01

    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.

  10. Detection and Identification of Salmonella enterica, Escherichia coli, and Shigella spp. via PCR-Electrospray Ionization Mass Spectrometry: Isolate Testing and Analysis of Food Samples

    PubMed Central

    Bell, Rebecca L.; Hellberg, Rosalee S.; Cheng, Chorng-Ming; Chen, Kai-Shun; Williams-Hill, Donna M.; Martin, William B.; Allard, Marc W.

    2012-01-01

    An assay to identify the common food-borne pathogens Salmonella, Escherichia coli, Shigella, and Listeria monocytogenes was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140 Salmonella enterica, 139 E. coli, 11 Shigella, and 36 Listeria patterns and 18 other Enterobacteriaceae organisms. This assay was tested to determine the scope of the assay's ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of S. enterica, E. coli, and Shigella species were analyzed. Overall, 100% of S. enterica, 99% of E. coli, and 73% of Shigella spp. were detected using this assay. The assay was also able to identify 30% of the S. enterica serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of S. enterica from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens. PMID:23001674

  11. Detection and Identification of Salmonella enterica, Escherichia coli, and Shigella spp. via PCR-electrospray ionization mass spectrometry: isolate testing and analysis of food samples.

    PubMed

    Pierce, Sarah E; Bell, Rebecca L; Hellberg, Rosalee S; Cheng, Chorng-Ming; Chen, Kai-Shun; Williams-Hill, Donna M; Martin, William B; Allard, Marc W

    2012-12-01

    An assay to identify the common food-borne pathogens Salmonella, Escherichia coli, Shigella, and Listeria monocytogenes was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140 Salmonella enterica, 139 E. coli, 11 Shigella, and 36 Listeria patterns and 18 other Enterobacteriaceae organisms. This assay was tested to determine the scope of the assay's ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of S. enterica, E. coli, and Shigella species were analyzed. Overall, 100% of S. enterica, 99% of E. coli, and 73% of Shigella spp. were detected using this assay. The assay was also able to identify 30% of the S. enterica serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of S. enterica from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens.

  12. PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions

    PubMed Central

    Acharya, Kamal R.; Dhand, Navneet K.; Whittington, Richard J.; Plain, Karren M.

    2017-01-01

    Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne’s disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne’s test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne’s disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples. PMID:28210245

  13. PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions.

    PubMed

    Acharya, Kamal R; Dhand, Navneet K; Whittington, Richard J; Plain, Karren M

    2017-01-01

    Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne's disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne's test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne's disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples.

  14. Pcr by Thermal Convection

    NASA Astrophysics Data System (ADS)

    Braun, Dieter

    The Polymerase Chain Reaction (PCR) allows for highly sensitive and specific amplification of DNA. It is the backbone of many genetic experiments and tests. Recently, three labs independently uncovered a novel and simple way to perform a PCR reaction. Instead of repetitive heating and cooling, a temperature gradient across the reaction vessel drives thermal convection. By convection, the reaction liquid circulates between hot and cold regions of the chamber. The convection triggers DNA amplification as the DNA melts into two single strands in the hot region and replicates into twice the amount in the cold region. The amplification progresses exponentially as the convection moves on. We review the characteristics of the different approaches and show the benefits and prospects of the method.

  15. Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

    PubMed Central

    Golparian, Daniel; Boräng, Stina; Sundqvist, Martin

    2015-01-01

    The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection of Neisseria gonorrhoeae. PMID:26468501

  16. Testing of the Bio-Seeq (Smiths Detection Handheld PCR Instrument): Sensitivity, Specificity, and Effect of Interferents on Yersinia Pestis Assay Performance

    DTIC Science & Technology

    2005-04-01

    HANDHELD PCR INSTRUMENT): SENSITIVITY, SPECIFICITY, AND EFFECT OF INTERFERENTS ON YERSINIA PESTIS ASSAY PERFORMANCE Kevin P. O’Connell Patricia E...Bio-Seeq® (Smiths Detection Handheld PCR Instrument): Sensitivity, Specificity, and Effect of Interferents on Yersinia pestis Assay 5b. GRANT NUMBER...DIR, ECBC, ATTN: AMSRD-ECB- RT -BM, APG, MD 21010-5424 NUMBER ECBC-TR-437 9. SPONSORING I MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORIMONITOR’S

  17. Application of a circulating-cathodic-antigen (CCA) strip test and real-time PCR, in comparison with microscopy, for the detection of Schistosoma haematobium in urine samples from Ghana.

    PubMed

    Obeng, B B; Aryeetey, Y A; de Dood, C J; Amoah, A S; Larbi, I A; Deelder, A M; Yazdanbakhsh, M; Hartgers, F C; Boakye, D A; Verweij, J J; van Dam, G J; van Lieshout, L

    2008-10-01

    In the detection of parasitic infection, the traditional methods based on microscopy often have low sensitivity and/or specificity compared with the newer, molecular tests. An assay based on real-time PCR and a reagent strip test for detecting circulating cathodic antigen (CCA) have both now been compared with urine filtration and microscopy, in the detection of Schistosoma haematobium infections. Urine samples, obtained from 74 'cases' in areas of Ghana with endemic S. haematobium and 79 'controls' from non-endemic areas, were each checked using the three methods. With the results of the filtration and microscopy taken as the 'gold standard', real-time PCR was found to be 100% specific and 89% sensitive whereas the CCA strips were 91% specific and 41% sensitive. With the samples found to contain > or =50 eggs/10 ml (indicating relatively intense infections), the sensitivities of the PCR and CCA were higher, at 100% and 62%, respectively. As expected, egg counts were negatively correlated with the number of amplification cycles needed, in the PCR, to give a signal that exceeded the background (r=-0.38; P<0.01). Although the real-time PCR and CCA strip tests are very different, both show promise in the detection of S. haematobium infections. The PCR has optimal specificity and high sensitivity but the specificity of the CCA strips and the sensitivity of both tools could still be improved. A more thorough re-evaluation of the sensitivity and specificity of microscopy and these newer diagnostic methods, with an estimation of the cost-effectiveness of each technique, is recommended.

  18. Comparison of performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus.

    PubMed

    Sábato, M Fernanda; Shiffman, Mitchell L; Langley, Michael R; Wilkinson, David S; Ferreira-Gonzalez, Andrea

    2007-08-01

    We evaluated the performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus (HCV) and performed a direct comparison of the systems on the same clinical specimens. Commercial HCV panels (genotype 1b) were used to evaluate linear range, sensitivity, and precision. The Roche COBAS TaqMan HCV test for research use only (RUO) with samples processed on the MagNA Pure LC instrument (Roche RUO-MPLC) and Abbott analyte-specific reagents (ASR) with QIAGEN sample processing (Abbott ASR-Q) showed a sensitivity of 1.0 log(10) IU/ml with a linear dynamic range of 1.0 to 7.0 log(10) IU/ml. The Roche ASR in combination with the High Pure system (Roche ASR-HP) showed a sensitivity of 1.4 log(10) IU/ml with a linear dynamic range of 2.0 to 7.0 log(10) IU/ml. All of the systems showed acceptable reproducibility, the Abbott ASR-Q being the most reproducible of the three systems. Seventy-six clinical specimens (50 with detectable levels of HCV RNA and various titers and genotypes) were tested, and results were compared to those of the COBAS Amplicor HCV Monitor v2.0. Good correlation was obtained for the Roche RUO-MPLC and Abbott ASR-Q (R(2) = 0.84 and R(2) = 0.93, respectively), with better agreement for the Abbott ASR-Q. However, correlation (R(2) = 0.79) and agreement were poor for Roche ASR-HP, with bias relative to concentration and genotype. Roche ASR-HP underestimated HCV RNA for genotypes 3 and 4 as much as 2.19 log(10) IU/ml. Our study demonstrates that Roche RUO-MPLC and Abbott ASR-Q provided acceptable results and agreed sufficiently with the COBAS Amplicor HCV Monitor v2.0.

  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. 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

  1. 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…

  2. 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

  3. Validation of a reference gene (BdFIM) for quantifying transgene copy numbers in Brachypodium distachyon by real-time PCR.

    PubMed

    Zhu, Hong; Wen, Feng; Li, Peng; Liu, Xiang; Cao, Jianmei; Jiang, Min; Ming, Feng; Chu, Zhaoqing

    2014-03-01

    Brachypodium distachyon has been proposed as a new model system for gramineous plants with a sequenced genome and an efficient transformation system. Many transgenic B. distachyon plants have been generated in recent years. To develop a reliable fast method for detecting transgenic B. distachyon and quantifying its transgene copy numbers, a species-specific reference gene is of great priority to be validated both in qualitative PCR and quantitative real-time PCR detection. In this study, we first proved that the BdFIM (B. distachyon fimbrin-like protein) gene is a suitable reference gene in qualitative PCR and quantitative real-time PCR for B. distachyon. Fourteen different B. distachyon varieties were tested by both qualitative and quantitative PCRs, and identical amplification products of BdFIM were obtained with all of them, while no amplification products were observed with samples from 14 other plant species, suggesting that BdFIM gene was specific to B. distachyon. The results of Southern blot analysis revealed that the BdFIM gene was low copy number in seven tested B. distachyon varieties. In conclusion, the BdFIM gene can be used as a reference gene, since it had species specificity, low heterogeneity, and low copy number among the tested B. distachyon varieties. Furthermore, the copy number of inserted sequences from transgenic B. distachyon obtained by real-time PCR methods and Southern blot confirmed that the BdFIM gene was an applicable reference gene in B. distachyon.

  4. Evaluation of a real-time PCR test for the detection and discrimination of theileria species in the African buffalo (Syncerus caffer).

    PubMed

    Chaisi, Mamohale E; Janssens, Michiel E; Vermeiren, Lieve; Oosthuizen, Marinda C; Collins, Nicola E; Geysen, Dirk

    2013-01-01

    A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.

  5. Fertilization potential and qualitative characteristics of human spermatozoa after short-term cryostorage at 5 degrees C in two different TEST-yolk buffer preparations.

    PubMed

    Zavos, P M; Correa, J R; Foster, C L; Massey, J B; Zarmakoupis-Zavos, P N

    1998-02-01

    Use of the media TEST-yolk buffer (TYB) in semenology today enables the short-term incubation and cryostorage of spermatozoa and its subsequent use in the various assisted reproductive technologies (ART). Preparation of TYB media involves the addition of egg yolk (20% v/v) to a physiological solution of the zwitterion buffers TES and Tris. The TYB is usually thermoprecipitated to remove the majority of the egg yolk globules and other macromolecules from the medium. However, removal of these egg yolk constituents could possibly eliminate or reduce essential factors that could enhance the sperm viability and fertilization potential after short-term dilution and storage. Improvements in the quality of the TYB could add greater benefits to those techniques employed in the various forms of ART. The objectives of the investigation were 1) to study the sperm qualitative characteristics following short-term cryostorage at 5 degrees C in either thermoprecipitated (T-TYB) or non-thermoprecipitated (NT-TYB), and 2) to compare the fertilizing potential of spermatozoa stored for 24 hours at 5 degrees C in the two TYB preparations. In Experiment 1, semen specimens from 15 patients were collected, assessed and split into two aliquots. Sperm specimens were processed by diluting 1:1 (v/v) with the T-TYB or NT-TYB, followed by centrifugation and reconstitution of the specimen to its initial volume with the corresponding TYB medium. Sperm specimens were cryostored for 1, 2, 24, 48 and 72 hours. Samples were taken at each interval and placed in a 37 degrees C water bath and allowed to warm for 15 minutes after each cryostorage interval. Semen specimens were assessed for percentage and grade of motility. The results of this study indicated that, although the NT-TYB yielded better results than the T-TYB, overall those differences were not statistically significant. In Experiment 2, the fertilization potential of spermatozoa recovered after 24 hours of cryostorage in the two TYB

  6. 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.

  7. 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

  8. Pre-Clinical Evaluation of a Real-Time PCR Assay on a Portable Instrument as a Possible Field Diagnostic Tool: Experiences from the Testing of Clinical Samples for African and Classical Swine Fever Viruses.

    PubMed

    Liu, L; Luo, Y; Accensi, F; Ganges, L; Rodríguez, F; Shan, H; Ståhl, K; Qiu, H-J; Belák, S

    2016-06-16

    African swine fever (ASF) and classical swine fever (CSF) are two highly infectious transboundary animal diseases (TADs) that are serious threats to the pig industry worldwide, including in China, the world's largest pork producer. In this study, a duplex real-time PCR assay was developed for the rapid detection and differentiation of African swine fever virus (ASFV) and classical swine fever virus (CSFV). The assay was performed on a portable, battery-powered PCR thermocycler with a low sample throughput (termed as 'T-COR4 assay'). The feasibility and reliability of the T-COR4 assay as a possible field method was investigated by testing clinical samples collected in China. When evaluated with reference materials or samples from experimental infections, the assay performed in a reliable manner, producing results comparable to those obtained from stationary PCR platforms. Of 59 clinical samples, 41 had results identical to a two-step CSFV real-time PCR assay. No ASFV was detected in these samples. The T-COR4 assay was technically easy to perform and produced results within 3 h, including sample preparation. In combination with a simple sample preparation method, the T-COR4 assay provides a new tool for the field diagnosis and differentiation of ASF and CSF, which could be of particular value in remote areas.

  9. 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.

  10. Test balloons? Small signs of big events: a qualitative study on circumstances facilitating adults' awareness of children's first signs of sexual abuse.

    PubMed

    Flåm, Anna Margrete; Haugstvedt, Eli

    2013-09-01

    This research examined caregivers' awareness of children's first signs of sexual abuse. The aim was to explore circumstances that facilitate adults' awareness of first signs in everyday natural settings. Data were obtained from a Norwegian university hospital's outpatient specialty mental health clinic. Included were all cases (N=20) referred during a two-year period for treatment after the disclosure of sexual abuse that was reported to the police and child protective service. Nonabusing caregivers' awareness of first signs were recollected in hindsight as part of therapy. Qualitative analysis was conducted to capture caregivers' experiences. As identified by caregivers, all children gave signs. Thereafter, children either stopped, delayed, or immediately disclosed sexual abuse. At first signs, each child had time and attention from trusted adults, connection to the abuser, and exhibited signs of reservation against that person or related activities. Then, if met with closed answers, first signs were rebuffed as once-occurring events. If met with open answers and follow-up questions, children continued to tell. Unambiguous messages were prompted only in settings with intimate bodily activity or sexual abuse related content. In sum, when trusted adults provided door-openings, children used them; when carefully prompted, children talked; when thoughtfully asked, children told. The study suggests that children's signs of sexual abuse can be understood as "test balloons" to explore understanding and whether anything is to be done. A disclosing continuation hinges on the trusted adult's dialogical attunement and supplementary door-openings. Divergent from an idea of behavioural markers, or purposeful versus accidental disclosures, this study calls for a broader attention: Moments of first signs are embedded in dialogue. A uniqueness at moments of first signs appears: Both to form such moments and to transform them into moments of meeting for joint exploration and

  11. 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…

  12. 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.

  13. Dengue Fever Testing

    MedlinePlus

    ... name: Dengue Antibodies (IgG, IgM); Dengue Virus by PCR Related tests: Arbovirus Testing , West Nile Virus Testing , ... testing are available: Molecular testing ( polymerase chain reaction, PCR )—this type of test detects the genetic material ...

  14. Early diagnosis of HIV in children below 18 months using DNA PCR test--assessment of the effectiveness of PMTCT interventions and challenges in early initiation of ART in a resource-limited setting.

    PubMed

    Gupta, Anil; Singh, Girraj; Kaushik, Pratap; Joshi, Bipin; Kalra, Kamlesh; Chakraborty, Sabyasachi

    2013-04-01

    This study was conducted to assess efficacy of the current Indian Prevention of Mother-to-Child Transmission (PMTCT) protocol in 217 HIV-exposed infants, and to assess challenges in the early initiation of antiretroviral treatment (ART) in 18 (8.3%) infants with HIV, as determined by the HIV-1 DNA polymerase chain reaction (PCR) test at ≥ 6 weeks to <18 months of age. The mother-to-child transmission (MTCT) rate in 154 mother-baby pairs fully compliant with the PMTCT protocol was 5.2%. However, if 25 pairs who were positive using dried blood spot (DBS) DNA PCR and who did not undergo whole blood testing are included in the analysis, then the overall MTCT rate would be 19.8%. The current protocol is 50% effective considering an MTCT rate of 35-40% without any intervention. ART was initiated in 10 (55.6%) HIV-infected children at a mean ± standard deviation (SD) age of 10.45 ± 4.9 (range: 4-17.5) months; delay resulted in opportunistic infections in one-third of the children. A single-dose nevirapine PMTCT regimen should be replaced by a triple antiretroviral regimen; DBS DNA PCR-positive infants may be given ART, and simultaneously a whole blood specimen should be taken to determine whether ART should be continued.

  15. Colorimetric Integrated PCR Protocol for Rapid Detection of Vibrio parahaemolyticus

    PubMed Central

    Cheng, Kewen; Pan, Daodong; Teng, Jun; Yao, Li; Ye, Yingwang; Xue, Feng; Xia, Fan; Chen, Wei

    2016-01-01

    Rapid detection of pathogens is of great significance for food safety and disease diagnosis. A new colorimetric method for rapid and easy detection of Vibrio parahaemolyticus (V. parahaemolyticus or Vp) has been developed in this research. A specific sequence was designed and integrated with the forward primer for molecular detection of Vp. This specific sequence was tested and treated as the horseradish peroxidase (HRP)-mimicking DNAzyme and could be amplified during the polymerase chain reaction (PCR) process. The products of PCR including the sequence of HRP-mimicking DNAzyme could produce the distinguished color in the presence of catalysis substrates. The optical signal of the catalysis reaction, which is in a linear relationship with the initial template of Vp, could be determined with the naked eye or measured with Ultraviolet-visible (UV-vis) for qualitative and quantitative detections, respectively. Based on the optical signal intensity, rapid and easy detection of Vp was successfully achieved with satisfied sensitivity and specificity. Furthermore, the detection of tdh, trh, tlh and toxR virulence genes of two Vp species (Vp 33847 and Vp 17802) were all performed successfully with this developed colorimetric integrated PCR protocol, which demonstrated potential applicability for the rapid detection of other bacteria. PMID:27690041

  16. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

    PubMed

    Farrell, D J

    1999-02-01

    Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

  17. Transfer and Evaluation of an Automated, Low-Cost Real-Time Reverse Transcription-PCR Test for Diagnosis and Monitoring of Human Immunodeficiency Virus Type 1 Infection in a West African Resource-Limited Setting

    PubMed Central

    Rouet, Francois; Ekouevi, Didier K.; Chaix, Marie-Laure; Burgard, Marianne; Inwoley, Andre; Tony, Thomas D'Aquin; Danel, Christine; Anglaret, Xavier; Leroy, Valeriane; Msellati, Philippe; Dabis, Francois; Rouzioux, Christine

    2005-01-01

    There is an urgent need for low-cost human immunodeficiency virus type 1 (HIV-1) viral load (VL) monitoring technologies in resource-limited settings. An automated TaqMan real-time reverse transcription-PCR (RT-PCR) assay was transferred to the laboratory of the Centre de Diagnostic et de Recherches sur le SIDA, Abidjan, Côte d'Ivoire, and assessed for HIV-1 RNA VL testing in 806 plasma samples collected within four ANRS research programs. The detection threshold and reproducibility of the assay were first determined. The quantitative results obtained with this assay were compared with two commercial HIV-1 RNA kits (the Versant version 3.0 and Monitor version 1.5 assays) in specimens harboring mainly the circulating recombinant form 02 strain (CRF02). The clinical evaluation of this test was done in different situations including the early diagnosis of pediatric infection and the monitoring of antiretroviral-treated patients. The quantification limit of our method was 300 copies/ml. The HIV-1 RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Versant kit (r = 0.901; P < 0.001) and the Monitor test (r = 0.856; P < 0.001) and homogeneously distributed according to HIV-1 genotypes. For the early diagnosis of pediatric HIV-1 infection, the sensitivity and specificity of the real-time PCR assay were both 100% (95% confidence intervals of 93.7 to 100.0 and 98.3 to 100.0, respectively), compared to the Versant results. Following initiation of antiretroviral treatment, the kinetics of HIV-1 RNA levels were very comparable, with a similar proportion of adults and children below the detection limit during follow-up with our technique and the Versant assay. The TaqMan real-time PCR ($12 per test) is now routinely used to monitor HIV-1 infection in our laboratory. This technology should be further evaluated in limited-resource countries where strains other than CRF02 are prevalent. PMID:15956387

  18. 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

  19. Evaluation of LAMP assay using phenotypic tests and conventional PCR for detection of blaNDM-1 and blaKPC genes among carbapenem-resistant clinical Gram-negative isolates.

    PubMed

    Solanki, Rachana; Vanjari, Lavanya; Ede, Nagapriyanka; Gungi, Akhila; Soory, Amarendranath; Vemu, Lakshmi

    2013-10-01

    Carbapenem-resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of the loop-mediated isothermal amplification (LAMP) assay for rapid and cost-effective detection of bla(NDM-1) and bla(KPC) genes among carbapenem-resistant Gram-negative bacteria in comparison with conventional PCR and existing phenotypic methods. A total of 60 carbapenem-resistant clinical isolates [Escherichia coli (15), Klebsiella pneumoniae (22), Acinetobacter baumannii (23)] were screened for the presence of carbapenemases (bla(KPC) and bla(NDM-1)) using phenotypic methods such as the modified Hodge test (MHT) and combined disc test (CDT) and molecular methods such as conventional PCR and LAMP assay. In all, 47/60 isolates (78.3%) were MHT positive while 48 isolates were positive by CDT [46.6% positive with EDTA, 30% with 3' aminophenylboronic acid (APB) plus EDTA and 1.6% with APB alone]. Isolates showing CDT positivity with EDTA or APB contained bla(NDM-1) and bla(KPC) genes, respectively. bla(NDM-1) was present as a lone gene in 28 isolates (46.7%) and present together with the bla(KPC) gene in 19 isolates (31.7%). Only one E. coli isolate had a lone bla(KPC) gene. The LAMP assay detected either or both bla(NDM-1) and bla(KPC) genes in four isolates that were missed by conventional PCR. Neither gene could be detected in 12 (20%) isolates. The LAMP assay has greater sensitivity, specificity and rapidity compared to the phenotypic methods and PCR for the detection of bla(NDM-1) and bla(KPC). With a turnaround time of only 2-3 h, the LAMP assay can be considered a point-of-care assay.

  20. Rapid detection and high occurrence of porcine rotavirus A, B, and C by RT-qPCR in diagnostic samples.

    PubMed

    Marthaler, Douglas; Homwong, Nitipong; Rossow, Kurt; Culhane, Marie; Goyal, Sagar; Collins, James; Matthijnssens, Jelle; Ciarlet, Max

    2014-12-01

    Rotaviruses are important cause of diarrhea in animals, including humans. Currently, rotavirus species A, B, C, E, and H (RVA-RVC, RVE, and RVH) have been identified in pigs. Traditionally, RVA has been considered the primary cause of diarrhea in pigs, and RVB and RVC had been described sporadically in pigs until recently. Qualitative porcine RVA, RVB, and RVC RT-PCR (RT-qPCR) assays were designed and 7508 porcine diarrheic samples, submitted to University of Minnesota, were tested to estimate the percentage of RVA, RVB, and RVC over a period of approximately 2 years (from 2009 to 2011). The individual RVA and RVC RT-qPCR assays were multiplex into a single RT-qPCR while the RVB RT-qPCR assay remained as an individual RT-qPCR. In total, 83% of the samples were positive for RVA, RVB, or RVC. As expected, RVA was detected at the highest overall percentage (62%). However, 33% and 53% of the samples were positive for RVB and RVC, respectively, indicating that both RVB and RVC are also epidemiologically important in the swine population. RVC was most predominant in young pigs (1-20 days of age), while RVA and RVB were most predominant in ≥21 day old pigs. As diagnostic tools, the developed RT-qPCR assays could successfully discriminate among infecting RV species, which could lead to better surveillance and epidemiological studies for ultimately better prevention and control strategies.

  1. PCR und Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Konrad, Regina; Busch, Ulrich

    Die vielfältigen Anwendungsmöglichkeiten der Polymerasekettenreaktion (polymerase chain reaction, PCR) machen sie zu einer der wichtigsten und am häufigsten eingesetzten Methoden in der molekularbiologischen Forschung und Diagnostik. Für diese Technologie wurde der Erfinder der Methode, Kary Mullis, 1993 mit dem Nobelpreis ausgezeichnet. Die PCR erlaubt einen hochsensitiven und spezifischen in-vitro-Nachweis von Desoxyribonukleinsäuren (DNA), da im Zuge der Reaktion Sequenzabschnitte gezielt vermehrt werden. Innerhalb weniger Stunden können aus einem einzigen Zielmolekül 1012 identische Moleküle entstehen [1].

  2. 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…

  3. Understanding & Conducting Qualitative Research.

    ERIC Educational Resources Information Center

    Stainback, Susan; Stainback, William

    In this book, which applies the state of the art in qualitative research to special education, qualitative research is used as a generic term for investigative methodologies described variously as ethnographic, naturalistic, anthropological, field research, or participant-observer research. Chapter 1 introduces and defines qualitative research and…

  4. The Qualitative Dimension.

    ERIC Educational Resources Information Center

    Lodge-Peters, Dianne S.

    The qualitative dimension of educational research methodology is explored, and the literature of qualitative methodology is reviewed so researchers may (1) understand more fully the qualitative dimension as it, in turn, fits within the parameters of educational research as a whole, and (2) have more informed access to the sometimes daunting array…

  5. 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…

  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. Rapid differentiation of Staphylococcus aureus, Staphylococcus epidermidis and other coagulase-negative staphylococci and meticillin susceptibility testing directly from growth-positive blood cultures by multiplex real-time PCR.

    PubMed

    Jukes, Leanne; Mikhail, Jane; Bome-Mannathoko, Naledi; Hadfield, Stephen J; Harris, Llinos G; El-Bouri, Khalid; Davies, Angharad P; Mack, Dietrich

    2010-12-01

    This study evaluated a multiplex real-time PCR method specific for the mecA, femA-SA and femA-SE genes for rapid identification of Staphylococcus aureus, Staphylococcus epidermidis and non-S. epidermidis coagulase-negative staphylococci (CoNS), and meticillin susceptibility testing directly in positive blood cultures that grew Gram-positive cocci in clusters. A total of 100 positive blood cultures produced: 39 S. aureus [12 meticillin-resistant S. aureus (MRSA), 31% of all the S. aureus]; 30 S. epidermidis (56.6% of the CoNS), 8 Staphylococcus capitis (15.1%), 3 Staphylococcus saprophyticus (5.7%), 4 Staphylococcus hominis (7.5%), 3 Staphylococcus haemolyticus (5.7%), 2 Staphylococcus warneri (3.8%), 1 Staphylococcus cohnii (1.9%) and 2 unidentified Staphylococcus spp. (3.8%); and 1 Micrococcus luteus in pure culture. Two blood cultures had no growth on subculture and five blood cultures grew mixed CoNS. For the 95 blood cultures with pure growth or no growth on subculture, there was very good agreement between real-time PCR and the BD Phoenix identification system for staphylococcal species categorization in S. aureus, S. epidermidis and non-S. epidermidis CoNS and meticillin-resistance determination (Cohen's unweighted kappa coefficient κ=0.882). All MRSA and meticillin-susceptible S. aureus were correctly identified by mecA amplification. PCR amplification of mecA was more sensitive for direct detection of meticillin-resistant CoNS in positive blood cultures than testing with the BD Phoenix system. There were no major errors when identifying staphylococcal isolates and their meticillin susceptibility within 2.5 h. Further studies are needed to evaluate the clinical benefit of using such a rapid test on the consumption of glycopeptide antibiotics and the alteration of empiric therapy in the situation of positive blood cultures growing staphylococci, and the respective clinical outcomes.

  8. An allele-specific PCR system for rapid detection and discrimination of the CYP2C19∗4A, ∗4B, and ∗17 alleles: implications for clopidogrel response testing.

    PubMed

    Scott, Stuart A; Tan, Qian; Baber, Usman; Yang, Yao; Martis, Suparna; Bander, Jeffrey; Kornreich, Ruth; Hulot, Jean-Sébastien; Desnick, Robert J

    2013-11-01

    CYP2C19 is involved in the metabolism of clinically relevant drugs, including the antiplatelet prodrug clopidogrel, which has prompted interest in clinical CYP2C19 genotyping. The CYP2C19∗4B allele is defined by both gain-of-function [c.-806C>T (∗17)] and loss-of-function [c.1A>G (∗4)] variants on the same haplotype; however, current genotyping and sequencing assays are unable to determine the phase of these variants. Thus, the aim of this study was to develop an assay that could rapidly detect and discriminate the related ∗4A, ∗4B, and ∗17 alleles. An allele-specific PCR assay, composed of four unique primer mixes that specifically interrogate the defining ∗17 and ∗4 variants, was developed by using samples (n = 20) with known genotypes, including the ∗4A, ∗4B, and/or ∗17 alleles. The assay was validated by testing 135 blinded samples, and the results were correlated with CYP2C19 genotyping and allele-specific cloning/sequencing. Importantly, among the six ∗4 carriers in the validation cohort, after allele-specific PCR testing both samples with a ∗1/∗4 genotype were reclassified to ∗1/∗4A, all three samples with a ∗4/∗17 genotype were reclassified to ∗1/∗4B, and a sample with a ∗4/∗17/∗17 genotype was reclassified to ∗4B/∗17. In conclusion, this rapid and robust allele-specific PCR assay can refine CYP2C19 genotyping and metabolizer phenotype classification by determining the phase of the defining ∗17 and ∗4 variants, which may have utility when testing CYP2C19 for clopidogrel response.

  9. 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.

  10. KRAS Mutation Test in Korean Patients with Colorectal Carcinomas: A Methodological Comparison between Sanger Sequencing and a Real-Time PCR-Based Assay

    PubMed Central

    Lee, Sung Hak; Chung, Arthur Minwoo; Lee, Ahwon; Oh, Woo Jin; Choi, Yeong Jin; Lee, Youn-Soo; Jung, Eun Sun

    2017-01-01

    Background Mutations in the KRAS gene have been identified in approximately 50% of colorectal cancers (CRCs). KRAS mutations are well established biomarkers in anti–epidermal growth factor receptor therapy. Therefore, assessment of KRAS mutations is needed in CRC patients to ensure appropriate treatment. Methods We compared the analytical performance of the cobas test to Sanger sequencing in 264 CRC cases. In addition, discordant specimens were evaluated by 454 pyrosequencing. Results KRAS mutations for codons 12/13 were detected in 43.2% of cases (114/264) by Sanger sequencing. Of 257 evaluable specimens for comparison, KRAS mutations were detected in 112 cases (43.6%) by Sanger sequencing and 118 cases (45.9%) by the cobas test. Concordance between the cobas test and Sanger sequencing for each lot was 93.8% positive percent agreement (PPA) and 91.0% negative percent agreement (NPA) for codons 12/13. Results from the cobas test and Sanger sequencing were discordant for 20 cases (7.8%). Twenty discrepant cases were subsequently subjected to 454 pyrosequencing. After comprehensive analysis of the results from combined Sanger sequencing–454 pyrosequencing and the cobas test, PPA was 97.5% and NPA was 100%. Conclusions The cobas test is an accurate and sensitive test for detecting KRAS-activating mutations and has analytical power equivalent to Sanger sequencing. Prescreening using the cobas test with subsequent application of Sanger sequencing is the best strategy for routine detection of KRAS mutations in CRC. PMID:28013534

  11. 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…

  12. 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.

  13. 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.

  14. 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

  15. Mycobacterium growth indicator tube testing in conjunction with the AccuProbe or the AMPLICOR-PCR assay for detecting and identifying mycobacteria from sputum samples.

    PubMed Central

    Ichiyama, S; Iinuma, Y; Yamori, S; Hasegawa, Y; Shimokata, K; Nakashima, N

    1997-01-01

    We have compared the ability of the Mycobacterium Growth Indicator Tube (MGIT) system, a new culture method with an oxygen-sensitive fluorescent sensor, to recover mycobacteria from sputum samples with the abilities of egg-based medium and the Septi-Chek AFB system. We have also assessed the clinical utility of the AccuProbe or the AMPLICOR-PCR assay to directly identify Mycobacterium tuberculosis complex and M. avium-M. intracellulare complex (MAC) from positive MGITs. From 382 sputum samples, 99 isolates of M. tuberculosis complex and 20 isolates of MAC were recovered. The MGIT system had the highest recovery rates for M. tuberculosis complex (97.0%) and MAC (100%), compared to recovery rates of 51.5 and 65.0%, respectively, with the egg-based medium and 81.8 and 85.0%, respectively, with the Septi-Chek AFB system. The shortest recovery times were also achieved with the MGIT system: 16.6 days for M. tuberculosis complex and 12.0 days for MAC, compared to 27.1 and 20.1 days, respectively, with the egg-based medium and 21.4 and 13.2 days, respectively, with the Septi-Chek AFB system. The AccuProbe identified 74 (77.1%) of the 96 M. tuberculosis complex-positive MGITs and 17 (85.0%) of the 20 MAC-positive vials. The AMPLICOR system correctly identified 94 (97.9%) of the 96 M. tuberculosis complex-positive MGITs and all 20 MAC-positive vials. Therefore, the MGIT system used in conjunction with the AMPLICOR system is a rapid and sensitive method for detecting and identifying M. tuberculosis complex and MAC isolates from sputum samples. PMID:9230374

  16. Development of Dual TaqMan Based One-Step rRT-PCR Assay Panel for Rapid and Accurate Diagnostic Test of MERS-CoV: A Novel Human Coronavirus, Ahead of Hajj Pilgrimage

    PubMed Central

    Hashemzadeh, Mohammad Sadegh; Rasouli, Rahimeh; Zahraei, Bentolhoda; Izadi, Morteza; Tat, Mahdi; Saadat, Seyed Hassan; Najarasl, Mohammad; Khansari Nejad, Behzad; Dorostkar, Ruhollah

    2016-01-01

    Background Coronaviruses (CoVs) are large ribonucleic acid (RNA) viruses causing primarily respiratory disease in humans. A novel human coronavirus, subsequently named middle east respiratory syndrome coronavirus (MERS-CoV), was first reported in Saudi Arabia in September of 2012. With increasing numbers of infections and deaths from MERS-CoV, development of a rapid and reliable kit was crucial to prevent further spread of MERS-CoV. Objectives In this study, we present two real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays for in-house rapid and sensitive diagnostic testing of MERS-CoV, detecting the regions upstream of the envelope gene (upE) and open reading frame (ORF) 1b, respectively, for initial screening and final confirmation of MERS-CoV infection, as recommended by the world health organization (WHO). Materials and Methods In this experimental study, acquiring patient samples was difficult; thus, according to WHO recommendations and standard protocols, we synthesized RNA sequences of upE and ORF1b genes as the template signatures and TaqMan based-diagnostic rRT-PCR assays were carried out using these synthetic genes for detection of MERS-CoV. In this research, we also inaugurated a cell-free system to transcribe these RNA sequences using the DNA templates synthesized. Results The upE and ORF1b based one-step rRT-PCR assays were optimized by testing several times via different synthetic RNAs, and validation results were highly successful. The sensitivity obtained for upE was fewer than ten copies of RNA template per reaction and for ORF1b was 50 or fewer copies per reaction. Conclusions This study showed that the developed rRT-PCR assays are rapid, reliable, reproducible, specific, sensitive, and simple tools for detection of MERS-CoV. Finally, a kit consisting of two assay signatures and controls was assembled, which can be distributed to public health laboratories in Iran to support international MERS-CoV surveillance and public

  17. Being a qualitative researcher.

    PubMed

    Holloway, Immy; Biley, Francis C

    2011-07-01

    This article, from a keynote address, is the result of some of the things which I learned about qualitative research during my many years of doing and teaching it. The main point I make is that qualitative researchers should present a good story which is based on evidence but focused on meaning rather than measurement. In qualitative inquiry, the researchers' selves are involved, their experiences become a resource. Researchers cannot distance themselves from the other participants, although they cannot fully present their meaning and experience. I also discuss voice, paradigm, and innovation as potentially problematic issues in qualitative research. These are terms often used but not always examined for their meaning in qualitative inquiry. If researchers are aware and sensitive, rather than overemotional or self-absorbed, qualitative research can be enlightening, person-centered, and humanistic.

  18. The qualitative research proposal.

    PubMed

    Klopper, H

    2008-12-01

    Qualitative research in the health sciences has had to overcome many prejudices and a number of misunderstandings, but today qualitative research is as acceptable as quantitative research designs and is widely funded and published. Writing the proposal of a qualitative study, however, can be a challenging feat, due to the emergent nature of the qualitative research design and the description of the methodology as a process. Even today, many sub-standard proposals at post-graduate evaluation committees and application proposals to be considered for funding are still seen. This problem has led the researcher to develop a framework to guide the qualitative researcher in writing the proposal of a qualitative study based on the following research questions: (i) What is the process of writing a qualitative research proposal? and (ii) What does the structure and layout of a qualitative proposal look like? The purpose of this article is to discuss the process of writing the qualitative research proposal, as well as describe the structure and layout of a qualitative research proposal. The process of writing a qualitative research proposal is discussed with regards to the most important questions that need to be answered in your research proposal with consideration of the guidelines of being practical, being persuasive, making broader links, aiming for crystal clarity and planning before you write. While the structure of the qualitative research proposal is discussed with regards to the key sections of the proposal, namely the cover page, abstract, introduction, review of the literature, research problem and research questions, research purpose and objectives, research paradigm, research design, research method, ethical considerations, dissemination plan, budget and appendices.

  19. 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

  20. Critiquing qualitative research.

    PubMed

    Beck, Cheryl Tatano

    2009-10-01

    The ability to critique research is a valuable skill that is fundamental to a perioperative nurse's ability to base his or her clinical practice on evidence derived from research. Criteria differ for critiquing a quantitative versus a qualitative study (ie, statistics are evaluated in a quantitative study, but not in a qualitative study). This article provides on guidelines for assessing qualitative research. Excerpts from a published qualitative research report are summarized and then critiqued. Questions are provided that help evaluate different sections of a research study (eg, sample, data collection methods, data analysis).

  1. Overview of qualitative research.

    PubMed

    Grossoehme, Daniel H

    2014-01-01

    Qualitative research methods are a robust tool for chaplaincy research questions. Similar to much of chaplaincy clinical care, qualitative research generally works with written texts, often transcriptions of individual interviews or focus group conversations and seeks to understand the meaning of experience in a study sample. This article describes three common methodologies: ethnography, grounded theory, and phenomenology. Issues to consider relating to the study sample, design, and analysis are discussed. Enhancing the validity of the data, as well reliability and ethical issues in qualitative research are described. Qualitative research is an accessible way for chaplains to contribute new knowledge about the sacred dimension of people's lived experience.

  2. 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.

  3. 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

  4. Barriers to and acceptability of provider-initiated HIV testing and counselling and adopting HIV-prevention behaviours in rural Uganda: a qualitative study.

    PubMed

    Kiene, Susan M; Sileo, Katelyn; Wanyenze, Rhoda K; Lule, Haruna; Bateganya, Moses H; Jasperse, Joseph; Nantaba, Harriet; Jayaratne, Kia

    2015-02-01

    In Uganda, a nationwide scale-up of provider-initiated HIV testing and counselling presents an opportunity to deliver HIV-prevention services to large numbers of people. In a rural Ugandan hospital, focus group discussions and key informant interviews were conducted with outpatients receiving provider-initiated HIV testing and counselling and staff to explore the HIV-prevention information, motivation and behavioural skills strengths and weaknesses, and community-level and structural barriers to provider-initiated HIV testing and counselling acceptability and HIV prevention among this population. Strengths and weakness occurred at all levels, and results suggest brief client-centred interventions during provider-initiated HIV testing and counselling may be an effective approach to increase prevention behaviours in outpatient settings.

  5. Acceptability Study on HIV Self-Testing among Transgender Women, Men who Have Sex with Men, and Female Entertainment Workers in Cambodia: A Qualitative Analysis

    PubMed Central

    Pal, Khuondyla; Ngin, Chanrith; Tuot, Sovannary; Chhoun, Pheak; Ly, Cheaty; Chhim, Srean; Luong, Minh-Anh; Tatomir, Brent; Yi, Siyan

    2016-01-01

    Background In Cambodia, HIV prevalence is high while HIV testing rates remain low among transgender women (TG women), men who have sex with men (MSM), and female entertainment workers (FEW). Introducing self-testing for HIV to these key populations (KPs) could potentially overcome the under-diagnosis of HIV and significantly increase testing rates and receipt of the results, and thus could decrease transmission. Therefore, this study aimed to determine the acceptability of HIV self-testing (HIVST) among these three categories of KPs. Methods This study was conducted through focus group discussions (FGDs) with TG women, MSM, and FEW in Phnom Penh city, Kampong Cham, Battambang, and Siem Reap provinces of Cambodia. Convenience sampling was used to recruit the participants. Two FGDs (six participants in each FGD) were conducted in each target group in each study site, totaling 24 FGDs (144 participants). Thematic analysis was performed to identify common or divergent patterns across the target groups. Results Almost all participants among the three groups (TG women, MSM, and FEW) had not heard about HIVST, but all of them expressed willingness to try it. They perceived HIVST as confidential, convenient, time-saving, and high-tech. Barriers to obtaining HIVST included cost, access, administration technique, embarrassment, and fear of pain. The majority preferred counseling before and after testing. Conclusions Participants showed high willingness to use and acceptability of HIVST due to its confidentiality/privacy and convenience even if it is not linked to a confirmatory test or care and treatment. Notwithstanding, to increase HIVST, the target groups would need affordable self-test kits, education about how to perform HIVST and read results, assurance about accuracy and reliability of HIVST, and provision of post-test counseling and facilitation of linkage to care and treatment. PMID:27829064

  6. 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…

  7. 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…

  8. 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…

  9. Part two: Qualitative research.

    PubMed

    Quick, J; Hall, S

    2015-01-01

    This second article in the series Spotlight on Research focuses on qualitative research, its applications, principles and methodologies. It provides an insight into how this approach can be used within the perioperative setting and gives advice for practitioners looking to undertake a qualitative research study.

  10. 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

  11. Value of repeat testing using Cepheid GeneXpert CT/NG for indeterminate PCR results when diagnosing Chlamydia trachomatis and Neisseria gonorrhoeae.

    PubMed

    Parcell, Benjamin J; Ratnayake, Lasantha; Kaminski, Geraldine; Olver, William J; Yirrell, David L

    2015-01-01

    Nucleic acid amplification tests (NAATs) are the most sensitive method for diagnosing chlamydia and gonorrhoea. We use the COBAS 4800 CT/NG combined assay (Roche Molecular Diagnostics, CA, USA), and whilst the majority of samples yield definitive results, a small proportion are reported as indeterminate. In these instances, it is usual practice to request repeat samples which delays diagnosis. This audit was twofold: first to establish the proportion of indeterminate results with current NAAT testing requiring re-sampling. Second, to determine whether a second NAAT such as Cepheid GeneXpert CT/NG assay (Cepheid, CA, USA) could be used on initial indeterminate samples to resolve indeterminate results, therefore reducing need for repeat sampling. During 2012, 144/21,931 (0.66%) samples were indeterminate for Neisseria gonorrhoeae, Chlamydia trachomatis or both, and a repeat sample was received in only 51.77% of patients with final results being delayed for more than 24 h. Over the next six months, there were 77/9472 (0.81%) indeterminate results. After an evaluation and introduction of the Cepheid assay, the number of indeterminate results fell to 9 (0.10%). Thus, use of the Cepheid assay significantly reduced indeterminate results, reduced reliance on a repeat sampling and significantly improved turnaround time, laboratory workflow and patient experience.

  12. 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.

  13. HIV Self-Testing among Men Who Have Sex with Men (MSM) in the UK: A Qualitative Study of Barriers and Facilitators, Intervention Preferences and Perceived Impacts

    PubMed Central

    Witzel, T. Charles; Rodger, Alison J.; Burns, Fiona M.; Rhodes, Tim; Weatherburn, Peter

    2016-01-01

    Introduction Innovative strategies, such as HIV self-testing (HIVST), could increase HIV testing rates and diagnosis. Evidence to inform the design of an HIVST intervention in the UK is scarce with very little European data on this topic. This study aims to understand values and preferences for HIVST interventions targeting MSM in the UK. We explore the acceptability of HIVST among MSM in the context of known barriers and facilitators to testing for HIV; assess preferences for, and the concerns about, HIVST. Methods Six focus group discussions (FGD) were conducted with 47 MSM in London, Manchester and Plymouth. HIVST as a concept was discussed and participants were asked to construct their ideal HIVST intervention. OraQuickTM and BioSureTM kits were then demonstrated and participants commented on procedure, design and instructions. FGDs were recorded and transcribed verbatim, then analysed thematically. Results Convenience and confidentiality of HIVST was seen to facilitate testing. Issues with domestic privacy problematised confidentiality. HIVST kits and instructions were thought to be unnecessarily complicated, and did not cater to the required range of abilities. The window period was the most important element of an HIVST, with strong preference for 4th generation testing. Kits which used a blood sample were more popular than those using saliva due to higher perceived accuracy although phobia of needles and/or blood meant some would only access HIVST if a saliva sample option was available. A range of access options was important to maintain convenience and privacy. HIVST kits were assumed to increase frequency of testing, with concerns related to the dislocation of HIVST from sexual health care pathways and services. Discussion Utility of HIVST arises from relatively high levels of confidentiality and convenience. Until 4th generation assays are available HIVST will be seen as supplementary in a UK context. PMID:27611777

  14. 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

  15. Low risk of contamination with automated and manual excision of dried blood spots for HIV DNA PCR testing in the routine laboratory.

    PubMed

    Driver, Glenn A; Patton, Janet C; Moloi, Jackie; Stevens, Wendy S; Sherman, Gayle G

    2007-12-01

    In low resource settings the inability to diagnose HIV in infants early presents a major obstacle to providing care for HIV-infected children. Dried blood spot samples offer a solution to the scarcity of skills available for venesecting young infants but pose challenges to laboratories because their processing requirements are distinct from that of the liquid blood samples widely used for viral detection assays. Different methods of excising 287 paired HIV-positive and HIV-negative dried blood spot samples (n=574) for testing by the Roche Amplicor HIV-1 DNA assay version 1.5 were assessed. A manual punch in conjunction with three different cleaning protocols (n=372) and an automated punch (BSD 1000 GenePunch) using a single cleaning protocol (n=202) was assessed for the risk of cross contamination between samples. A single false positive result obtained using the automated punch may have been attributable to contamination during the excision step of the assay. Excision of dried blood spot samples is associated with a very low risk of cross contamination regardless of the instrument or cleaning intervention used. The process of excising dried blood spot samples should not compromise the results of HIV viral detection assays performed on dried blood spots in routine laboratories which is encouraging for increasing access to an accurate diagnosis of HIV in infants.

  16. 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.

  17. 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

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

    2011-05-01

    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.

  18. Qualitative research in thanatology.

    PubMed

    Carverhill, Philip A

    2002-04-01

    A new research paradigm has been emerging which holds significant potential for the field of death studies. The qualitative project is a diverse collection of methodologies that focuses its interests on the words, narratives, and stories of individuals and groups. Part of its appeal may lie in the inherent closeness of fit between qualitative inquiry and applied work with the dying and the bereaved. The author introduces the individual articles in this special issue and outlines the development of the project as well as some current issues in qualitative research in thanatology.

  19. Multiplex PCR Testing Detection of Higher-than-Expected Rates of Cervical Mycoplasma, Ureaplasma, and Trichomonas and Viral Agent Infections in Sexually Active Australian Women▿

    PubMed Central

    McIver, Christopher J.; Rismanto, Nikolas; Smith, Catherine; Naing, Zin Wai; Rayner, Ben; Lusk, M. Josephine; Konecny, Pamela; White, Peter A.; Rawlinson, William D.

    2009-01-01

    Knowing the prevalence of potential etiologic agents of nongonococcal and nonchlamydial cervicitis is important for improving the efficacy of empirical treatments for this commonly encountered condition. We describe four multiplex PCRs (mPCRs), designated VDL05, VDL06, VDL07, and VDL09, which facilitate the detection of a wide range of agents either known to be or putatively associated with cervicitis, including cytomegalovirus (CMV), enterovirus (EV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2) (VDL05); Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma genitalium, and Mycoplasma hominis (VDL06); Chlamydia trachomatis, Trichomonas vaginalis, Treponema pallidum, and group B streptococci (VDL07); and adenovirus species A to E (VDL09). The mPCRs were used to test 233 cervical swabs from 175 women attending a sexual-health clinic in Sydney, Australia, during 2006 and 2007. The agents detected alone or in combination in all cervical swabs (percentage of total swabs) included CMV (6.0), EV (2.1), EBV (2.6), VZV (4.7), HSV-1 (2.6), HSV-2 (0.8), HSV-2 and VZV (0.4), U. parvum (57.0), U. urealyticum (6.1), M. genitalium (1.3), M. hominis (13.7), C. trachomatis (0.4), T. vaginalis (3.4), and group B streptococci (0.4). Adenovirus species A to E and T. pallidum were not detected. These assays are adaptable for routine diagnostic laboratories and provide an opportunity to measure the true prevalence of microorganisms potentially associated with cervicitis and other genital infections. PMID:19261782

  20. 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

  1. Are women ready for the new cervical screening protocol in England? A systematic review and qualitative synthesis of views about human papillomavirus testing

    PubMed Central

    Hendry, M; Pasterfield, D; Lewis, R; Clements, A; Damery, S; Neal, R D; Adke, R; Weller, D; Campbell, C; Patnick, J; Sasieni, P; Hurt, C; Wilson, S; Wilkinson, C

    2012-01-01

    Background: A new protocol for human papillomavirus (HPV) testing within the UK cervical screening programme commenced in April 2011, creating new patient experiences. This is the first review to synthesise a substantial body of international evidence of women's information needs, views and preferences regarding HPV testing. We aimed to inform the development of educational materials to promote informed choice, reduce anxiety and improve disease control. Methods: We searched 12 bibliographic databases. Two reviewers independently screened papers and assessed study quality; disagreements were resolved by discussion. Results were extracted verbatim and authors' findings treated as primary data. Studies were synthesised collaboratively using framework methods. Results: We synthesised findings from 17 studies. Women had overwhelmingly negative concerns; an HPV diagnosis was daunting, had associated problems of disclosure of a sexually transmitted infection (STI), impacted on relationships and provoked fear of stigmatisation. Nevertheless, many thought HPV testing could be a preferable alternative to repeat cytology. Knowledge was poor; women struggled to interpret limited information in the context of existing knowledge about STIs and cervical cancer. Conclusion: Women are likely to be poorly informed, have limited understanding and many unanswered questions. This could increase anxiety and reduce ability to make informed choices, presenting a substantial challenge for those who design and provide information. PMID:22699825

  2. New qualitative detection methods of genetically modified potatoes.

    PubMed

    Watanabe, Takahiro; Kuribara, Hideo; Mishima, Takashi; Kikuchi, Hiroyuki; Kodama, Takashi; Futo, Satoshi; Kasama, Kikuko; Toyota, Akie; Nouno, Masanori; Saita, Ayako; Takahashi, Kunihiko; Hino, Akihiro; Akiyama, Hiroshi; Maitani, Tamio; Kubo, Misao

    2004-09-01

    In Japan, 8 lines of genetically modified (GM) potato (2 lines of NewLeaf potato; NL, 3 lines of NewLeaf Plus potato; NLP, and 3 lines of NewLeaf Y potato; NLY) have already been authorized as safe for use in foods and feeds. We have developed polymerase chain reaction (PCR) methods for the qualitative detection of the GM potatoes for the screening and the identification of NL, NLP and NLY. The gene encoding uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) was used as a taxon specific gene. We designed the primer pair to detect the cryIIIA genes as a screening method for GM potatoes because the gene should be inserted in all 8 lines of the GM potatoes. For identification of NL, NLP and NLY, we further designed three specific primer pairs for the different recombinant DNAs (r-DNA) specifically introduced into NL, NLP, or NLY. In addition, to identify the 3 lines of NLY that have been introduced with the same r-DNA, the three line-specific primer pairs for the border sequence between the r-DNA and genomic DNA of NLY 3 lines were designed. Six lines of GM potato used as the test material were specifically identified using the each primer pair under the same PCR condition. The detection limits of all the GM potatoes should be approximately 0.1%. Furthermore, the specificity and reproducibility of the methods were confirmed in a six-laboratory collaborative study.

  3. Qualitative Case Study Guidelines

    DTIC Science & Technology

    2013-11-01

    methods in public relations and marketing communications. New York, Routledge 166-185 13. Denzin , N. K. (1978) The Research Act: A Theoretical...Introduction to Sociological Methods. 2nd ed. New York, McGraw-Hill 14. Denzin , N. K. and Lincoln, Y. S. (2011) The SAGE Handbook of Qualitative...The Art of Science. In: Denzin , N. K. and Lincoln, Y. S. (eds.) Handbook of Qualitative Research. Thousand Oaks, Sage 19. GAO (1990) Case Study

  4. 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.

  5. 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

  6. PCR for capsular typing of Haemophilus influenzae.

    PubMed Central

    Falla, T J; Crook, D W; Brophy, L N; Maskell, D; Kroll, J S; Moxon, E R

    1994-01-01

    A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine. Images PMID:7814470

  7. Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR.

    PubMed

    de Morais, Rayana Carla Silva; Gonçalves, Suênia da Cunha; Costa, Pietra Lemos; da Silva, Kamila Gaudêncio; da Silva, Fernando José; Silva, Rômulo Pessoa E; de Brito, Maria Edileuza Felinto; Brandão-Filho, Sinval Pinto; Dantas-Torres, Filipe; de Paiva-Cavalcanti, Milena

    2013-04-01

    Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.

  8. The use of triangulation in qualitative research.

    PubMed

    Carter, Nancy; Bryant-Lukosius, Denise; DiCenso, Alba; Blythe, Jennifer; Neville, Alan J

    2014-09-01

    Triangulation refers to the use of multiple methods or data sources in qualitative research to develop a comprehensive understanding of phenomena (Patton, 1999). Triangulation also has been viewed as a qualitative research strategy to test validity through the convergence of information from different sources. Denzin (1978) and Patton (1999) identified four types of triangulation: (a) method triangulation, (b) investigator triangulation, (c) theory triangulation, and (d) data source triangulation. The current article will present the four types of triangulation followed by a discussion of the use of focus groups (FGs) and in-depth individual (IDI) interviews as an example of data source triangulation in qualitative inquiry.

  9. 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...

  10. 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...

  11. Doctors' perspectives on PSA testing illuminate established differences in prostate cancer screening rates between Australia and the UK: a qualitative study

    PubMed Central

    Pickles, Kristen; Carter, Stacy M; Rychetnik, Lucie; Entwistle, Vikki A

    2016-01-01

    Objectives To examine how general practitioners (GPs) in the UK and GPs in Australia explain their prostate-specific antigen (PSA) testing practices and to illuminate how these explanations are similar and how they are different. Design A grounded theory study. Setting Primary care practices in Australia and the UK. Participants 69 GPs in Australia (n=40) and the UK (n=29). We included GPs of varying ages, sex, clinical experience and patient populations. All GPs interested in participating in the study were included. Results GPs' accounts revealed fundamental differences in whether and how prostate cancer screening occurred in their practice and in the broader context within which they operate. The history of prostate screening policy, organisational structures and funding models appeared to drive more prostate screening in Australia and less in the UK. In Australia, screening processes and decisions were mostly at the discretion of individual clinicians, and varied considerably, whereas the accounts of UK GPs clearly reflected a consistent, organisationally embedded approach based on local evidence-based recommendations to discourage screening. Conclusions The GP accounts suggested that healthcare systems, including historical and current organisational and funding structures and rules, collectively contribute to how and why clinicians use the PSA test and play a significant role in creating the mindlines that GPs employ in their clinic. Australia's recently released consensus guidelines may support more streamlined and consistent care. However, if GP mindlines and thus routine practice in Australia are to shift, to ultimately reduce unnecessary or harmful prostate screening, it is likely that other important drivers at all levels of the screening process will need to be addressed. PMID:27920082

  12. Validation of quantitative and qualitative methods for detecting allergenic ingredients in processed foods in Japan.

    PubMed

    Sakai, Shinobu; Adachi, Reiko; Akiyama, Hiroshi; Teshima, Reiko

    2013-06-19

    A labeling system for food allergenic ingredients was established in Japan in April 2002. To monitor the labeling, the Japanese government announced official methods for detecting allergens in processed foods in November 2002. The official methods consist of quantitative screening tests using enzyme-linked immunosorbent assays (ELISAs) and qualitative confirmation tests using Western blotting or polymerase chain reactions (PCR). In addition, the Japanese government designated 10 μg protein/g food (the corresponding allergenic ingredient soluble protein weight/food weight), determined by ELISA, as the labeling threshold. To standardize the official methods, the criteria for the validation protocol were described in the official guidelines. This paper, which was presented at the Advances in Food Allergen Detection Symposium, ACS National Meeting and Expo, San Diego, CA, Spring 2012, describes the validation protocol outlined in the official Japanese guidelines, the results of interlaboratory studies for the quantitative detection method (ELISA for crustacean proteins) and the qualitative detection method (PCR for shrimp and crab DNAs), and the reliability of the detection methods.

  13. New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Herthnek, David; Bölske, Göran

    2006-01-01

    Background Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. Results Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. Conclusion We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used. PMID:17020599

  14. "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

  15. Comparison of the novel human papillomavirus 4 auto-capillary electrophoresis test with the hybrid capture 2 assay and with the PCR HPV typing set test in the detection of high-risk HPV including HPV 16 and 18 genotypes in cervical specimens.

    PubMed

    Hong, Jin Hwa; Song, Seung Hun; Kim, Jong Kee; Han, Jeong Hyun; Lee, Jae Kwan

    2009-08-01

    The aim of this study was to compare the novel human papillomavirus (HPV) detection method, the HPV 4 Auto-capillary Electrophoresis (ACE) test with the hybrid capture (HC) 2 assay for the detection of high-risk HPVs. In addition, we compared the HPV 4 ACE test with the polymerase chain reaction HPV Typing Set test for the detection of HPV 16 and HPV 18 genotypes. One hundred ninety-nine cervical swab samples obtained from women with previous abnormal Pap smears were subjected to testing with the three HPV tests. The HPV 4 ACE test and the HC 2 assay showed substantial agreement for detection of high-risk HPVs (85.4%, kappa=0.71). The HPV 4 ACE test also showed substantial agreement with the PCR HPV Typing Set test in the detection of HPV 16 and HP V 18 genotypes (89.9%, kappa=0.65). In correlation with cytologic results, the sensitivities and specificities of the HPV 4 ACE test and HC 2 assay were 92.9% vs. 92.9% and 48.1% vs. 50.8%, respectively, when high-grade squamous intraepithelial lesions were regarded as abnormal cytologies. The novel HPV 4 ACE test is a valuable tool for the detection of high-risk HPVs and for genotyping of HPV 16 and HPV 18.

  16. “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

  17. 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.

  18. PCR in forensic genetics.

    PubMed

    Morling, Niels

    2009-04-01

    Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more and more advanced, special investigations in cases concerning crime, paternity, relationship, disaster victim identification etc. The present review gives an update on the use of DNA investigations in forensic genetics.

  19. 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.

  20. Presumptive and Confirmatory Drug Tests

    ERIC Educational Resources Information Center

    Anderson, Craig

    2005-01-01

    The majority of drug testings are first done with some kind of qualitative presumptive tests. After the qualitative presumptive tests are performed, a confirmatory test is necessary which demonstrates to the students the rigor needed to conclusively identify a substance.

  1. Validation of iQ-Check E. coli O157:H7 real-time PCR test kit for detection of Escherichia coli O157:H7 in selected foods.

    PubMed

    Lauer, Wendy F; Tymciu, Sylvie; Sidi, Caroline D; Sonigo, Pierre

    2009-01-01

    iQ-Check E. coli O157:H7 (Bio-Rad Laboratories, Hercules, CA) is a real-time PCR kit for detection of E. coli O157:H7 from selected foods. Specific fluorescent oligonucleotide probes are used to detect target DNA during the amplification, by hybridizing to the amplicons. These fluorescent probes are linked to a fluorophore which fluoresces only when hybridized to the target sequence. Three foods (ground beef, apple cider, fresh spinach) were selected to compare the performance of iQ-Check E. coli O157:H7 to the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) reference method for ground beef and the U.S. Food and Drug Administration/Bacteriological Analytical Manual reference method for apple cider and fresh spinach. Three protocols were tested in this study: a shortened 8 h primary enrichment in buffered peptone water (BPW), a 24 h enrichment in BPW, and an enrichment in appropriate reference method enrichment broth. The iQ-Check E. coli O157:H7 method was able to identify more true/confirmed positive samples than the reference method. Inclusivity and exclusivity rates of the method were 100%. iQ-Check E. coli O157:H7 performed as expected when minor procedural variations were introduced, validating the ruggedness of the method. There was no significant difference observed in performance over the shelf life of the kit.

  2. Escherichia coli and Enterococcus spp. in rainwater tank samples: comparison of culture-based methods and 23S rRNA gene quantitative PCR assays.

    PubMed

    Ahmed, W; Richardson, K; Sidhu, J P S; Toze, S

    2012-10-16

    In this study, culture-based methods and quantitative PCR (qPCR) assays were compared with each other for the measurement of Escherichia coli and Enterococcus spp. in water samples collected from rainwater tanks in Southeast Queensland, Australia. Among the 50 rainwater tank samples tested, 26 (52%) and 46 (92%) samples yielded E. coli numbers as measured by EPA Method 1603 and E. coli 23S rRNA gene qPCR assay, respectively. Similarly, 49 (98%) and 47 (94%) samples yielded Enterococcus spp. numbers as measured by EPA Method 1600 and Enterococcus spp. 23S rRNA gene qPCR assay, respectively. The mean E. coli (2.49 ± 0.85) log(10) and Enterococcus spp. (2.72 ± 0.32) log(10) numbers as measured by qPCR assays were significantly (P < 0001) different than E. coli (0.91 ± 0.80) log(10) and Enterococcus spp. (1.86 ± 0.60) log(10) numbers as measured by culture-based method. Weak but significant correlations were observed between both EPA Method 1603 and the E. coli qPCR assay (r = 0.47, P = 0.0009), and EPA Method 1600 and the Enterococcus spp. qPCR assay (r = 0.42, P = 0.002). Good qualitative agreement was found between the culture-based method and the Enterococcus spp. qPCR assay in terms of detecting fecal pollution in water samples from the studied rainwater tanks. More research studies, however, are needed to shed some light on the discrepancies associated with the culture-based methods and qPCR assays for measuring fecal indicator bacteria.

  3. Individualised Qualitative Evaluation.

    ERIC Educational Resources Information Center

    O'Sullivan, Denis

    1987-01-01

    The author discusses student evaluation in relation to adult and continuing education programs offered by the Department of Adult Education, University College, Cork. He highlights the need for a more individualized and interactive approach to evaluation, allowing the student to benefit from qualitative feedback in the process of being evaluated.…

  4. First Semester Qualitative Analysis

    ERIC Educational Resources Information Center

    DeLap, James H.

    1969-01-01

    Describes a two-hour laboratory course entitled "Chemical Periodicity offered first semester of the freshman year. Three cation groups, one anion group, and a final unkown salt are qualitatively analyzed. Course fosters scientific thinking in experimentation by encouraging student-initiated schemes of analyses rather than "cookbook schemes. (RR)

  5. 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…

  6. Advances in Qualitative Research.

    ERIC Educational Resources Information Center

    1998

    This document contains five papers from a symposium on advances in qualitative research in human resource development (HRD). "Case Study and Its Virtuoso Possibilities" (Verna J. Willis) asserts that the case study method is particularly well suited for research in HRD because its creative and investigative possibilities have not yet…

  7. 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…

  8. 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)

  9. Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

    PubMed

    Chaouachi, Maher; Alaya, Akram; Ali, Imen Ben Haj; Hafsa, Ahmed Ben; Nabi, Nesrine; Bérard, Aurélie; Romaniuk, Marcel; Skhiri, Fethia; Saïd, Khaled

    2013-01-01

    KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

  10. Lyme Disease Tests

    MedlinePlus

    ... Borrelia Antibodies, IgM/IgG; Borrelia DNA Detection by PCR Related tests: CSF Analysis All content on Lab ... fluid (CSF) . In special cases, polymerase chain reaction (PCR) testing may be performed on a sample because ...

  11. 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...

  12. Investigation of false-positive reactions for CBH351 maize in screening PCR analysis.

    PubMed

    Monma, Kimio; Moriuchi, Rie; Sagi, Naoki; Ichikawa, Hisatsugu; Satoh, Kazue; Tobe, Takashi; Kamata, Kunihiro

    2006-02-01

    Examination for CBH351 maize was conducted by the qualitative polymerase chain reaction (PCR) method in maize grain and maize processed foods obtained in the Tokyo area. The numbers of samples possibly positive in the screening test were 7 of 22 (31.8%) for maize grain samples, 4 of 14 (28.6%) for semi-processed foods, 11 of 30 (36.7%) for canned products, 3 of 30 (10.0%) for maize snacks, 3 of 4 (75%) for tacos and 1 of 3 (33.3%) for tortillas. However, CBH351 maize was not detected in the confirmation test. Therefore, the results of the screening test were false-positive. Since the reaction might have been caused by the base sequences of the 3'-end of primers CaM03-5' and CBH02-3' used in the screening test, a new primer pair was designed. The PCR products obtained with the new primer pair TMC2-5'--TMS2-3' were specific for CBH351 and were not obtained with barley, wheat, rice, RRS, Bt11, or Event176. Thus, the new primer pair shows high specificity. CBH351 maize was detected from samples containing at least 0.05% CBH 351 maize DNA by using this primer pair.

  13. 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.

  14. Pregnancy test

    MedlinePlus

    ... eds. Henry's Clinical Diagnosis and Management by Laboratory Methods . 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:chap 25. Read More Ectopic pregnancy HCG blood test - qualitative HCG blood test - quantitative HCG in urine Miscarriage ...

  15. Misuse of PCR assay for diagnosis of mollusc protistan infections.

    PubMed

    Burreson, Eugene M

    2008-06-19

    Polymerase chain reaction (PCR) assays are useful tools for pathogen surveillance, but they are only proxy indications of pathogen presence in that they detect a DNA sequence. To be useful for detection of actual infections, PCR assays must be thoroughly tested for sensitivity and specificity, and ultimately validated against a technique, typically histology, which allows visualization of the parasite in host tissues. There is growing use of PCR assays for pathogen surveillance, but too often the assumption is made that a positive PCR result verifies an infection in a tested host. This assumption is valid only if the assay has been properly validated for the geographic area and for the hosts examined. Researchers should interpret unvalidated PCR assay results with caution, and editors and reviewers should insist that robust validations support all assertions that PCR results confirm infections.

  16. 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 ...

  17. 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

    2015-05-05

    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.

  18. Qualitative science policy.

    PubMed

    Mitcham, Carl

    2007-12-01

    Qualitative research struggles against a tide of quantitative methods. To assist in this struggle, it is useful to consider the historical and philosophical origins of quantitative methods as well as criticisms that have been raised against them. Although these criticisms have often been restricted to discussions in the philosophy of science, they have become increasingly prominent in debates regarding science policy. This article thus reviews current science policy debates concerning scientific autonomy and the linear model of science-society relationships. Then, having considered the multiple meanings of quality, it argues for a science policy reassessment of quantitative research, for deeper engagements between science policy and the social sciences, and finally, for a more explicit alliance between science policy and qualitative methods.

  19. Interviews in qualitative research.

    PubMed

    Peters, Kath; Halcomb, Elizabeth

    2015-03-01

    Interviews are a common method of data collection in nursing research. They are frequently used alone in a qualitative study or combined with other data collection methods in mixed or multi-method research. Semi-structured interviews, where the researcher has some predefined questions or topics but then probes further as the participant responds, can produce powerful data that provide insights into the participants' experiences, perceptions or opinions.

  20. Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA.

    PubMed

    Tang, Hui; Cai, Qingchun; Li, Hu; Hu, Peng

    2016-06-16

    Quantitative real-time PCR (qPCR) has been widely implemented for clinical hepatitis B viral load testing, but a lack of standardization and relatively poor precision hinder its usefulness. Droplet digital PCR (ddPCR) is a promising tool that offers high precision and direct quantification. In this study, we compared the ddPCR QX100 platform by Bio-Rad with the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA) to detect serial plasmid DNA dilutions of known concentrations as well as HBV DNA extracted from patient serum samples. Both methods showed a high degree of linearity and quantitative correlation. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays. Patient sample quantifications by ddPCR and qPCR were highly agreeable based on the Bland-Altman analysis. Collectively, our findings demonstrate that ddPCR offers improved analytical sensitivity and specificity for HBV measurements and is suitable for clinical HBV detection.

  1. Battling Malaria in Rural Zambia with Modern Technology: A Qualitative Study on the Value of Cell Phones, Geographical Information Systems, Asymptomatic Carriers and Rapid Diagnostic Tests to Identify, Treat and Control Malaria

    PubMed Central

    Isaksson, Arvid Lissel

    2014-01-01

    During the last decade much progress has been made in reducing malaria transmission in Macha, Southern Province, Zambia. Introduction of artemisinin combination therapies as well as mass screenings of asymptomatic carriers is believed to have contributed the most. When an endemic malaria situation is moving towards a non-endemic situation the resident population loses acquired immunity and therefore active case detection and efficient surveillance is crucial to prevent epidemic outbreaks. Our purpose was to evaluate the impact of cell phone surveillance and geographical information systems on malaria control in Macha. Furthermore, it evaluates what screening and treatment of asymptomatic carriers and implementation of rapid diagnostic tests in rural health care has led to. Ten in-depth semi-structured interviews, field observations and data collection were performed at the Macha Research Trust and at surrounding rural health centers. This qualitative method was inspired by rapid assessment procedure. The cell phone surveillance has been easily integrated in health care, and its integration with Geographical Information Systems has provided the ability to follow malaria transmission on a weekly basis. In addition, active case detection of asymptomatic carriers has been fruitful, which is reflected in it soon being applied nationwide. Furthermore, rapid diagnostic tests have provided rural health centers with reliable malaria diagnostics, thereby decreasing excessive malaria treatments and selection for drug resistance. This report reflects the importance of asymptomatic carriers in targeting malaria elimination, as well as development of effective surveillance systems when transmission decreases. Such an approach would be cost-efficient in the long run through positive effects in reduced child mortality and relief in health care. PMID:28299110

  2. Battling Malaria in Rural Zambia with Modern Technology: A Qualitative Study on the Value of Cell Phones, Geographical Information Systems, Asymptomatic Carriers and Rapid Diagnostic Tests to Identify, Treat and Control Malaria.

    PubMed

    Nygren, David; Isaksson, Arvid Lissel

    2014-02-04

    During the last decade much progress has been made in reducing malaria transmission in Macha, Southern Province, Zambia. Introduction of artemisinin combination therapies as well as mass screenings of asymptomatic carriers is believed to have contributed the most. When an endemic malaria situation is moving towards a non-endemic situation the resident population loses acquired immunity and therefore active case detection and efficient surveillance is crucial to prevent epidemic outbreaks. Our purpose was to evaluate the impact of cell phone surveillance and geographical information systems on malaria control in Macha. Furthermore, it evaluates what screening and treatment of asymptomatic carriers and implementation of rapid diagnostic tests in rural health care has led to. Ten in-depth semi-structured interviews, field observations and data collection were performed at the Macha Research Trust and at surrounding rural health centers. This qualitative method was inspired by rapid assessment procedure. The cell phone surveillance has been easily integrated in health care, and its integration with Geographical Information Systems has provided the ability to follow malaria transmission on a weekly basis. In addition, active case detection of asymptomatic carriers has been fruitful, which is reflected in it soon being applied nationwide. Furthermore, rapid diagnostic tests have provided rural health centers with reliable malaria diagnostics, thereby decreasing excessive malaria treatments and selection for drug resistance. This report reflects the importance of asymptomatic carriers in targeting malaria elimination, as well as development of effective surveillance systems when transmission decreases. Such an approach would be cost-efficient in the long run through positive effects in reduced child mortality and relief in health care.

  3. Learning and Teaching Qualitative Research with Qualitative Data Analysis Software.

    ERIC Educational Resources Information Center

    Mahlamaki-Kultanen, Seija

    This study examined the way qualitative data analysis software and virtual teaching methods can support the learning of qualitative research. Study methodology was based on phenomenology, and data were gathered in a pilot course on qualitative research methodology in which 22 adult part time graduate students participated. The course was built…

  4. A novel, sensitive and specific real-time PCR for the detection of traces of allergenic Brazil nut (Bertholletia excelsa) in processed foods.

    PubMed

    Röder, Martin; Filbert, Helene; Holzhauser, Thomas

    2010-11-01

    Food-allergic individuals have to strictly avoid the offending food because no causative immunotherapies are available. Thus, reliable labelling of allergenic ingredients or precautionary labelling of cross-contacts with potential allergens is of major importance. Verification of compliance with labelling requirements and identification of cross-contacts demand test methods that enable the specific and sensitive detection of the analyte. Brazil nut (Bertholletia excelsa) is such a food commodity with allergenic potential. We describe the development of a novel qualitative real-time polymerase chain reaction (PCR) specific for Brazil nut DNA and its comparison with a qualitative commercially available lateral flow device (LFD) that detects Brazil nut protein. Specificity was investigated with 58 foods, and no false-positive reactions were observed in real-time PCR. The sensitivity was investigated with spiked chocolate and incurred dough samples as well as cookies baked thereof. The simultaneous spiking of matrices with identical amounts of Brazil nut and peanut between 5 and 100,000 mg/kg allowed the verification of the spike quality with two peanut-specific enzyme-linked immunosorbent assay. The real-time PCR detected Brazil nut in all three matrices down to the lowest investigated spike level of 5 mg/kg. The real-time PCR results from the analysis of 15 retail samples were confirmed by LFD results and were in concordance with the labelling of products. The real-time PCR showed unparalleled specificity, and primary data indicated potentially quantitative features in spiked and retail samples. Because of entirely reproducible chemistry of this real-time PCR, this is the first generally available Brazil nut-specific detection method with an appropriate sensitivity to help avoid severe allergic reactions for Brazil nut-allergic individuals.

  5. 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.

  6. Ecosystems and People: Qualitative Insights

    EPA Science Inventory

    Both qualitative and quantitative techniques are crucial in researching human impacts from ecological changes. This matches the importance of ?mixed methods? approaches in other disciplines. Qualitative research helps explore the relevancy and transferability of the foundational ...

  7. 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 ...

  8. Development and validation of a rotor-gene real-time PCR assay for detection, identification, and quantification of Chlamydia trachomatis in a single reaction.

    PubMed

    Jalal, Hamid; Stephen, Hannah; Curran, Martin D; Burton, Janet; Bradley, Michelle; Carne, Christopher

    2006-01-01

    A multitarget real-time PCR (MRT-PCR) for detection of Chlamydia trachomatis DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of C. trachomatis DNA in a single reaction, (ii) detection of all genovars of C. trachomatis without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median C. trachomatis load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different C. trachomatis-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of C. trachomatis and in the quantitative format for study of the pathogenesis of C. trachomatis-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload.

  9. Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples.

    PubMed

    Mokhtari, W; Nsaibia, S; Gharbi, A; Aouni, M

    2013-02-01

    Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen. A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T(m) = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C(q)) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea. The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples.

  10. A novel nested PCR for the diagnosis of calicivirus infections in the cat.

    PubMed

    Marsilio, Fulvio; Di Martino, Barbara; Decaro, Nicola; Buonavoglia, Canio

    2005-01-05

    A novel nested PCR (nPCR) assay is reported on the diagnosis of the feline calicivirus (FCV) infection. The test was performed on 47 ocular and 40 pharyngeal swabs collected from 47 cats with respiratory syndrome; among the 87 samples examined, 18 ocular and 23 pharyngeal swabs were positive in nPCR. The nPCR sensitivity was compared to other diagnostic techniques such as virus isolation on cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR). The nPCR was more sensitive than the virus isolation and RT-PCR; therefore, it can be used for calicivirosis diagnosis in cats.

  11. Microfluidics-Based PCR for Fusion Transcript Detection.

    PubMed

    Chen, Hui

    2016-01-01

    The microfluidic technology allows the production of network of submillimeter-size fluidic channels and reservoirs in a variety of material systems. The microfluidic-based polymerase chain reaction (PCR) allows automated multiplexing of multiple samples and multiple assays simultaneously within a network of microfluidic channels and chambers that are co-ordinated in controlled fashion by the valves. The individual PCR reaction is performed in nanoliter volume, which allows testing on samples with limited DNA and RNA. The microfluidics devices are used in various types of PCR such as digital PCR and single molecular emulsion PCR for genotyping, gene expression, and miRNA expression. In this chapter, the use of a microfluidics-based PCR for simultaneous screening of 14 known fusion transcripts in patients with leukemia is described.

  12. Blood grouping based on PCR methods and agarose gel electrophoresis.

    PubMed

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  13. Approximate Qualitative Temporal Reasoning

    DTIC Science & Technology

    2001-01-01

    i.e., their boundaries can be placed in such a way that they coincide with the cell boundaries of the appropriate partition of the time-line. (Think of...respect to some appropriate partition of the time-line. For example, I felt well on Saturday. When I measured my temperature I had a fever on Monday and on...Bittner / Approximate Qualitative Temporal Reasoning 49 [27] I. A. Goralwalla, Y. Leontiev , M. T. Özsu, D. Szafron, and C. Combi. Temporal granularity for

  14. 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

  15. Miniaturized technology for DNA typing: cassette PCR.

    PubMed

    Manage, Dammika P; Pilarski, Linda M

    2015-01-01

    With the smaller size, low cost, and rapid testing capabilities, miniaturized lab-on-a-chip devices can change the way medical diagnostics are currently performed in the health-care system. We have demonstrated such a device that is self-contained, simple, disposable, and inexpensive. It is capable of performing DNA amplification on an inexpensive instrument suitable for near point of care settings. This technology will enable on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices. Our device, a gel capillary cassette, termed cassette PCR, contains capillary reaction units each holding a defined primer set, with arrays of capillary reaction units for simultaneously detecting multiple targets. With the exception of the sample to be tested, each capillary reaction unit holds all the reagents needed for PCR in a desiccated form that can be stored at room temperature for up to 3 months and even longer in colder conditions. It relies on capillary forces for sample delivery of microliter volumes through capillaries, hence avoiding the need for pumps or valves. In the assembled cassette, the wax architecture supporting the capillaries melts during the PCR and acts as a vapor barrier as well as segregating capillaries with different primer sets. No other chip sealing techniques are required. Cassette PCR accepts raw samples such as urine, genital swabs, and blood. The cassette is made with off-the-shelf components and contains integrated positive and negative controls.

  16. Occurrence of a Sequence in Marine Cyanophages Similar to That of T4 g20 and Its Application to PCR-Based Detection and Quantification Techniques†

    PubMed Central

    Fuller, Nicholas J.; Wilson, William H.; Joint, Ian R.; Mann, Nicholas H.

    1998-01-01

    Viruses are ubiquitous components of marine ecosystems and are known to infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus Synechococcus. A conserved region from the cyanophage genome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant similarity to a gene encoding a capsid assembly protein (gp20) from the enteric coliphage T4. The results of a comparison of gene 20 sequences from three cyanomyoviruses and T4 allowed us to design two degenerate PCR primers, CPS1 and CPS2, which specifically amplified a 165-bp region from the majority of cyanomyoviruses tested. A competitive PCR (cPCR) analysis revealed that cyanomyovirus strains could be accurately enumerated, and it was demonstrated that quantification was log-linear over ca. 3 orders of magnitude. Different calibration curves were obtained for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial inaccuracies in estimates of phage abundance in natural assemblages. Further sequence analysis of cyanomyovirus gene 20 homologs would be necessary in order to design primers which do not exhibit phage-to-phage variability in priming efficiency. It was demonstrated that PCR products of the correct size could be amplified from seawater samples following 100× concentration and even directly without any prior concentration. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophages in natural assemblages. Further optimization of procedures may ultimately lead to a sensitive assay which can be used to analyze natural cyanophage populations both quantitatively (by cPCR) and qualitatively following phylogenetic analysis of amplified products. PMID:9603813

  17. Comparison of the immunofluorescence assay with RT-PCR and nested PCR in the diagnosis of canine distemper.

    PubMed

    Jóźwik, A; Frymus, T

    2005-05-01

    Two pairs of primers were prepared, both localized within the sequences of the nucleoprotein gene (NP) of canine distemper virus (CDV). A number of experiments were done to optimize the conditions of RT-PCR and nested PCR methods. The nucleic acids of the Onderstepoort, Rockborn, Snyder Hill and Lederle strains of CDV could be detected with these primers. However, they did not react with the sequences of the Edmonston strain of the measles virus. The detection limit for RT-PCR was 10 TCID50 and for nested PCR 0.1 TCID50 of CDV. The RT-PCR was able to demonstrate the nucleic acid of CDV in the blood of all seven puppies vaccinated with a modified live virus. Blood samples of 23 dogs clinically suspected of distemper were examined by RT-PCR combined with nested PCR, and the results were compared with the detection of the CDV antigen in the smears from the mucous membranes by the direct immunofluorescence (IF) test. Of the 23 dogs, 12 were positive in nested PCR, six in the IF assay, and only two in single RT-PCR. It is concluded that nested PCR seems to be the most sensitive method for ante-mortem diagnosis of canine distemper, especially in its subacute or chronic forms.

  18. 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…

  19. 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...

  20. Detection of bovine Herpesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue-based diagnostic test of bovine encephalitis.

    PubMed

    Ferrari, H F; Luvizotto, M C R; Rahal, P; Cardoso, T C

    2007-12-01

    The aim of this study was to evaluate the application of PCR technique for the detection of BoHV-5 in routinely formalin-fixed, paraffin-embedded brain tissues in 20 naturally infected calves affected by fatal meningoencephalitis. Brains were divided into two halves, one kept fresh for virus isolation and PCR assay, targeting the glycoprotein C gene from BoHV-5 genome. The other half brain, corresponding to posterior cortex region, was submitted to formalin fixation and embedded into paraffin blocks for microscopic evaluation and total DNA isolation. Most of the slides showed severe multifocal non-supurative encephalitis with neuronal degeneration, neurophagia, and no acidophilic intranuclear inclusions could be found in neurons and glial. The 20 fresh samples were confirmed, by virus isolation and PCR assay, as having the BoHV-5 virus and, respective glicoprotein C sequence, while 15 of 20 formalin-fixed, paraffin-embedded samples were considered positive for the same analysis. The results revealed the first description of PCR efficiency, applied to formalin-fixed, paraffin-embedded brain collected from naturally infected calves, improving the detection of BoHV-5 from archival samples in South America.

  1. Conducting qualitative research within Clinical Trials Units: avoiding potential pitfalls.

    PubMed

    Cooper, Cindy; O'Cathain, Alicia; Hind, Danny; Adamson, Joy; Lawton, Julia; Baird, Wendy

    2014-07-01

    The value of using qualitative research within or alongside randomised controlled trials (RCTs) is becoming more widely accepted. Qualitative research may be conducted concurrently with pilot or full RCTs to understand the feasibility and acceptability of the interventions being tested, or to improve trial conduct. Clinical Trials Units (CTUs) in the United Kingdom (UK) manage large numbers of RCTs and, increasingly, manage the qualitative research or collaborate with qualitative researchers external to the CTU. CTUs are beginning to explicitly manage the process, for example, through the use of standard operating procedures for designing and implementing qualitative research with trials. We reviewed the experiences of two UK Clinical Research Collaboration (UKCRC) registered CTUs of conducting qualitative research concurrently with RCTs. Drawing on experiences gained from 15 studies, we identify the potential for the qualitative research to undermine the successful completion or scientific integrity of RCTs. We show that potential problems can arise from feedback of interim or final qualitative findings to members of the trial team or beyond, in particular reporting qualitative findings whilst the trial is on-going. The problems include: We make recommendations for improving the management of qualitative research within CTUs.

  2. Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer.

    PubMed

    Saito, Keiko; Drgon, Tomás; Robledo, José A F; Krupatkina, Danara N; Vasta, Gerardo R

    2002-11-01

    Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.

  3. Hepatitis C Test

    MedlinePlus

    ... Hepatitis C Antibody; Anti-HCV; HCV-PCR; HCV-RNA; Hepatitis C Viral Load Formal name: Viral Hepatitis C Antibody Screen; Viral Hepatitis C RNA by PCR; Hepatitis C Virus Genotype Related tests: ...

  4. Comparison between ICT and PCR for diagnosis of Chlamydia trachomatis.

    PubMed

    Khan, E R; Hossain, M A; Paul, S K; Mahmud, C; Hasan, M M; Rahman, M M; Nahar, K; Kubayashi, N

    2012-04-01

    Chlamydia trachomatis is an obligate intracellular gram-negative bacterium which is the most prevalent cause of bacterial sexually transmitted infections (STI). The present study was carried to diagnose genital Chlamydia trachomatis infection among women of reproductive age, attending Mymensingh Medical College Hospital, during July 2009 to June 2010 by Immunochromatographic test (ICT) and Polymerase chain reaction (PCR). A total of 70 females were included in this study. Out of 70 cases 56 were symptomatic and 14 asymptomatic. Endocervical swabs were collected from each of the cases and examined by Immunochromatographic test (ICT) for antigen detection and Polymerase chain reaction (PCR) for detection of endogenous plasmid-based nucleic acid. A total 29(41.4%) of the cases were found positive for C. trachomatis either by ICT or PCR. Of the 56 symptomatic cases, 19(33.9%) were found ICT positive and 17(30.4%) were PCR positive. Among 14 asymptomatic females, 2(14.3%) were ICT positive and none were PCR positive. Though PCR is highly sensitive but a total of twelve cases were found ICT positive but PCR negative. It may be due to presence of plasmid deficient strain of C trachomatis which could be amplified by ompA based (Chromosomal gene) multiplex PCR.

  5. Situating methodology within qualitative research.

    PubMed

    Kramer-Kile, Marnie L

    2012-01-01

    Qualitative nurse researchers are required to make deliberate and sometimes complex methodological decisions about their work. Methodology in qualitative research is a comprehensive approach in which theory (ideas) and method (doing) are brought into close alignment. It can be difficult, at times, to understand the concept of methodology. The purpose of this research column is to: (1) define qualitative methodology; (2) illuminate the relationship between epistemology, ontology and methodology; (3) explicate the connection between theory and method in qualitative research design; and 4) highlight relevant examples of methodological decisions made within cardiovascular nursing research. Although there is no "one set way" to do qualitative research, all qualitative researchers should account for the choices they make throughout the research process and articulate their methodological decision-making along the way.

  6. Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis.

    PubMed

    Keid, L B; Soares, R M; Vasconcellos, S A; Salgado, V R; Megid, J; Richtzenhain, L J

    2010-07-17

    The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

  7. 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

  8. 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.

  9. Qualitative research in transfusion medicine.

    PubMed

    Arnold, E; Lane, S

    2011-10-01

    Transfusion medicine research has traditionally employed quantitative methods to answer clinical research questions. Increasingly, qualitative research methods are being used in the field to address a wide variety of research questions in areas such as blood donation, transfusion practices and policy development. This article describes the key characteristics, methodologies and methods of qualitative research and draws on examples to show how qualitative research approaches have been applied in the field of transfusion medicine. It is hoped that this overview will inform and encourage the application of qualitative research in the field of transfusion medicine.

  10. Individual Protection Testing (Task 5 - Qualitative Fit Test Simulants)

    DTIC Science & Technology

    1990-04-01

    Threshold, Index Disposition Compound ppm (20 C) Code(a) Acetaldehyde 0.3 4,300,000 8 Acetic acid 1.0 15,000 A Acetic anhydride 0.36 14,611 A Acetone 140...8.1 0.15 0.002 Isoamyl acetate 81 2190 41 0.6 Methyl salicylate 3.7 100 1.9 0.03 Nonanoic acid 5.5 148 2.8 0.04 Skatole 1.2 32 0.61 0.01 The following...derivative of salicylic acid other than aspirin (Erickson, 1982). Its use in the fragrance industry amounts to about 90,000 lb/year (Opdyke, 1978). In

  11. Mentoring Qualitative Research Authors Globally: "The Qualitative Report" Experience

    ERIC Educational Resources Information Center

    Chenail, Ronald J.; St. George, Sally; Wulff, Dan; Duffy, Maureen; Laughlin, Martha; Warner, Kate; Sahni, Tarmeen

    2007-01-01

    Authoring quality qualitative inquiry is a challenge for most researchers. A lack of local mentors can make writing even more difficult. To meet this need, The "Qualitative Report (TQR)" has helped authors from around the world develop their papers into published articles. "TQR" editorial team members will discuss the history of the journal, their…

  12. COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

    PubMed Central

    GÖKMEN, Tülin GÜVEN; SOYAL, Ayben; KALAYCI, Yıldız; ÖNLEN, Cansu; KÖKSAL, Fatih

    2016-01-01

    SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM), serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT) and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP) of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively). The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01%) than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis. PMID:27680169

  13. 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.

  14. 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

  15. Multiplex PCR identification of Taenia spp. in rodents and carnivores.

    PubMed

    Al-Sabi, Mohammad N S; Kapel, Christian M O

    2011-11-01

    The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.

  16. [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.

  17. 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.

  18. 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.

  19. 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…

  20. Applications of competitor RNA in diagnostic reverse transcription-PCR.

    PubMed

    Kleiboeker, Steven B

    2003-05-01

    Detection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.

  1. 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.

  2. 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.

  3. Beyond PICO: the SPIDER tool for qualitative evidence synthesis.

    PubMed

    Cooke, Alison; Smith, Debbie; Booth, Andrew

    2012-10-01

    Standardized systematic search strategies facilitate rigor in research. Current search tools focus on retrieval of quantitative research. In this article we address issues relating to using existing search strategy tools, most typically the PICO (Population, Intervention, Comparison, Outcome) formulation for defining key elements of a review question, when searching for qualitative and mixed methods research studies. An alternative search strategy tool for qualitative/mixed methods research is outlined: SPIDER (Sample, Phenomenon of Interest, Design, Evaluation, Research type). We used both the SPIDER and PICO search strategy tools with a qualitative research question. We have used the SPIDER tool to advance thinking beyond PICO in its suitable application to qualitative and mixed methods research. However, we have highlighted once more the need for improved indexing of qualitative articles in databases. To constitute a viable alternative to PICO, SPIDER needs to be refined and tested on a wider range of topics.

  4. Comparison of standard, quantitative and digital PCR in the detection of enterotoxigenic Bacteroides fragilis.

    PubMed

    Purcell, Rachel V; Pearson, John; Frizelle, Frank A; Keenan, Jacqueline I

    2016-09-30

    Gut colonization with enterotoxigenic Bacteroides fragilis (ETBF) appears to be associated with the development of colorectal cancer. However, differences in carriage rates are seen with various testing methods and sampling sites. We compared standard PCR, SYBR green and TaqMan quantitative PCR (qPCR) and digital PCR (dPCR) in detecting the B. fragilis toxin (bft) gene from cultured ETBF, and from matched luminal and faecal stool samples from 19 colorectal cancer patients. Bland-Altman analysis found that all three quantitative methods performed comparably in detecting bft from purified bacterial DNA, with the same limits of detection (<1 copy/μl). However, SYBR qPCR under-performed compared to TaqMan qPCR and dPCR in detecting bft in clinical stool samples; 13/38 samples were reported positive by SYBR, compared to 35 and 36 samples by TaqMan and dPCR, respectively. TaqMan qPCR and dPCR gave bft copy numbers that were 48-fold and 75-fold higher for the same samples than SYBR qPCR, respectively (p < 0.001). For samples that were bft-positive in both fecal and luminal stools, there was no difference in relative abundance between the sites, by any method tested. From our findings, we recommend the use of TaqMan qPCR as the preferred method to detect ETBF from clinical stool samples.

  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.

  6. 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)

  7. A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis.

    PubMed

    Boonyayatra, S; Fox, L K; Besser, T E; Sawant, A; Gay, J M; Raviv, Z

    2012-01-01

    The focus of the current research was to develop real-time PCR assays with improved sensitivity and the capacity to simultaneously speciate the 3 most common mycoplasma mastitis agents: Mycoplasma bovis, Mycoplasma californicum, and Mycoplasma bovigenitalium. Real-time PCR was chosen because it provides rapid results. Partial 16S rRNA gene sequencing was used as the gold standard for evaluating candidate real-time PCR assays. To ascertain the real-time PCR assay specificity, reference strains of Mycoplasma species, Acholeplasma axanthum, and common gram-positive and gram-negative mastitis pathogens were tested. No cross-reactions were observed. Mycoplasma spp. isolated from bovine milk samples (n=228) and other organ sites (n=40) were tested by the real-time PCR assays and the partial 16S rRNA gene sequencing assay. Overall accuracy of this novel real-time PCR was 98.51%; 4 of 228 isolates identified as M. bovis by the partial 16S rRNA gene sequencing assay were identified as both M. bovis and M. californicum by real-time PCR. Subsequent amplicon sequencing suggested the presence of both M. bovis and M. californicum in these 4 samples. Using a cycle threshold of 37, the detection limits for real-time PCR were 10 copies of DNA template for both M. bovis and M. bovigenitalium, and 1 copy for M. californicum. This real-time PCR assay is a diagnostic technique that may be used as a screening tool or as a confirmation test for mycoplasma mastitis.

  8. Qualitative research: adding drive and dimension to clinical research.

    PubMed

    Rusinová, Katerĭna; Pochard, Frédéric; Kentish-Barnes, Nancy; Chaize, Marine; Azoulay, Elie

    2009-01-01

    Qualitative research and its methods stem from the social sciences and can be used to describe and interpret complex phenomena that involve individuals' views, beliefs, preferences, and subjective responses to places and people. Thus, qualitative research explores the many subjective factors that may influence patient outcomes, staff well-being, and healthcare quality, yet fail to lend themselves to the hypothesis-testing approach that characterizes quantitative research. Qualitative research is valuable in the intensive care unit to explore organizational and cultural issues and to gain insight into social interactions, healthcare delivery processes, and communication. Qualitative research generates explanatory models and theories, which can then serve to devise interventions, whose efficacy can be studied quantitatively. Thus, qualitative research works synergistically with quantitative research, providing new impetus to the research process and a new dimension to research findings. Qualitative research starts with conceptualizing the research question, choosing the appropriate qualitative strategy, and designing the study; rigorous methods specifically designed for qualitative research are then used to conduct the study, analyze the data, and verify the findings. The researcher is the data-collecting instrument, and the data are the participants' words and behaviors. Data coding methods are used to describe experiences, discover themes, and build theories. In this review, we outline the rationale and methods for conducting qualitative research to inform critical care issues. We provide an overview of available qualitative methods and explain how they can work in close synergy with quantitative methods. To illustrate the effectiveness of combining different research methods, we will refer to recent qualitative studies conducted in the intensive care unit.

  9. Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

    PubMed

    Greenspoon, S A; Sykes, K L V; Ban, J D; Pollard, A; Baisden, M; Farr, M; Graham, N; Collins, B L; Green, M M; Christenson, C C

    2006-12-20

    Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the

  10. Qualitative Process Theory.

    DTIC Science & Technology

    1984-07-01

    write a heat flow process that violates energy conservation and transfers " caloric fluid" between the source and destination. The assumptions made about...removed in ease of ex1-0ClCits. Seco nd, if’ thle program is drawNing concilsions that rely criticaillyoi atClrsum in, then1 it IIos’t test ss

  11. 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.

  12. Qualitative tools and experimental philosophy

    PubMed Central

    Andow, James

    2016-01-01

    Abstract Experimental philosophy brings empirical methods to philosophy. These methods are used to probe how people think about philosophically interesting things such as knowledge, morality, and freedom. This paper explores the contribution that qualitative methods have to make in this enterprise. I argue that qualitative methods have the potential to make a much greater contribution than they have so far. Along the way, I acknowledge a few types of resistance that proponents of qualitative methods in experimental philosophy might encounter, and provide reasons to think they are ill-founded.

  13. Optimized PCR assay for detection of white spot syndrome virus (WSSV).

    PubMed

    Nunan, Linda M; Lightner, Donald V

    2011-01-01

    A rapid PCR assay for detection of white spot syndrome virus (WSSV) was developed based on the nested PCR procedure described by Lo et al. (1996) and outlined as the recommended PCR diagnostic assay in the Manual of Diagnostic Tests for Aquatic Animals published by the Office of International Epizootics (OIE, 2009). The optimized procedure incorporated the second step primers used in the nested WSSV PCR. By adjusting the annealing temperature and shortening the cycling times, this modified assay is substantially faster and as sensitive as the recommended OIE protocol. The modified PCR test was compared directly to the two-step nested PCR protocol and a modified nested procedure. The sensitivity of the published assay was determined by template dilutions of semi-purified WSSV virions that had been quantitated using real-time PCR for detection of WSSV. Various isolates were tested using the modified procedure, to ensure that the assay was able to detect WSSV from different geographical locations.

  14. PCR detection of Babesia ovata from questing ticks in Japan.

    PubMed

    Sivakumar, Thillaiampalam; Tattiyapong, Muncharee; Okubo, Kazuhiro; Suganuma, Keisuke; Hayashida, Kyoko; Igarashi, Ikuo; Zakimi, Satoshi; Matsumoto, Kotaro; Inokuma, Hisashi; Yokoyama, Naoaki

    2014-04-01

    Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n=1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (β-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100 fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described β-tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata.

  15. Prospective comparison of the diagnostic potential of real-time PCR, double-sandwich enzyme-linked immunosorbent assay for galactomannan, and a (1-->3)-beta-D-glucan test in weekly screening for invasive aspergillosis in patients with hematological disorders.

    PubMed

    Kawazu, Masahito; Kanda, Yoshinobu; Nannya, Yasuhito; Aoki, Katsunori; Kurokawa, Mineo; Chiba, Shigeru; Motokura, Toru; Hirai, Hisamaru; Ogawa, Seishi

    2004-06-01

    The establishment of an optimal noninvasive method for diagnosing invasive aspergillosis (IA) is needed to improve the management of this life-threatening infection in patients with hematological disorders, and a number of noninvasive tests for IA that target different fungal components, including galactomannan, (1-->3)-beta-d-glucan (BDG), and Aspergillus DNA, have been developed. In this study, we prospectively evaluated the diagnostic potential of three noninvasive tests for IA that were used in a weekly screening strategy: the double-sandwich enzyme-linked immunosorbent assay (ELISA) for galactomannan (Platelia Aspergillus), a real-time PCR assay for Aspergillus DNA (GeniQ-Asper), and an assay for BDG (beta-glucan Wako). We analyzed 149 consecutive treatment episodes in 96 patients with hematological disorders who were at high risk for IA and diagnosed 9 proven IA cases, 2 probable IA cases, and 13 possible invasive fugal infections. In a receiver-operating characteristic (ROC) analysis, the area under the ROC curve was greatest for ELISA, using two consecutive positive results (0.97; P = 0.036 for ELISA versus PCR, P = 0.055 for ELISA versus BDG). Based on the ROC curve, the cutoff for the ELISA could be reduced to an optical density index (O.D.I.) of 0.6. With the use of this cutoff for ELISA and cutoffs for PCR and BDG that give a comparable level of specificity, the sensitivity/specificity/positive predictive value/negative predictive value of the ELISA and the PCR and BDG tests were 1.00/0.93/0.55/1.00, 0.55/0.93/0.40/0.96, and 0.55/0.93/0.40/0.96, respectively. In conclusion, among these weekly screening tests for IA, the double-sandwich ELISA test was the most sensitive at predicting the diagnosis of IA in high-risk patients with hematological disorders, using a reduced cutoff of 0.6 O.D.I.

  16. Prospective Comparison of the Diagnostic Potential of Real-Time PCR, Double-Sandwich Enzyme-Linked Immunosorbent Assay for Galactomannan, and a (1→3)-β-d-Glucan Test in Weekly Screening for Invasive Aspergillosis in Patients with Hematological Disorders

    PubMed Central

    Kawazu, Masahito; Kanda, Yoshinobu; Nannya, Yasuhito; Aoki, Katsunori; Kurokawa, Mineo; Chiba, Shigeru; Motokura, Toru; Hirai, Hisamaru; Ogawa, Seishi

    2004-01-01

    The establishment of an optimal noninvasive method for diagnosing invasive aspergillosis (IA) is needed to improve the management of this life-threatening infection in patients with hematological disorders, and a number of noninvasive tests for IA that target different fungal components, including galactomannan, (1→3)-β-d-glucan (BDG), and Aspergillus DNA, have been developed. In this study, we prospectively evaluated the diagnostic potential of three noninvasive tests for IA that were used in a weekly screening strategy: the double-sandwich enzyme-linked immunosorbent assay (ELISA) for galactomannan (Platelia Aspergillus), a real-time PCR assay for Aspergillus DNA (GeniQ-Asper), and an assay for BDG (β-glucan Wako). We analyzed 149 consecutive treatment episodes in 96 patients with hematological disorders who were at high risk for IA and diagnosed 9 proven IA cases, 2 probable IA cases, and 13 possible invasive fugal infections. In a receiver-operating characteristic (ROC) analysis, the area under the ROC curve was greatest for ELISA, using two consecutive positive results (0.97; P = 0.036 for ELISA versus PCR, P = 0.055 for ELISA versus BDG). Based on the ROC curve, the cutoff for the ELISA could be reduced to an optical density index (O.D.I.) of 0.6. With the use of this cutoff for ELISA and cutoffs for PCR and BDG that give a comparable level of specificity, the sensitivity/specificity/positive predictive value/negative predictive value of the ELISA and the PCR and BDG tests were 1.00/0.93/0.55/1.00, 0.55/0.93/0.40/0.96, and 0.55/0.93/0.40/0.96, respectively. In conclusion, among these weekly screening tests for IA, the double-sandwich ELISA test was the most sensitive at predicting the diagnosis of IA in high-risk patients with hematological disorders, using a reduced cutoff of 0.6 O.D.I. PMID:15184460

  17. 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.

  18. Joint association analysis of bivariate quantitative and qualitative traits.

    PubMed

    Yuan, Mengdie; Diao, Guoqing

    2011-11-29

    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.

  19. 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.

  20. Multiplex PCR to detect four different tomato-infecting pathogens.

    PubMed

    Quintero-Vásquez, Gabriela Alejandra; Bazán-Tejeda, María Luisa; Martínez-Peñafiel, Eva; Kameyama-Kawabe, Luis; Bermúdez-Cruz, Rosa María

    2013-07-01

    This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200-800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.

  1. Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project.

    PubMed

    Hein, Ingeborg; Flekna, Gabriele; Krassnig, Martina; Wagner, Martin

    2006-09-01

    A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).

  2. Specific PCR product primer design using memetic algorithm.

    PubMed

    Yang, Cheng-Hong; Cheng, Yu-Huei; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2009-01-01

    To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150-300 bps and 500-800 bps, and two different methods of calculating T(m) for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near-optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma-pd/.

  3. Rapid PCR amplification of DNA utilizing Coriolis effects.

    PubMed

    Mårtensson, Gustaf; Skote, Martin; Malmqvist, Mats; Falk, Mats; Asp, Allan; Svanvik, Nicke; Johansson, Arne

    2006-08-01

    A novel polymerase chain reaction (PCR) method is presented that utilizes Coriolis and centrifugal effects, produced by rotation of the sample disc, in order to increase internal circulatory rates, and with them temperature homogenization and mixing speeds. A proof of concept has been presented by testing a rapid 45-cycle PCR DNA amplification protocol. During the repeated heating and cooling that constitutes a PCR process, the 100 microL samples were rotated at a speed equivalent to an effective acceleration of gravity of 7,000 g. A cycle time of 20.5 s gave a total process time of 15 min to complete the 45 cycles. A theoretical and numerical analysis of the resulting flow, which describes the increased mixing and temperature homogenization, is presented. The device gives excellent reaction speed efficiency, which is beneficial for rapid PCR.

  4. Development and evaluation of event-specific quantitative PCR method for genetically modified soybean A2704-12.

    PubMed

    Takabatake, Reona; Akiyama, Hiroshi; Sakata, Kozue; Onishi, Mari; Koiwa, Tomohiro; Futo, Satoshi; Minegishi, Yasutaka; Teshima, Reiko; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

    2011-01-01

    A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (C(f)), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined C(f) values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.

  5. Molecular characterization of Corynebacterium pseudotuberculosis isolates using ERIC-PCR.

    PubMed

    Guimarães, Alessandro de Sá; Dorneles, Elaine Maria Seles; Andrade, Giovanna Ivo; Lage, Andrey Pereira; Miyoshi, Anderson; Azevedo, Vasco; Gouveia, Aurora Maria Guimarães; Heinemann, Marcos Bryan

    2011-12-15

    Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.

  6. Evaluation of Rice Resistance to Southern Rice Black-Streaked Dwarf Virus and Rice Ragged Stunt Virus through Combined Field Tests, Quantitative Real-Time PCR, and Proteome Analysis

    PubMed Central

    Wang, Zhenchao; Yu, Lu; Jin, Linhong; Wang, Wenli; Zhao, Qi; Ran, Longlu; Li, Xiangyang; Chen, Zhuo; Guo, Rong; Wei, Yongtian; Yang, Zhongcheng; Liu, Enlong; Hu, Deyu; Song, Baoan

    2017-01-01

    Diseases caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) considerably decrease grain yield. Therefore, determining rice cultivars with high resistance to SRBSDV and RRSV is necessary. In this study, rice cultivars with high resistance to SRBSDV and RRSV were evaluated through field trials in Shidian and Mangshi county, Yunnan province, China. SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to quantitatively detect virus gene expression levels in different rice varieties. The following parameters were applied to evaluate rice resistance: acre yield (A.Y.), incidence of infected plants (I.I.P.), virus load (V.L.), disease index (D.I.), and insect quantity (I.Q.) per 100 clusters. Zhongzheyou1 (Z1) and Liangyou2186 (L2186) were considered the most suitable varieties with integrated higher A.Y., lower I.I.P., V.L., D.I. and I.Q. features. In order to investigate the mechanism of rice resistance, comparative label-free shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomic approaches were applied to comprehensively describe the proteomics of rice varieties’ SRBSDV tolerance. Systemic acquired resistance (SAR)-related proteins in Z1 and L2186 may result in the superior resistance of these varieties compared with Fengyouxiangzhan (FYXZ). PMID:28241456

  7. 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.

  8. Qualitative research as methodical hermeneutics.

    PubMed

    Rennie, David L

    2012-09-01

    The proportion of publications of qualitative research in mainstream psychology journals is small. Thus, in terms of this important criterion, despite its recent rapid growth, qualitative research is marginalized in psychology. The author suggests that contributing to this situation is the lack of a coherent and unifying methodology of qualitative research methods that elucidates their credibility. He groups the many qualitative research methods into 3 main kinds, then applies to them 4 propositions offered as such a methodology: (1) Qualitative research is hermeneutical, entailing application of the method of the hermeneutic circle to text about experience and/or action. (2) Implicit in the use of the hermeneutic circle method is the activity of educing and articulating the meaning of text, an activity that modifies and interacts with C. S. Peirce's (1965, 1966) logical operations of abduction, theorematic deduction, and induction. (3) The cycling of these 4 moments enables demonstration, achieved rhetorically, of the validity of the understandings resulting from the exegesis of the text under study. (4) This demonstrative rhetoric is enhanced when researchers disclose reflexively those aspects of their perspectives they judge to have most relevant bearing on their understandings. The author compares abduction as formulated here with other recent uptakes of it. As an installment on the generality of the methodology, he explores its fit with the descriptive phenomenological psychological method, conversation analysis, and thematic analysis.

  9. 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.

  10. 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)

  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.

  12. 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 ...

  13. Doing Qualitative Research in Education Settings.

    ERIC Educational Resources Information Center

    Hatch, J. Amos

    This book, designed for novice researchers, provides a step-by-step guide to the development of a research project. It emphasizes learning how to do qualitative work and provides specific examples from real studies. The chapters are: (1) "Deciding To Do a Qualitative Study"; (2) "Designing Qualitative Studies"; (3) "Collecting Qualitative Data";…

  14. Considerations for Readers of Qualitative Research. Editorial.

    ERIC Educational Resources Information Center

    Ferguson, Dianne L.; Halle, James W.

    1995-01-01

    This article distinguishes between "using qualitative methods" and "doing qualitative research." It highlights the qualitative approaches of the authors of five articles in this issue and considers the challenges of this type of qualitative research manuscript for the editorial process. (DB)

  15. 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.

  16. Comparative PCR-based fingerprinting of Vibrio cholerae isolated in Malaysia.

    PubMed

    Shuan Ju Teh, Cindy; Thong, Kwai Lin; Osawa, Ro; Heng Chua, Kek

    2011-01-01

    Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.

  17. 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.

  18. 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.

  19. Improved PCR Amplification of Broad Spectrum GC DNA Templates.

    PubMed

    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.

  20. 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 m